Post on 14-Aug-2020
LC-ESI-MS-MS Analysis
of Non-enzymatic Posttranslational
Protein Modifications
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnbe rg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Dima Aldiab
aus Damaskus
Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Tag der mündlichen Prüfung: 30. 5. 2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatterin: Prof. Dr. Monika. Pischetsrieder
Zweitberichterstatter: Prof. Dr. Thomas. Drewello
Danksagung
Mein ganz herzlicher Dank gilt meiner Doktormutter Frau Prof. Dr. M. Pischetsrieder
für die Betreuung der Arbeit und für die freundliche und wissenschaftliche
Unterstützung während des Aufenthaltes in Deutschland. Mein Dank gilt weiterhin
dem Hochschulministerium in Syrien sowie der Tishreen-Universität, mit deren
Unterstützung die Durchführung dieser Abeit in Deutschland erst ermöglicht wurde.
Des Weiteren möchte ich mich bei Herrn Prof. Dr. T. Drewello für die Übernahme
des Zweitgutachtens bedanken, sowie Herrn Prof. Dr. M. Heinrich und Herrn PD Dr.
T. Göen für die Übernahme der Doktorprüfung.
Außerdem bedanke ich mich bei Herrn Dr. R. Waibel sowie dessen Mitarbeiterinnen
für die Aufnahme der NMR-Spektren.
Sehr herzlich danke ich Frau C. Meißner für die freundliche Hilfe während des
Aufenthaltes in Deutschland.
Für die Freundschaft und Hilfsbereitschaft bedanke ich mich herzlich bei meinen
jetztigen und ehemaligen Kollegen im Arbeitkreis. Liebe Kollegen, ich danke euch:
Andrea Wühr, Artur Kessler, Bianca Meyer, Florian Baum, Gregor Vollmer, Ingrid
Weigel, Jasmin Meltretter, Kerstin Augner, Leonie Atzenbeck, Martin Tutsch,
Mellanie Deckert, Nina Zänglein, Nadine Schneider, Rainer Bäuerlein, Sabrina
Gensberger, Sarah Elschenbroich, Stefan Mittelmaier, Tanja Sauer, Tobias Hoch,
Ulla Müller und Viola Breyer.
Der größte Dank geht an meinen Mann Eyad und meine Eltern, sowie an meine
Freunde in Syrien.
Schließlich gilt ein besonderer Dank meiner verstorbenen Mutter. Danke Mama,
ohne deine Liebe und Unterstützung wäre diese Arbeit und viele andere wichtige
Dinge nicht möglich gewesen.
I
Table of contents
1 Introduction .................................... ....................................................................... 1
1.1 The Maillard reaction ........................................................................................ 1
1.2 The phases of the Maillard reaction ................................................................. 2
1.3 The effects of Maillard reaction ........................................................................ 4
1.3.1 Nutrition and food safety ........................................................................... 4
1.3.2 Advanced Glycation End-Products (AGEs) ............................................... 5
1.3.3 Physiological effects of dietary MRPs ....................................................... 6
1.3.4 Beneficial Maillard products ...................................................................... 8
1.4 Prevention of the Maillard reaction ................................................................... 9
1.5 Milk ................................................................................................................. 10
1.5.1 Milk proteins ............................................................................................ 11
1.5.1.1 Casein .............................................................................................. 12
1.5.1.2 Whey protein .................................................................................... 13
1.5.2 Heat treatment of milk ............................................................................. 14
1.5.3 Effects of heat treatment on milk ............................................................. 15
1.5.3.1 Effects of heat treatment on milk protein.......................................... 15
1.5.3.2 Effect of heat treatment on lactose .................................................. 17
1.5.3.2.1 Lactose isomerisation/degradation ........................................... 17
1.5.3.2.2 Lactose and Maillard reaction ................................................... 17
1.5.3.3 Effects of heat treatment on milk fat ................................................ 20
1.5.3.4 Effects of heat treatments on vitamins ............................................. 21
1.5.3.5 Other effects of heat treatment ........................................................ 21
1.5.3.5.1 Flavor ........................................................................................ 21
1.5.3.5.2 Allergenicity .............................................................................. 21
1.5.4 Indicators for milk heat treatment ............................................................ 22
1.6 Liquid Chromatography Electro Spray Ionization Tandem Mass Spectrometry (LC-ESI-MS-MS) .................................................................................................. 23
1.7 Aims of the work: ............................................................................................ 26
2 Results and discussion .......................... ............................................................ 27
2.1 Selection of the marker compounds for protein modification .......................... 27
2.2 Standards synthesis ....................................................................................... 33
2.2.1 Synthesis of LacLys ................................................................................ 33
2.2.1.1 Affinity chromatography ................................................................... 36
2.2.1.1.1 Boronic acid affinity chromatography ........................................ 37
2.2.1.2 Hydrophilic interaction liquid chromatography (HILIC) ..................... 38
2.2.1.2.1 ZIC-HILIC column ..................................................................... 39
2.2.1.3 Identification of LacLys by 1H-NMR ................................................. 42
2.2.1.4 Determination of the purity of LacLys by 1H-NMR............................ 43
2.2.2 Synthesis of CML .................................................................................... 44
2.2.3 Synthesis of MeSO ................................................................................. 45
II
2.2.4 Trial to synthesize lysine aldehyde ......................................................... 47
2.2.5 Trial to synthesize Cys-SOH ................................................................... 48
2.2.6 OH-Trp .................................................................................................... 49
2.2.7 Discussion ............................................................................................... 49
2.3 Method development for the analysis of glycation and oxidation products in milk by LC-ESI-MS-MS ........................................................................................ 51
2.3.1 Introduction ............................................................................................. 51
2.3.2 ESI-MS-MS conditions ............................................................................ 52
2.3.2.1 Flow-dependent MS parameters ...................................................... 53
2.3.2.2 Compound-dependent MS parameters ............................................ 53
2.3.3 MS2 fragmentation of the investigated analytes ..................................... 55
2.3.4 Chromatographic seperation of CML, LacLys, MeSO and 5-OH-Trp using a C18 column ................................................................................................... 57
2.3.5 Glycation of α-LA in a milk model mixture ............................................... 59
2.3.6 Oxidation of α-LA in a milk model mixture ............................................... 64
2.3.7 Discussion ............................................................................................... 66
2.4 Detection of glycation and oxidation products in milk ..................................... 68
2.4.1 Analysis of MeSO and LacLys ................................................................ 69
2.4.1.1 Formation of MeSO during sample work-up .................................... 71
2.4.2 Analysis of 5-OH-Trp .............................................................................. 72
2.4.3 Analysis of CML ...................................................................................... 73
2.4.4 Separation of 5-OH-Trp, MeSO, CML and LacLys using a ZIC-HILIC column ............................................................................................................. 74
2.4.5 Comparison of the signal intensities using C18 or ZIC-HILIC columns ... 75
2.4.6 Analysis of milk samples by LC-MS-MS using a ZIC-HILIC column ....... 76
2.4.7 Discussion ............................................................................................... 78
2.5 Analysis of ornithine in milk proteins .............................................................. 81
2.5.1 Introduction ............................................................................................. 81
2.5.2 LC-ESI-MS-MS analysis of ornithine ....................................................... 82
2.5.3 Discussion ............................................................................................... 85
2.6 Method optimization for the detection of MeSO, CML and LacLys in proteins of processed milk ..................................................................................................... 86
2.6.1 Optimization of protein hydrolysis ........................................................... 86
2.6.2 Results .................................................................................................... 87
2.6.3 Discussion ............................................................................................... 93
2.7 Method validation for the quantification of LacLys, CML and MeSO in milk proteins ................................................................................................................ 97
2.7.1 Introduction ............................................................................................. 97
2.7.2 Quantitative analysis by standard addition .............................................. 98
2.7.3 Results .................................................................................................. 100
2.7.3.1 Within-day repeatability .................................................................. 100
2.7.3.2 Between-day repeatability .............................................................. 101
2.7.3.3 Recovery ........................................................................................ 102
2.7.3.4 Limit of detection and limit of quantitation ...................................... 105
2.7.4 Discussion ............................................................................................. 106
2.8 Quantitation .................................................................................................. 109
2.8.1 Introduction ........................................................................................... 109
2.8.2 Results .................................................................................................. 109
2.8.3 Discussion ............................................................................................. 111
3 Materials and methods ........................... .......................................................... 114
III
3.1 Materials and apparatus ............................................................................... 114
3.1.1 General materials .................................................................................. 114
3.1.2 Apparatus .............................................................................................. 114
3.1.3 Materials ............................................................................................... 115
Synthesis ................................................................................................... 115
Hydrolysis .................................................................................................. 116
Materials and equipments for liquid chromatography and mass spectrometry ................................................................................................................... 116
Oxidation and glycation in milk models ...................................................... 117
3.2 Buffers and solutions .................................................................................... 117
Milk resembling Phosphate Buffer Saline (PBS), pH 6.8 ............................... 117
Solutions for enzymatic hydrolysis (Hasenkopf et al) ................................. 118
Solutions for enzymatic hydrolysis (Delatour et al) .................................... 118
Solutions for the ninhydrin assay ................................................................... 119
Solutions to isolate lysyl oxidase .................................................................... 119
Standard`s concentration to determine the fragments of LacLys, CML, MeSO and 5-OH-Trp ................................................................................................. 120
Aqueous mobile phases for liquid chromatography ....................................... 120
Solutions for affinity chromatography ............................................................. 120
3.3 Methods ....................................................................................................... 121
3.3.1 Lactulosyllysine synthesis ..................................................................... 121
3.3.1.1. Trial to synthesize lactulosyllysine ................................................ 121
3.3.1.2 Chromatographic conditions for the separation of FMOC-LacLys . 121
3.3.1.3 Removal of the blocking group of FMOC-LacLys .......................... 122
3.3.1.4 Trial to purifiy LacLys using m-Aminoboronic acid ......................... 122
3.3.1.5 Synthesis of FMOC-LacLys ........................................................... 122
3.3.1.5.1 Fractionation of FMOC-LacLys on a ZIC-HILIC column ......... 123
3.3.1.5.2 Determination of the purity of LacLys by 1H-NMR .................. 123
3.3.2 Synthesis of Cbz-CML .......................................................................... 123
Removal of the blocking group of Cbz-CML .............................................. 124
3.3.3 Synthesis of methionine sulfoxide (MeSO) ........................................... 124
3.3.4 Trial to synthesize lysine aldehyde ....................................................... 124
3.3.5 Trial to synthesize cysteine sulfenic acid (Cys-SOH) ............................ 126
3.3.6 The conditions of liquid chromatography for the seperation of CML, MeSO, LacLys and 5-OH-Trp on a C18 column ............................................ 126
3.3.7 The conditions of liquid chromatography for the seperation of CML, MeSO, LacLys and 5-OH-Trp on a ZIC-HILIC column ................................... 127
3.3.8 Stimulation of glycation in a milk model mixture .................................... 127
3.3.9 Stimulation of oxidation in a milk model mixture ................................... 127
3.3.10 Preparation of milk samples for LC-MS-MS analysis .......................... 128
3.3.10.1 Milk defatting ................................................................................ 128
3.3.10.2 Removal of lactose and minerals from milk and milk model mixture ................................................................................................................... 128
3.3.10.3 Enzymatic protein hydrolysis (Hasenkopf et al) ........................... 128
3.3.10.4 Purification of protein hydrolyzate by ultrafiltration ....................... 128
3.3.10.5 Enzymatic protein hydrolysis (Delatour et al) ............................... 129
3.3.10.6 Acidic protein hydrolysis (Delatour et al) ...................................... 129
3.3.10.7 Purification of protein hydrolyzate by solid phase extraction ........ 130
3.3.10.7.1 The standards MeSO, LacLys and CML on a SPE column .. 131
3.3.11 Trial to detect ornithine in milk protein ................................................ 131
IV
3.3.12 Investigation the effect of sodium borohydride on MeSO .................... 131
3.3.13 Ninhydrin assay .................................................................................. 132
3.3.14 Method validation ................................................................................ 132
3.3.14.1 Within-day repeatability (intra-day variation) ................................ 132
3.3.14.2 Between-day repeatability (inter-day variation) ............................ 133
3.3.14.3 Recovery ...................................................................................... 134
3.3.14.4 LOD and LOQ .............................................................................. 135
LOD and LOQ of LacLys and MeSO ..................................................... 135
LOD and LOQ of CML ........................................................................... 136
3.3.15 Qantitation ........................................................................................... 137
3.3.15.1 Determination protein content by Kjeldahl ................................... 138
4 Summary ......................................... ................................................................... 139
5 Zusammenfassung ................................. ........................................................... 144
Bibliography ...................................... ................................................................... 150
List of Tables .................................... .................................................................... 159
List of Figures ................................... ................................................................... 161
V
Nomenclature
AP amadori product
AGEs advanced glycation end products
amu atomic mass unit
BSA bovine seum albumin
Caco2 colon adenocarcinoma cell
Cbz-Lys carbobenzyloxylysine
CAD collision activated dissociation gas
CUR curtain gas flow
CML Nε-Carboxymethyllysine
cps count per second
CRP C-reactive protein
CYP1A2 cytochrome P450 1A2
Cys-SOH cysteine sulfenic acid
CE collision energy
CV coefficient of variation
CXP collision cell exit potential
DM diabetes mellitus
DMF dimethylformamide
DNP 2, 4 dinitrophenylhydrazine
1DG 1-desoxyglucosone
3DG 3-desoxyglucosone
DP declustering potential
EP entrance potential
ESI electrospray ionization
ESRD end stage renal disease
EtOH ethanol
FMOC-Lys flurenylmethyloxycarbonyl lyisne
Gal galactose
HA formula hypoallergenic formula
VI
1H-NMR Proton Nuclear Magnetic Resonance
h hour
H2O2 hydrogen peroxide
HILIC hidrophilic interaction liquid chromatography
HMF hydroxymethylfurfural
HMW high molecular weight
HPLC high performance iquid chromatography
IBD inflammatory bowel disease
igG immunoglobulin G
IQ imidazaquinoxaline
IS ion spray voltage
KD kidney disease
Lac lactose
LacLys lactulosyllysine
LC liquid chromatography
LC-ESI-MS-MS liquid chromatography electrospray ionization
tandem mass spectrometry
LDL low density lipoprotein
LF lactoferrin
LMW low molecular weight
LOX losyl oxidase
LP Lactoperoxidase
m multiplett (NMR)
m/z mass to charge
MAOs Mono Amino Oxidase enzymes
MAP-kinase Mitogen-Activated Protein kinase
mAU milli absorbance unit
Me methionine
MeOH methanol
MeSO methionine sulfoxide
MeSOO methionine sulfone
MGO methylglyoxal
min minute
MRM multireaction monitoring
VII
MRPs Maillard reaction products
MS-MS tandem mass spectrmetry
NAT2 N-acetyltransferase
NF-kappaB nuclear factor kappa of activated B cells
NFPA nonafluoropentanoic acid
NPLC normal phase liquid chromatography
5-OH-Trp 5-Hydroxytryptophan
O2. superoxide
OH. hydroxyl radicals
PBS phosphate buffer saline
Pd palladium
PP proteose-peptones
ppm part per million (NMR)
RAGE receptors for advanced glyacation end products
ROS reactive oxygen species
RP reversed phase
RT retention time
s singlet (NMR)
S/N signal to noise
t triplet (NMR)
Temp temperature
TFA trifluoroacetic acid
TIC total ion chromatogram
TNF alpha tumor necrosis factor alpha
UHT ultra high temperature
VCAM vascular cell adhesion molecule
WP whey protein
α-LA alpha lactalbumin
β-LG beta lactoglobin
δ chemical shift (NMR)
J J-coupling (NMR)
CHAPTER 1. INTRODUCTION 1
1 Introduction
1.1 The Maillard reaction
The Maillard reaction or non-enzymatic browning comprises a series of chemical
reactions between amino groups and carbonyl compounds leading to the formation
of a variety of Maillard reaction products (MRPs). Controlled browning is often used
to develop desirable flavor, odor or color in food including coffee, bread and soy
bean sauce (Friedman 1996).
First studies of the Maillard reaction were focused on the reaction of
monosaccharides, especially glucose and fructose, and disaccharides such as
maltose and lactose. However, work has also been carried out on polysaccharides
(Gerrard 2002). Additionally, many studies elucidated the role of fat and
carbohydrate breakdown products such as methylglyoxal in Maillard chemistry
(Thornalley 1994).
Maillard reactions were first reported in 1912. Since then, food scientists studied the
mechanism of browning and its effects on food appearance, safety and nutritional
quality, while in medical science the relationship between browning reactions in vivo,
diseases and aging has only be explored for about 30 years (Friedman 1996).
Initially it was discovered that human hemoglobin contains protein-bound Amadori-
products that are particularly formed in diabetic patients with elevated blood glucose
levels. Measurement of fructosylated hemoglobin is now widely used as an index of
glycemia in diabetes (Schleicher 1991).
CHAPTER 1. INTRODUCTION 2
1.2 The phases of the Maillard reaction
The Maillard reaction can be divided into three phases. The primary reactions were
described by L.C.Maillard himself and are shown in figure 1. The condensation
between amine and carbonyl groups leads to the formation of labile Schiff-base,
which undergoes tautomerism yielding the relatively stable aminoketose, the
Amadori product (AP). Formation of AP is reversible and it is considered an
important intermediate in Maillard chemistry (Gerrard 2002). The products which
formed in this phase show neither color nor absorbance in the UV range (Weigel
2004).
Figure 1 : The early phase of the Maillard reaction
In the intermediate phase, Amadori products undergo many characteristic reactions
such as enolisation, elimination, autoxidation, decarboxylation, or aldol reaction
leading to a huge variety of products in varying yields (Gerrard 2002). These
products can show yellow to brown color and may absorb light in the UV range
(Weigel 2004). Many products were isolated from this stage such as
hydroxymethylfurfural (HMF), which has been used as indicator for heat damage
(Friedman 1996; Fernandez-Artigas, Guerra-Hernandez et al. 1999). Rearrangement
of the Amadori product can also produce highly reactive dicarbonyl compounds such
as methylglyoxal (MGO), 3-desoxyglucosone (3DG) and 1-desoxyglucosone (1DG)
CHAPTER 1. INTRODUCTION 3
which react again with free amino groups leading to cross-linking (Niwa, Katsuzaki et
al. 1997).
Many low molecular weight compounds formed during the early and intermediate
phases react with each other to form heterogeneous polymers during the late phase.
These products have a dark brown color and are called melanoidins. Additionally,
many Maillard products have been characterized that remain protein-bound such as
pentosidine, pyrraline. These moieties could be formed in human body between
protein-bound amines and sugars and called advanced glycation end-products or
AGEs (Gerrard 2002). The relationship between the early and late phases of the
Maillard reaction is exemplified in figure 2.
Figure 2 : Phases of the Maillard reaction
CHAPTER 1. INTRODUCTION 4
1.3 The effects of Maillard reaction
1.3.1 Nutrition and food safety
The loss of nutritional quality and food safety during the Maillard reaction is attributed
to the degradation of amino acids, a decrease in protein digestibility and to the
production of antinutritional and toxic compounds. Some studies showed that the
loss of nutritional quality of heat-treated casein, casein-glucose and casein-starch
mixtures can be related to decreased protein digestibility rather than to the
destruction of essential amino acids (Friedman 1992). The blockage of the ε-amino
group of lysine during the Maillard reaction will decrease the activity of trypsin and
reduce the global protein digestibility. Additionally, the intermediate products of the
Maillard reaction can react with amino acids leading to enzyme-resistant cross-links.
Furthermore, some heterocyclic compounds formed in the later stage of Maillard
reaction can inhibit carboxypeptidase. These changes impair the intestinal
absorption of amino acids and as a result impair the nutritional quality of protein
leading to growth inhibition (Mauron 1990).
Mutagenic and carcinogenic products in cooked, protein-rich food are formed by
several mechanisms including carbohydrate caramelization, protein pyrolysis, amino
acid-creatinine reaction and the Maillard reaction. Mutagens formation is dependent
on heating-time and processing temperature. Heterocyclic amines are the most
potent mutagens formed by the Maillard reaction. They induce a variety of tumors in
rodents. Of special interest is their formation in heat processed fish and meat. Many
reactions take place during these processes such as cyclization and dehydration to
give pyrole, pyrazine, and pyridine derivatives. In the next step, the heterocycles
pyrazine and pyridine undergo further transformation with the participation of
aldehydes and creatinine to produce heterocyclic amines such as
imidazaquinoxalines (IQ). The high temperature recommended to destroy pathogens
in meat may lead to the formation of greater amounts of the heterocyclic amines. It
was found that the heterocyclic amines are activated in the human organism by
cytochrome P450 1A2 (CYP1A2) and by N-acetyltransferase 2 (NAT2) to give
mutagens and carcinogens (Friedman 1996).
CHAPTER 1. INTRODUCTION 5
Another class of the heterocyclic amines, called carbolines, are formed when free or
protein-bound tryptophan is exposed to heat under food processing conditions.
These products show binding to the benzodiazepine receptor, premutagencity,
inhibitory activity towards mono amino oxidase enzymes (MAOs) and psychtropic
effects (Ronner, Lerche et al. 2000).
1.3.2 Advanced Glycation End-Products (AGEs)
The Maillard reaction takes place in the body which makes the formation and the
generation of AGEs in vivo an inevitable process. AGEs stimulate cellular responses
mediated by specific receptors (RAGE) (Drinda, Franke et al. 2002) and can lead to
tissue damage through alterations of tissue protein structure and function, such as
crosslinkage. Cross-linked proteins exhibit decreased solubility and increased
proteolysis resistance (Smith, Taneda et al. 1994).
The accumulation of AGEs such as pentosidine, pyrraline and CML (figure 3) in
different tissues has been implicated in the process of aging (Ahmed, Frye et al.
1997) and in the pathogenesis of complications associated with diabetes mellitus
such as nephropathy, retinopathy and neuropathy (Monnier, Sell et al. 1992). AGEs
were also found at elevated level in the serum and synovial tissue of patients with
rheumatoid arthritis (Drinda, Franke et al. 2002). Furthermore, Alzheimer`s disease
(Smith, Taneda et al. 1994), cataract (Lyons, Silvestri et al. 1991), and
atherosclerosis (Nagai, Hayashi et al. 2002) were related to the formation of AGEs.
Figure 3 : Some structures of AGEs
CHAPTER 1. INTRODUCTION 6
1.3.3 Physiological effects of dietary MRPs
The structural similarities between MRPs and AGEs lead to question the impact of
dietary MRPs consumption on the induction of systemic effects similar to the effects
of the analogues AGEs. The effects of MRPs depend on their absorption,
metabolism, and excretion. It is still little known about the effects of small intestinal
enzymes and those of large intestinal bacteria on melanoidins; additionally, the
metabolism of MRPs is still under investigation. The intestinal absorption of MRPs
was investigated in different animals showing that most of the ingested MRPs were
not absorbed but excreted in the feces, probably due to the resistance to enzymatic
and acidic hydrolysis in the gastrointestinal tract. The absorbed MRPs were either
excreted in the urine or retained in different tissue over time. Exemplarily, rats fed
heated egg white-glucose mixture excreted 3.2% of the ingested MRPs in urine and
73.6% in the feces (Valle-Riestra 1970). In another study, the metabolic transit of
casein-14C-glucose derived melanoidins was investigated after fractionation into high
molecular weight melanoidins (HMW >10 kDa) and low molecular weight
melanoidins (LMW< 10 kDa). 27% of LMW melanoidins were excreted with the urine,
while 61% were excreted in the feces. Just 4.3 % of the HMW melanoidins were
excreted in the urine versus 87 % in the feces indicating that the absorption of HMW
melanoidins is lower than this one of LMW melanoidins. Both of them are used or
retained in the body only in little amount, since the radioactivity remained in the
carcass was very low (Finot and Magnenat 1981).
The experiments have been expanded to involve humans. It was demonstrated for
example that the concentration of both, CML and fluorescent AGEs, in the plasma of
healthy omnivores were lower compared with those in the plasma of healthy
vegetarians (vegetarian diet duration 7-8 years). The anticipated mechanism
involves an enhanced dietary AGEs flux in vegetarians caused by a higher intake of
both whole grain products and technological processed grain products (Sebekova,
Krajcoviova-Kudlackova et al. 2001). One study on human has shown that a diet rich
in MRPs, which was prepared from egg white and fructose, induced an elevation of
AGEs concentration in serum and urine. Serum level of AGEs increased proportional
to the ingested amount of MRPs, while the urinary excretion correlates inversely with
the degree of kidney disease (KD) (Koschinsky, He et al. 1997). Additionally, the
serum concentrations of glycated LDL were higher in the patients with both diabetes
CHAPTER 1. INTRODUCTION 7
mellitus (DM) and end stage renal disease (ESRD) followed by those with only
ESRD compared to the healthy control. Thus, it can be assumed that a diet rich in
MRPs increases the risk factors for disease development in patient with KD (Bucala,
Makita et al. 1994). Some of the consumed MRPs can survive the digestive process
and transported into the circulation resulting in elevation of AGEs concentration in
serum. Consequently, they induce an increase in the level of the inflammatory
mediators such as CRP, VCAM and TNFα as well as glycated LDL (Vlassara, Cai et
al. 2002). In vitro, the thermally generated AGEs promote a dose-dependent
activation of MAP kinase in Caco2 cells through the binding to RAGE. This activation
may result in cellular reactions such as inflammatory response, cellular proliferation,
tumor growth and metastasis (Zill, Bek et al. 2003).
Maillard products which cannot pass into the circulation can affect the mucosa of the
large bowel. It is well known that the activation of nuclear transcription factor NF-κB
in both, mucosal macrophage and T lymphocytes, plays an important role in the
pathogenesis of inflammatory bowel diseases (IBD) such as Crohn`s disease and
ulcerative colitis. Therefore, the aim of several new treatment strategies of IBD is to
achieve a specific blockage of NF-κB, but all of these drugs must be cautiously used
since NF-κB is involved in cell growth and apoptosis. The activation of NF-κB can be
induced by variable stimuli like oxidative stress, cytokines, bacteria and viruses
(Dijkstra, Moshage et al. 2002). Additionally, it can be induced by MRPs via the
generation of H2O2. For example, one study demonstrated that the stimulation of
macrophages with coffee-derived or lysine-ribose-derived melanoidins induced a 13
and 18 fold increase of NF-κB activation, respectively (Muscat, Pelka et al. 2007).
Thus, the consumption of MRPs-rich diet may affect the intestinal immune function
and participate in the pathogenesis of IBD.
The consumption of food-derived MRPs is implicated in the progression of many
other diseases. Exemplarily, the long-term dietary intake of MRPs by rats induced an
increase in the renal and hepatic magnesium concentration and a decrease in the
femoral magnesium concentration which could affect the bone health (Delgado-
Andrade, Seiquer et al. 2007). MRPs cause also a hypertrophy of organs like liver or
stomach and they affect the activities of the intestinal pancreatic enzymes such as
chymotrypsin. Additionally, they have adverse effects on the mineral metabolism
(Ca, Mg, Cu, and Zn) and variable effects on both allergic response and cholesterol
CHAPTER 1. INTRODUCTION 8
metabolism. The extent of the severity of these effects is influenced by the severity
of heat treatment, by the content and the structure of different Maillard and cross-
linked products as well as by the health and susceptibility of the consumer (Finot,
Aeschbacher et al. 1990).
1.3.4 Beneficial Maillard products
Beside the undesirable effects of MRPs, they may also have health promoting
effects such as antimutagenic activity. The available information about the mutagenic
effect of MRPs is contradictory. Some studies describe MRPs as mutagenic, others
as slight or not mutagenic. Wagner et al have reported that MRPs from heated
fructose-cysteine mixtures have antimutagenic effect in the presence of H2O2 and S9
(Wagner, Reichhold et al. 2007). S9 are microsomes derived from the membrane of
endoplasmic reticulum and considered as source for metabolizing enzymes,
especially cytochrome P450. The antimutagenic mechanism of the Maillards
products can include inhibition or reduction of the hepatic microsomal activity, or
scavenging of free radicals by melanoidins. Antimutagens are classified into
desmutagens and bioantimutagens. Desmutagens inactivate mutagens by chemical
or enzymatc modifications, while bioantimutagens suppress the mutagenesis after
DNA modification by the mutagen (Kada, Inoue et al. 1986). Furthermore,
melanoidins may be able to reduce the absorption of mutagens (Powrie, Wu et al.
1986).
Melanoidins, which are formed during the late phase of the Maillard reaction,
possess further antioxidative activity. This antioxidative activity is related in most
cases to poorly characterized melanoidins. Scavenging of reactive oxygen species
such as hydroxyl radicals, binding of metal ion and decomposing of primary radical
to non radical compounds could be the important mechanisms of their antioxidative
effect (Morales and Jimenez-Perez 2004). It is well known that oxidized LDL plays
an important role in the development of atherosclerosis. Some studies have shown
that glucose-lysine, arginine or glycine derived Maillard products can inhibit copper-
induced oxidation of human LDL in vitro. Thus, they could reduce the risk of
cardiovascular disease in vivo (Dittrich, El-Massry et al. 2003).
CHAPTER 1. INTRODUCTION 9
Other studies have demonstrated that fructose-tryptophan derived Maillard products
prevent lipid oxidation in sardin, while glucose-tryptophan derived Maillard products
inhibit linoleic acid oxidation (Friedman 1996) .
Maillard reaction products show antibiotic effects against many strains of gram-
positive bacteria, such as bacillus subtilus, lactobacillus and staphylococcus. The
growth of different Escherichia strains was inhibited to different extent, while the
strains of salmonella were not at all inhibited. Products with higher molecular weight
(higher than 1000 Da) had a greater inhibitory activity than those with lower
molecular weight (Einarsson, Snygg et al. 1983). Potential targets of the antibacterial
activity of MRPs include bacterial membrane, genetic material, bacterial enzymes
and reduced oxygen uptake as a result of its interaction with iron (Einarsson, Eklund
et al. 1988; Friedman 1996). A variety of casein-lactose derived melanoidins have
shown a strong inhibition of the adherence of urease to gastric mucin and so
suppressed Helicobacter pylori colonization, which induces gastric pathologies in an
animal model and in human subjects (Hiramoto, Itoh et al. 2004). Additionally,
melanoidins can stimulate the growth of the nutritional gut flora in vitro such as
bifidobacteria, lactobacilli, bacteroides and clostridia. Thus, melanoidins may affect
the growth of these bacteria in vivo which improve the health of lower gut (Ames,
Wynne et al. 1999).
1.4 Prevention of the Maillard reaction
Many approaches can be used to prevent or minimize food browning and its
antinutritional and toxicological consequences.
Thiols inhibit browning reactions and, under certain conditions, they may be as
effective as sodium sulfite in preventing nonenzymatic browning. Acetylation of
lysine amino group by transglutaminase will transform it to amide group and thus will
prevent its participation in the browning reaction. Some studies revealed that oxygen
seems to be required for non-enzymatic browning, consequently, antioxidants should
suppress the browning in food (Friedman 1996). Finally a variety of substances such
as aminoguanidine, acetylsalicylic acid, or pyridoxine have been reported as
inhibitors of the Maillard reaction (Matsuura, Aradate et al. 2002).
CHAPTER 1. INTRODUCTION 10
1.5 Milk
The presence of lactose and proteins as well as the neutral pH value render milk as
a suitable medium for the Maillard reaction during processing and storage. This
leads to alteration of the nutritional value of milk concurrently with the formation of
various products like lactulosyllysine and CML.
Milk is produced in mammary glands in mammals. There are different sources of
milk for human nutrition such as cattle, goats and sheep, but cow is the major source
of milk in the world. Nowadays, the word milk refers to cow milk. Milk and milk
products represent important dietary components because of their high nutritional
value (Belitz. H. D 2009).
Milk is a complex liquid food with 87 % water. It contains numerous nutrients such as
saturated and unsaturated lipids, proteins including caseins and whey proteins,
carbohydrates mainly lactose as well as different vitamins and minerals. The
important milk components for human nutrition and their concentrations are
illustrated in table 1.
In addition to its nutritional value, some studies suggested physiological functions of
milk proteins that may promote health. It has been reported for example that some
milk peptides have antihypertensive effects through the inhibition of angiotensin-
converting enzymes (Nakamura, Yamamoto et al. 1995). Other peptides derived
from κ-casein have antithrombotic effect due to its ability to inhibit both, the
aggregation of ADP-activated platelets and the binding of fibrinogen to ADP-
activated platelets (Jolles, Levy-Toledano et al. 1986).
Other studies reported that milk consumption may stimulate insulin release, an effect
which may be caused by whey proteins (Nilsson, Holst et al. 2007). Fresh milk is a
good source of glutathione which is a tripeptide of cysteine, glycine and glutamic
acid. Glutathione acts as an antioxidant, whereby it reduces the levels of reactive
oxygenspecies (ROS) in cells (Haug, Hostmark et al. 2007).
Milk is also a source of selenium and the vitamins A and E. These three nutrients
exhibit also antioxidant activity and promote the human health (Lindmark-Mansson
and Akesson 2000). Many factors can affect milk composition such as cattle breed,
age, stage of lactation, health status of the udder, energy balance and feeding
CHAPTER 1. INTRODUCTION 11
regime. For example, milk concentration of selenium, vitamin A and E reflex their
intake by feed (Haug, Hostmark et al. 2007).
Cow milk differs from human milk in many aspects. It has about 3 and 7 times higher
levels of minerals and casein, respectively, than human milk. Additionally, in human
milk there is no β-lactoglobulin (β-LG), immunoglobulin G (IgG) is replaced by
immunoglobulin A (igA), while lactoferrin (LF) and lysozyme present the two major
proteins. Despite all of these differences, cow milk is still the major ingredient for
infant formulas (Wit 1998)
milk component
content in 1L whole milk
milk component
content in 1L whole milk
total fat 33 g magnesium 0.1 g
total saturated fatty acids
19 g zinc 4 mg
oleic acid 8 g selenium 37 µg
omega 6 fatty acid
1.2 g vit E 0.6 mg
omega 3 fatty acid 0.75 g vit A 280 µg
lactose 53 g vit B12 4.4 µg
proteins 32 g riboflavin 1.83 mg
calcium 1.1 g folate 50 µg
Table 1 : Milk components and their concentrations in whole milk (Haug, Hostmark et al. 2007)
1.5.1 Milk proteins
The total protein content of one liter milk is about 33 g which is divided approximately
into 80% of caseins and 20% of whey proteins. These proteins represent a source
for essential amino acids and have a high nutritional value.
CHAPTER 1. INTRODUCTION 12
1.5.1.1 Casein
Up to 10% of the total casein fraction is present as monomers, the other part exists
in colloidal particles known as casein micelles. The principal casein fractions are αS-
casein, β-casein, γ-casein and κ-casein. These fractions are present in water in
colloidal form, except of κ-casein which is soluble. Caseins differ in their properties
from whey proteins and among each other. Casein micelles consist of small units
called submicelles which bind together through calcium phosphate bridges. Each
submicelle contains about 30 casein molecule. There are two different types of
submicelles. Submicelles with κ-casein occupy the surface of the micelles and
prevent the aggregation of submicelles by steric repulsion. Submicelles without κ-
casein are localized in the interior position of the micelles. Casein is not very
susceptible to coagulation. It coagulates only when heated at high temperature for
long time (e.g. 5 hours at 120 °C).
However, temperature and low pH can strongly affect the structure of the micelles.
For example, caseins coagulate at pH 4.6; this coagulation is enhanced by
increasing heat treatment. On the other hand, the coagulation temperature drops
with decreasing pH (Belitz. H. D 2009).
One biological function of casein is to carry calcium and phosphate. Two thirds of the
total minerals in milk are bound to casein micelles, in which 44% of the total
inorganic phosphate and 90% of the calcium in milk are bound as colloidal calcium
phosphate. The components of casein micelles are shown in table 2. Another
function of casein, especially of αS-casein, is the production of the clot in the
stomach of infants for efficient digestion (Wit 1998).
micelle component %
micelle component %
casein 93.20 phosphate (org) 2.3
Ca 2.9 phosphate (anorg)
2.9
Mg 0.10 citrate 0.40
Na 0.10 K 0.30
Table 2: Composition of casein micelles (Belitz. H. D 2009)
CHAPTER 1. INTRODUCTION 13
1.5.1.2 Whey protein
Whey is the liquid which remains after casein has been precipitated during cheese
production. Whey proteins are globular proteins which represent about 20 % of the
total protein content in milk. Whey proteins are rapidly digested proteins which
provide high concentrations of essential amino acid, especially lysine and
tryptophan. Therefore they have a high nutritional value. The term whey proteins
comprises different proteins, namely β-lactoglobulin (β-LG), α-lactalbumin (α-LA),
bovine serum albumin (BSA), immunoglobulin G (IgG), lactoferrin (LF),
lactoperoxidase (LP), proteose-peptone (PP) and different enzymes (Haug,
Hostmark et al. 2007). These proteins differ in their biological function as well as in
their abundance in milk, as seen in table 3.
whey proteins Concentration in milk
g/L Biological function
β-LG 3.2 carier for provitamin A
α-LA 1.2 lactose synthesis
BSA 0.4 fatty acid transfer
IgG 0.8 passive immunity
LF 0.2 bacteriostatic agent
LP 0.03 antibacterial agent
Enzymes 0.03
Proteose-peptones 1.3 opioid activity
Table 3: Composition, abundance and biological function of whey proteins. Adapted from (Wit 1998)
There are clear differences in the sizes of casein micelles and whey proteins. The
radius of casein micelle is 50 nm. The radius of whey proteins vary between 1.8 nm
for α-LA, 3.5 nm as diad axis of β-LG. Radius of BSA is 4 nm and tetrad axis of IgG
is 6 nm (Wit 1998).
Whey proteins have high nutritional value due to the high content of essential amino
acids. Additionally, whey proteins are widely used in food industry because of their
CHAPTER 1. INTRODUCTION 14
functional properties. For example, they are used in confectionery, dairy and bakery
products as replacement of egg protein to induce and stabilize foaming, as well as
for their emulsifying properties. Moreover, desalted whey is used to produce infant
formulas. In this case, cow milk is supplemented with whey proteins to reduce the
ratio between casein and whey protein from 80/20 to 40/60 in infant formula.
Furthermore, the content of lactose, fat, minerals and vitamins of infant formuls is
adapted to human milk (Wit 1998).
Besides the nutritional and functional properties of whey proteins, some studies have
reported that milk whey proteins may promote bone formation in healthy adult
women (Aoe, Toba et al. 2001). Additionally, they inhibit the formation of gastric
mucosal ulcerative lesions in the stomach of rats caused by absolute ethanol
ingestion (Rosaneli, Bighetti et al. 2002). Other studies reported that whey proteins
exhibit antiviral activity (Andersen, Jenssen et al. 2003) in animal studies in addition
to anti cancer effect in the patients with metastatic carcinoma (Kennedy, Konok et
al. 1995). Furthermore, different effects on the metabolism and the immune system
were observed (Krissansen 2007).
1.5.2 Heat treatment of milk
Milk heat treatment is classified into several categories:
1. Thermization: In this process, milk is heated under conditions milder than
pasteurization (57 °C) which reduces the number of bacteria and preserves
milk taste.
2. Pasteurization: For pasteurization, either high temperature is applied for short
time like 85 °C for 2-3 s and 72-75 °C for 15-30 s, or low temperature for long
time while stirring (e.g. 63-66 °C for 30-32 min). Pasteurization destroys all
pathogens that are present in milk.
3. Ultrahigh temperature treatment (UHT) eliminates all spores and
microorganisms. It is applied either indirectly by coils or plates at 136-138 °C
for 5-8 s, or directly by live steam injection at 140-145 °C for 2-4 s followed by
aseptic packaging.
4. Bactotherm process is a combination of centrifugal sterilization in bactofuges
at 65-70 °C and UHT treatment of the separated sedi ment followed by
recombination.
CHAPTER 1. INTRODUCTION 15
5. Sterilization: Sterilized milk is usually produced in 2 steps consisting of
heating in an autoclave at 107-115 °C for 20-40 min followed by heating in
bottle at 120-130 °C for 8-12 min (Belitz. H. D 200 9).
Heat treatment in general is applied to kill all disease-causing microorganisms
ensuring safety of milk consumption as well as to increase the products shelf live.
Additionally, heat treatment is important to achieve the functional uses of whey
proteins. However, during thermally treatment, several milk constitutes can be
negatively affected.
1.5.3 Effects of heat treatment on milk
1.5.3.1 Effects of heat treatment on milk protein
During heat treatment of milk, many reactions can take place which involve only
protein or protein with the other macronutrients. These reactions include:
• Protein reactions: Lysinoalanine (LAL) is formed by the reaction of lysine with
dehydroalanine. The latter is produced by β-elimination of cysteine and serine
(Mauron 1990). The content of LAL in sterilized and condensed milk was 13
and 18 fold higher than the content in fresh milk (Haagsma and Slump 1978).
• Protein-oxidized lipid interactions: During these reactions, unsaturated lipids
are oxidized resulting in different products. These products can be classified
into primary products (hydroperoxide), secondary products (aldehyde, ketone,
carbonyls) and stable products like carboxylic acids. Methionine is rapidly
oxidized by hydroperoxides to methionine sulfoxide, while lysine reacts with
the secondary products like aldehyde and ketone in a Maillard-type reaction
(Nielsen, Loliger et al. 1985).
• Protein-polyphenol interactions: These processes include the reaction of
lysine or methionine with quinones to form lysine-quinones adducts and
methionine sulfoxide, respectively (Mauron 1990). Such reaction might be
relevant to choco-milk products.
• Protein-lactose reactions: This reaction is called Maillard reaction or protein
glycation and it is considered as the most frequent reaction in milk leading to
protein damage during processing.
CHAPTER 1. INTRODUCTION 16
• Protein oxidation: In parallel to glycation, milk proteins are subjected to
various oxidation reactions under processing conditions (Mauron 1990). In
milk products, tryptophan, methionine and cysteine are particularly sensitive
to oxidative modification. For example, tryptophan oxidation can lead to an
important number of derivatives generated by opening of the indole ring.
Oxidation converts methionine to methionine sulfoxide and further to
methionine sulphone under stronger oxidative conditions (Guy and Fenaille
2006). Cysteine could be oxidized to cysteine sulfenic acid (Meltretter, Seeber
et al. 2007). Other residues, such as lysine, proline, arginine, histidine and
tyrosine can also be subjected to oxidation. Oxidation of the latter amino acids
is often characterized with the formation of carbonyl groups within their
structures (Guy and Fenaille 2006). Carbonyl formation is also a result of the
reaction between the amino group and the reactive dicarbonyl compounds
generated from Maillard reaction and/or lipid oxidation. In addition, free radical
produced by metal-catalyzed oxidation in infant formulas can yield carbonyl
groups. Therefore, the protein carbonyl (PRC) content is used as an indicator
for protein oxidation (Fenaille, Parisod et al. 2005). Lysine aldehyde is an
example for a carbonyl product formed in milk proteins during heat treatment
(Meltretter, Seeber et al. 2007).
• Denaturation: Heat treatment can change the secondary and tertiary
structures of milk proteins, mainly whey proteins causing denaturation
(Mauron 1990). The order of heat stability of whey protein is : α-LA > β-LG >
BSA > IgG (Mortier L 2000). During denaturation sulfhydryl groups are
cleaved, especially in β-LG, leading to the formation of sulfur compounds, like
methyl sulfide, which contribute to the characteristic flavor of milk (Calvo
1992).
The previous interactions lead to the degradation of essential amino acids as well as
to a decrease of the protein digestibility. Consequently they reduce the nutritional
value of milk (Mauron 1990).
CHAPTER 1. INTRODUCTION 17
1.5.3.2 Effect of heat treatment on lactose
1.5.3.2.1 Lactose isomerisation/degradation
Lactose is the main natural disaccharide in milk consisting of glucose and galactose.
During heat treatment of milk, lactose is subjected to isomerisation and degradation.
It has been shown that the activation energy for isomerisation at low temperature
(<100 °C) is higher than the early Miallard reactio n indicating that the participation of
lactose in the early Miallard reaction is more favored than its isomerisation. On the
other hand, lactose isomerisation and degradation is more important at high
temperatures (120 °C). It was found for example tha t 80% of lactose has undergone
isomerisation at sterilization temperature versus 20% of lactose participated in the
Maillard reaction. The products of lactose isomerisation are lactulose and epilactose.
Lactulose is a disaccharide of galacatose and fructose which is normally not found in
raw milk, while epilactose is a disaccharide of galacatose and mannose (van Boekel
1998).
Epilactose is detected in small amounts only in sterilized milk. Lactulose is detected
in higher amounts in heated milk like sterilized and UHT milk as well as in
pasteurized milk (Olano, Calvo et al. 1989). In condensed milk, for example, about
10 % lactose is isomerized to lactulose (Belitz. H. D 2009). Lactulose is widely used
as indicator for heat treatment of milk, especially to distinguish UHT milk from
sterilized milk, because its content in sterilized milk is 5 times higher than in UHT
milk (Olano, Calvo et al. 1989; Luzzana, Agnellini et al. 2003).
Many products arise from lactulose degradation such as galactose, formic acid and
others. Formic acid is formed in relatively large amounts and is mainly responsible
for the pH decrease in heated milk. The degradation products of lactose can undergo
further Maillard reaction (van Boekel 1998). Studies have shown that although the
amount of galactose increases proportionally to the severity of heating, this marker
could not clearly differentiate between different types of heat treatment (Olano, Calvo
et al. 1989).
1.5.3.2.2 Lactose and Maillard reaction
In the early stage, lactose reacts with the ε-amino group of protein-bound lysine
forming the Schiff’s base which is transformed into lactulosyllysine (LacLys) via
CHAPTER 1. INTRODUCTION 18
Amadori product rearrangement (figure 1). In the advanced stage, LacLys is
fragmented to give CML, erythronic acid and galacatose (van Boekel 1998).
Figure 4 : Early stage of the Maillard reaction in milk
CML is formed only in a little amount in heated milk and is described as a useful
indicator for the advanced stage of the Maillard reaction in milk products (Fenaille,
Parisod et al. 2006).
Later, it has been shown that CML is formed by two additional mechanisms. During
autoxidative glycation, CML is formed by the reaction between lysine and glyoxal
which results from metal-catalysed glucose autoxidation. In the third mechanism,
which is also called Namiki pathway, the Schiff’s base is degraded resulting in C-2
fragments like glyoxal and glycoaldehyde, which subsequently react with lysine to
form CML (Glomb and Monnier 1995; Ferreira, Ponces Freire et al. 2003). Figure 5
illustrates the three pathways for the formation of CML.
CHAPTER 1. INTRODUCTION 19
Figure 5: Formation of CML through different pathways (Ferreira, Ponces Freire et al. 2003)
Besides the oxidative cleavage, LacLys is subjected to further degradation (figure 6),
mainly via 2, 3-enolization leading to 1-deoxyosone, which is a very reactive
intermediate. 1-Deoxyosone then undergoes further reactions like enolization or
cyclization leading to 3-furanone and β-pyranone, respectively. β-Pyranone
isomerizes to cyclopentenone and both of them may convert into
galactosylisomaltose. These products may further react with proteins to form cross-
linked proteins.
There are two other breakdowns routes for degradation of LacLys. The 3-
deoxyosone-pathway via 1,2-enolization is favored under acidic conditions and leads
to the formation of HMF, pyrraline as well as pentosidine. Both lysylpyrraline and
pentosidine are formed in very little amounts in milk and could be used as indicators
for severe heat treatment. The third breakdown route is the 4-deoxosone-pathway
which is favored under more alkaline conditions leading to 4-deoxyaminoreductone.
The last two degradation routes are probably not important in milk because of its
neutral pH (van Boekel 1998). In the final stage, melanoidins are formed which are
bound to milk proteins. Melanoidins in milk are not very well characterized (van
Boekel 1998).
CHAPTER 1. INTRODUCTION 20
Figure 6: Degradation of Amadori product under acidic, neutral or alkaline conditions
1.5.3.3 Effects of heat treatment on milk fat
Milk fat are present as globules surrounded by a complex membrane called milk fat
globule membrane (MFGM) which contains a mixture of unsaturated phospholipids,
proteins, glycoproteins, cholesterol, enzymes and other minor components. These
globules are emulsified in the aqueous phase. Milk fat contributes to the flavor and
color of milk. However, it is one of the least chemically stable compounds in milk
which can readily undergo autoxidation, especially under the conditions of
processing and storage. Unsaturated fatty acids are the main target of oxidation, in
particular unsaturated phospholipids in the MFGM. Milk cholesterol can also be
oxidized under the conditions of heat treatment and drying forming more than 60
cholesterol products. Some of them can induce the onset of atherosclerosis (Guy
and Fenaille 2006).
CHAPTER 1. INTRODUCTION 21
Lipid oxidation deteriorates the fat itself and produces oxidative fragments, some of
which are volatile and can cause a rancidic off-flavor. Furthermore, some lipid
oxidation products like dicarbonyl compounds can react readily with food proteins to
form Advanced Lipidoxidation End-products (ALEs). Consequently, lipid oxidation
has a negative impact on color, flavor, functional/physical properties and nutritional
value (Guy and Fenaille 2006).
1.5.3.4 Effects of heat treatments on vitamins
During heat treatment, the water-soluble vitamins B1, B6, B12, folic acid and vitamin C
are prone to degradation. About 10-30% of these vitamins are lost in UHT milk.
Sterilization destroys ca 50% of the vitamins B1, B6, folic acid and up to 100% of
vitamin C and B12 (Belitz. H. D 2009). Fat soluble vitamins are also susceptible to
degradation. For example, UHT milk has lost up to 52% of α-tocopherol after 3
months storage at 40 °C. α-Tocopherol levels in milk powder decreased by 17% after
storage for 20 days at 20 °C (Guy and Fenaille 2006 ).
1.5.3.5 Other effects of heat treatment
1.5.3.5.1 Flavor
The flavor of fresh milk is caused by a numerous natural components. During heat
treatment, milk flavor is altered due to the formation of new compounds. These new
compounds can be generated from proteins, lipids, lactose and others. For instance,
sulfur compounds which give milk the cooked flavor are formed due to denaturation
of proteins, mainly of β-LG, and subsequent cleavage of sulfhydryl compounds.
Fat are also important contributors to milk flavor due to the formation of methyl
ketones, aldehyde and hexanal during heat treatment of milk. Additionally, lactose
degradation products like benzaldehyde and furanoids can contribute to the flavor of
heated milk (Calvo 1992). Strong heat treatment of milk, like sterilization, results in
the accumulation of volatile Maillard products like methylpropanal which affect milk
flavor (Belitz. H. D 2009).
1.5.3.5.2 Allergenicity
It was observed that milk is less antigenic in vivo after heat treatment. The antigenic
sites of protein may be selectively altered by the reaction with reducing sugar under
CHAPTER 1. INTRODUCTION 22
mild heat treatment. This chemical and structural modification could be responsible
for the observed reduction in antigenicity (Friedman 1996). In contrast, another study
conculded that the modified proteins, especially those that are resistant to digestion
tend to increase the allergenicity of milk, because they may represent the major
allergens in food (Fenaille, Parisod et al. 2005).
1.5.4 Indicators for milk heat treatment
Two types of chemical reactions may be used as indicators to evaluate the heat
treatment of milk. The first group includes degradation, denaturation and inactivation
of heat labile components like whey proteins and enzymes. The activity of negative
alkaline phosphatase and lactoperoxidase, for example, are used as indicators for
monitoring the heat damage of pasteurized and highly pasteurized milk, respectively.
The whey protein nitrogen index (WPNI), which expresses the amount of
undenaturated whey proteins, is also used as indicator for heat treatment.
The second group includes the formation of new substances like lactulose and the
Amadori product (Mortier L 2000). The Amadori product (LacLys) can be determined
by the furosine method, which is based on the acidic hydrolysis of LacLys at 110 °C
for 24 hours leading to the formation of furosine and pyridosine as well as to
regeneration of some lysine. However, when food is subjected to severe heat
treatment, the Amadori product is transformed into advanced glycation end-products.
Therefore furosine is not suitable as indicator for severely heated milk. Instead
advanced end-product such as CML must be applid as indicator for heat treatment
(Charissou, Ait-Ameur et al. 2007).
CHAPTER 1. INTRODUCTION 23
1.6 Liquid Chromatography Electro Spray Ionization
Tandem Mass Spectrometry (LC-ESI-MS-MS)
Liquid chromatography is a physical separation method base on the distribution of
the analytes between two phases. The first one is the stationary phase and the
second phase is a liquid phase that moves through the stationary phase in a defined
direction. Many detectors are in routine use together with liquid chromatography
such as UV, fluorescence, conductivity as well as mass spectrometry. Mass
spectrometry allows the differentiation of compounds which have similar retention
time independent on their chromatographic resolution (Ardrey 2003).
Mass spectrometry is an excellent qualitative tool for the determination of a
compound`s molecular weight. Moreover, tandem mass spectrometry (MS-MS)
induces characteristic fragments of the analyte which allows the elucidation of its
structure. Additionally, MS-MS is considered a very selective and sensitive detector
for the quantitation of analytes even in complex matrixes such as blood, plasma and
food. The greater selectivity of MS-MS compared to simple MS is the dual mass
selection, first by the selection of precursor ion from different ions in the matrixes,
and second by the selection of products ions, which arose from the pre-selected
precursor ion.
Many techniques are used to induce the ionization of the analytes in the ion source
under atmospheric pressure such as atmospheric pressure chemical ionization
(APCI) and electro spray ionization (ESI). For ESI, the eluate from the HPLC, is
directed into the ion source of the mass spectrometer through a capillary maintained
at high voltage and atmospheric pressure. The application of high voltage on the
electrospray needle disperses the liquid stream forming a mist of highly charged
droplets which are desolvated during their pass through the atmospheric pressure
region. Desolvation is assisted by a stream of drying gas, usually nitrogen.
The ionization in ESI takes place directly in the solution during spraying. The lower
temperature which is applied compared to APCI, makes ESI a softer ionization
technique, since no/or hardly any thermal decomposition of thermally labile
molecules is formed during ionization. Ionic, ionizable and polar compounds are
easily ionized by ESI, while non polar molecules have very poor response in ESI.
CHAPTER 1. INTRODUCTION 24
The ionization is followed by the evaporation of the solvent assisted by heat at
elevated flow rates (Zimmer 2003).
In positive-ion mass spectrometry, ions are formed by the addition of various
species, mostly protons, to the analyte molecule. In negative-ion mode, the
molecules are usually deprotonated to form ions. The number of charges on the ion
is related to the number of added species. Thus, the measured mass to charge ratio
(m/z) of one ion is dependent on the molecular weight of the analyte itself as well as
on the number of the added species, as shown by the following equation:
m/z = (M+nH)/n
M is the molecular weight of the analyte, H is the molecular weight of the added
species (1 for hydrogen, 18 for ammonium ion and 23 for sodium) and n is the
number of charges which are carried by the ion. Ions are generated outside of the
mass analyzer and are then directed into its entrance.
The triple quadrupole is the most widely used mass analyzer in MS-MS. It consists of
three sets of quadrupole rods: two mass analyzer, MS1 and MS3 (Q1 and Q3), and
the collision cell (Q2) (Ardrey 2003). Figure 7 illustrates the set up of a triple
quadrupole tandem mass spectrometer.
The molecular ion [M+H]+ (precursor ion) is selected in the first mass analyzer (Q1),
and is then transmitted into the collision cell (Q2), where fragmentation is carried out.
In the collision cell, the ions collide with a wall of gas molecules, usually nitrogen,
which is called collision gas or collisionally activated dissociation gas (CAD gas).
The generated fragment ions f+1, f+2, f+3, f+4 (product ions) are focused into the
second mass analyzer (Q3), where usually only the product ions of the highest
intensity are chosen and monitored (f+1,f+
3).
This technique is called selected reaction monitoring (SRM), because it monitors the
fragmentation of a selected precursor ion to a selected product ion. It is considered
as one of the most widely used MS-MS experiments. The term multiple reactions
monitoring (MRM) refers to simultaneous monitoring of several pairs of products ions
and precursor ions derived from different analytes (Zimmer 2003).
Other widely used MS-MS experiments are the product ion-scan, the precursor ion
scan and the constant neutral loss scan.
A single product ion is usually inadequate to identify the analyte of interest. If
chromatography is involved, the combination of retention time and a single product
ion may be considered adequate, especially if this ion is generated by soft ionization
CHAPTER 1. INTRODUCTION 25
technique. However, in order to ensure the identity of the analyte, it is recommended
to monitor at least two product ions of the analyte (Ardrey 2003). In such cases, the
product ion which gives a chromatographic peak with the highest intensity is called
the quantifier fragment (f+3), because it is used for quantification. The other
fragments are used to confirm the analyte`s identity and are called the qualifier
fragments (like f+1). Mass spectrometry is commonly used for the quantitation of low
molecular weight molecules below 1000-2000 Da (Zimmer 2003).
Figure 7: Schematic illustration of tandem mass spectrometry in MRM mode [adapted from (Zimmer 2003)]
CHAPTER 1. INTRODUCTION 26
1.7 Aims of the work:
Untargeted peptide mapping indicate that lactulosyllysine, ε-carboxymethyllysine,
methionine sulfoxide, lysine aldehyde and cysteine sulfenic acid or
hydroxytryptophan are the major protein modifications which are formed during the
heating of a milk model. So far however, the relevance of these products in heated
milk is not clear. Therefore, the aims of the present work were the quantification of
the major protein modification in heated milk by LC-ESI-MS-MS analysis of the
modified amino acids after enzymatic protein hydrolysis. For this purpose the
following steps were carried out:
• The synthesis of standard compounds lactulosyllysine, ε-carboxymethyllysine,
methionine sulfoxide, lysine aldehyde and cysteine sulfenic acid, which
represent the major modified amino acids in milk.
• Development of a method to detect lactulosyllysine, ε-carboxymethyllysine,
methionine sulfoxide, lysine aldehyde, cysteine sufenic acid as well as 5-
hydroxytryptophan and ornithine in milk proteins via LC-ESI-MS-MS in MRM
mode.
• Development of a method to release lactulosyllysine, ε-carboxymethyllysine
and methionine sulfoxide from milk proteins by enzymatic hydrolysis.
• Validation of the method
• Quantification of lactulosyllysine, ε-carboxymethyllysine and methionine
sulfoxide in different heated milk products.
CHAPTER 2. RESULTS AND DISCUSSION 27
2 Results and discussion
2.1 Selection of the marker compounds for protein
modification
The goal of the present study was the development of a LC-ESI-MS-MS method to
separate and quantify the major protein modifications, which are formed during
processing of milk. Tryptophan, methionine and cysteine are particularly sensitive to
oxidation in milk products. Other residues like lysine could also be subjected to
oxidation. Furthermore, lysine is one of the preferential sites for glycation. Thus,
lactulosyllysine (LacLys) and ε-carboxymethyllysine (CML) are described as
indicators for early and advanced stage of the Maillard reaction, respectively.
LacLys is the first stable product of the reaction between lactose and protein-bound
lysine in the early Maillard reaction. Several methods are in routine use to detect
LacyLys in milk products; among those, the furosine method is the most commen
one. This method is based on acid hydrolysis (HCl 6N) of LacLys at 110 °C for 24
hours which leads to the formation of furosine and pyridosine as well as to
regeneration of some lysine. Furosine can then easily be determined either by ion-
exchange chromatography with ninhydrin detection (Henle, Walter et al. 1991) or by
HPLC-UV detection at 280 nm (Guerra-Hernandez, Corzo et al. 1999).
The problem of the furosine method is the conversion factor which is used to
calculate concentrations of LacLys and other Amadori products. This factor is
uncertain and tightly dependent on the reaction conditions. Baptista et al have found,
for example, that the acid hydrolysis of LacLys yielded approximately 32 %, 40%,
and 28% of furosine, lysine and pyridosine, respectively (Baptista. Jose 2004).
Another literature reported 50 % yield of furosine (Penndorf, Biedermann et al.
2007). Furthermore, furosine formation differs depending on the sugar involved in
the formation of the Amadori product. After acidic hydrolysis, for instance, the molar
yield of furosine was 32% from fructosyllysine, 34 % from lactulosyllysine and 42%
from tagatosyllysine (Krause, Knoll et al. 2003).
CHAPTER 2. RESULTS AND DISCUSSION 28
For indirect estimation, LacLys is oxidized by periodic acid which yields CML,
erytronic acid and galacatose. The content of CML is then determined by GC-MS
(Badoud, Fay et al. 1991). However, this method may lead to overestimation of
LacLys, due to CML is already present in the milk proteins.
Another indirect method was used to estimate LacLys in milk products. This method
induced the formation of hydroxymethylfurfural (HMF) from LacLys by heating in the
presence of oxalic acid. Subsequently, HMF was determined by colorimetric
methods. However, HMF can also be formed during lactose degradation (van Boekel
1998).
Figure 8 : Degradation of LacLys via oxidative cleavage, acid hydrolysis and heating in the presence of oxalic acid
CHAPTER 2. RESULTS AND DISCUSSION 29
Recently, MALDI-TOF-MS has been used as a new specific method for the detection
of LacLys as well as other Maillard products after partial enzymatic hydrolysis
(Meltretter, Seeber et al. 2007). Absolute quantification however was not possible by
any of these methods. Therefore, it is important to develop a method for direct
analysis of LacLys.
CML is a well characterized and extensively studied MRP in vivo as well in food and
it is widely used as an indicator for the advanced Maillard reaction. At present,
different analytical approaches have been employed for the determination of CML in
milk and dairy products. For example, CML was measured in infant formula by
ELISA (Birlouez-Aragon, Pischetsrieder et al. 2004). CML was also determined after
acidic hydrolysis by GC-MS (Charissou, Ait-Ameur et al. 2007). In recent years,
complete protein hydrolysis followed by LC-ESI-MS-MS in MRM mode with isotopic
dilution assay has been widely used for the determination of CML in different milk
types like pasteurized, UHT, skimmed and condensed milk (Hegele, Buetler et al.
2008) as well as in raw milk, infant formula, evaporated whole milk and cheese
(Assar, Moloney et al. 2009).
Methionine can be oxidized during heating of milk as well as by the treatment with
H2O2, which is used to sterilize whey, milk and milk containers. Additionally,
methionine is oxidized by H2O2 which is generated during lipid oxidation. Oxidation
converts methionine mainly to methionine sulfoxide (MeSO), while stronger oxidative
conditions lead to the formation of methionine sulfone (figure 9) (Guy and Fenaille
2006). The oxidation of methionine to MeSO, which has a more polar side chain,
may decrease the bioavailability of the sulphur amino acid and may affect the
functionality of milk protein resulting in a change of the emulsifying capacity (Baxter,
Lai et al. 2007). MeSO may be used as good marker for oxidation, but it is not often
quantified, mainly because of its lability during protein hydrolysis (Guy and Fenaille
2006).
CHAPTER 2. RESULTS AND DISCUSSION 30
Figure 9: Products of methionine oxidation
Lysine aldehyde is identified as the main carbonyl compound in proteins oxidized by
by metal catalysis. Metal-catalysed oxidation involves the generation of H2O2 and
subsequent reduction of Fe3+ and Cu2+ to Fe2+ and Cu1+. Both, Fe2+ and Cu1+, bind
to specific metal binding sites on proteins and react with H2O2 to generate the highly
reactive OH. radicals. The latter attacks neighbouring amino acids forming carbonyl
compounds (Requena, Chao et al. 2001; Akagawa, Sasaki et al. 2006). Additionally,
lysine aldehyde is formed by Strecker-type degredation during the Maillard reaction
(Suyama, Akagawa et al. 2002)
Figure 10: Lysine oxidation to form lysine aldehyde
Lysine aldehyde is also formed by the action of lysyl oxidase (LOX). LOX catalyzes
the oxidative deamination of the ε-amino group of lysine residues, especially in
collagen and elastin, to yield lysine aldehyde which is also referred to as allysine.
Once generated, allysine condenses with another allysine molecule or with the ε-
amino group of lysine leading to crosslinkage and decreased protein functionality
(Akagawa, Wako et al. 1999; Sell, Strauch et al. 2007). Lysine aldehyde and other
carbonyl compounds are measured conveniently by the use of 2, 4-
dinitrophenylhydrazine (DNP) which reacts with carbonyl groups to generate
dinitrophenylhydrazone. The latter displays characteristic UV-absorbance at 360-
CHAPTER 2. RESULTS AND DISCUSSION 31
390nm. Specific methods were developed to detect lysine aldehyde such as the use
of MALDI-TOF-MS after partial enzymatic hydrolysis, where lysine aldehyde is
identified by the mass difference of -1Da (Meltretter, Seeber et al. 2007). Another
specific method was reported which has used sodium borohydride to reduce lysine
aldehyde to hydroxyaminocaproic acid prior to the analysis by GC-MS (Requena,
Chao et al. 2000).
It is well known that many amino acids in proteins are susceptible to oxidation by
several forms of reactive oxygen species (ROS), such as superoxide (O2.), hydroxyl
radical (OH.) and hydrogen peroxide (H2O2) (Reddie, Seo et al. 2008). Some studies
indicate the formation of hydroxytryptophan (OH-Trp) in whey proteins. The oxidation
of the thiol group of cysteine can result in a wide range of products. The major
product of the reaction with H2O2 is cysteine sulfenic acid (Cys-SOH), which is a
highly reactive product. It is assumed that cysteine sulfenic acid is a transient
intermediate to form more stable products such as cysteine sulfinic acid and cysteine
sulfonic acid. Cysteine sulfenic acid may also bind to neighouring cysteine to form a
disulfide bond giving cystine (Saurin, Neubert et al. 2004). Furthermore, two
molecules of sulfenic acid can yield the thiosulfinate derivative (figure 11). However,
despite of its high reactivity, cysteine sulfenic acid was identified in some enzymes
(Claiborne, Miller et al. 1993). Milk proteins are also suceptible to oxidation during
processing and storage. Some studies indicate the formation of cysteine sulfenic
acid in whey proteins via MALDI-TOF-MS (Meltretter, Seeber et al. 2007).
Additionally, formation of ornithine from arginine may occur in food during heat
treatment.
CHAPTER 2. RESULTS AND DISCUSSION 32
Figure 11: Some cysteine oxidation products
The aims of the present work were the quantification of the compounds LacLys,
CML, MeSO, 5-OH-Trp, Cys-SOH, lysine aldehyde and ornithine in milk by LC-ESI-
MS-MS analysis in MRM mode after complete protein hydrolysis. Since most of
these modified amino acids are not commercially available, the first step of this work
was to develop methods to synthesize the reference compounds in order to use
them as standards for identification and quantification.
CHAPTER 2. RESULTS AND DISCUSSION 33
2.2 Standards synthesis
2.2.1 Synthesis of LacLys
LacLys was synthesized according to a method described in literature to synthesize
Nε-(1-deoxy-D-fructose-1-yl)-L-lysine which is the Amadori product from glucose
(fructosyllysine, FL) (Smith and Thornalley 1992; Vinale, Fogliano et al. 1999). In
order to prevent lactosylation at the α-amino group of lysine, Nα-
fluorenylmethyloxycarbonyl-lysine (Nα-FMOC-Lys) was used. After refluxing FMOC-
Lys with lactose, the reaction mixture was analyzed by LC-MS using a RP-18
column.
The total ion current (TIC) of the synthesized product showed two peaks, one after
11.34 min which is unreacted FMOC-Lys. The second peak appeared after 10.7 min
indicating a higher polarity than FMOC-Lys. Lactosylation of whey proteins causes a
decrease in the retention time of the modified whey proteins compared to the native
one due to the highly hydrophilic lactose moieties (Czerwenka, Maier et al. 2006).
Additionally, both, FMOC-Lys and the unknown compound, had the same UV-
absorption maximum at 272 nm. Furthermore, the molecular ion [M+1]+ of the
unknown compound appeared at m/z 693 corresponding to m/z of FMOC-LacLys.
These data refer to a possible formation of FMOC-LacLys which should be
confirmed by 1H-NMR. However, using a RP-18 column it was difficult to isolate this
unknown compound from the reaction mixture due to its similar retention time
compared to FMOC-Lys as well as due to its low yield (about 10% of FMOC-Lys has
undergone reaction).
CHAPTER 2. RESULTS AND DISCUSSION 34
Figure 12: The chromatogram of the reaction mixture of FMOC-lys and lactose shows the formation of a new product with m/z of 693 indicating the formation of FMOC-LacLys
After the removal of the blocking group (FMOC), the resulting yellowish powder was
analyzed by mass spectrometry and showed the molecular ion [M+1]+ of m/z 471.2
corresponding to protonated lactulosyllysine (MW 470.211). Additionally, MS2 of the
putative lactulosyllysine yielded the fragments m/z 471.2→ 225.1, m/z 471.2→ 128.1
and m/z 471.2→ 84.1, which are identical to the fragments of fructosyllysine, which
is the analogous product of LacLys (Hegele, Parisod et al. 2008).
Figure 13 : MS2 spectrum of the synthesized LacLys
The observed fragments can be explained by the following path way. The
protonation of the alcohol function on C5 of fructose moiety of LacLys (m/z 471.2) is
followed by a loss of water and possibly nucleophilic attack of Nε of lysine to the
same carbocation leading to the formation of a six-membered ring (the fragment m/z
471.2→453). Another water moiety is then cleaved from the intermediately formed
ring with the alcohol function on C6 of the fructose moiety giving the fragment m/z
CHAPTER 2. RESULTS AND DISCUSSION 35
471.2→435. The fragment m/z 471.2→ 435 then either undergoes rearrangement of
a hydrogen atom and elimination of six-memberd ring involving the Nε of lysine giving
the qualifier fragment m/z 471.2→ 128.1. Alternatively, it eliminates galactose and
formaldehyde to yield the product ion m/z 471.2→ 225.1 which showed the highest
intensity of the LacLys fragments (the quantifier fragment). The product ion m/z
471.2→ 84.1 is generated from the fragment m/z 471.2→ 225.1 by the elimination of
both 3-hydroxypyridine and formic acid (Hegele, Parisod et al. 2008). The following
figure shows the fragmentation pathway of the protonated LacLys and the resulting
ion products.
Figure 14: Fragmentation pathway of lactulosyllysine
CHAPTER 2. RESULTS AND DISCUSSION 36
The synthesized LacLys showed a complex 1H-NMR spectrum due to the presence
of lysine, lactose and morpholine. This resulted in difficulties in interpretation of the
spectrum as well as the determination of the product purity. Therefore, trials were
carried out to purify LacLys as well as to improve its yield. In order to increase the
solubility of lactose, which shoud lead to a higher reaction yield, lactose was
dissolved in 1 ml water before it was added to the reaction mixture. Additionally, the
reaction mixture was refluxed for 5, 7 and 8 hours. At last, the precipitated lactose
was removed by filtration.
As a result, no real improvement was noted. Only 14% of FMOC-Lys reacted with
lactose. This result was not satisfied, considering that the synthesis required further
steps. Therefore, it was attemped to purify LacLys using affinity chromatography.
2.2.1.1 Affinity chromatography
Affinity chromatography separates biochemical mixtures on the basis of reversible
interactions between the analyte of interest and a specific binding molecule. It
enables the purification of a substance present at a low concentration in a crude
sample. The target substance can interact with a specific functional group as a result
of electrostatic or hydrophobic interaction, van der Waals forces and hydrogen
bonding. This interaction can be reversed, either specifically by a competitive
substance, or non specifically by changing the pH, ionic strength or the polarity.
The stages of affinity chromatography are summarized in figure 15 and include:
1- Equilibration of the affinity medium with the binding buffer to ensure that the
target molecule interacts with a specific binding substance, while all other
molecules are washed though the column.
2- The sample is applied, the target molecule binds specifically to the binding
molecule and the unbound substances elute through the column with the
binding buffer.
3- Conditions are changed to recover the target molecule, either specifically or not
specifically by the elution buffer.
4- Affinity medium is re-equilibrated using the binding buffer.
CHAPTER 2. RESULTS AND DISCUSSION 37
Figure 15: Illustration of affinity chromatography. [Adapted according to the handbook (Affinity Chromatography) from Amersham Biosciences]
2.2.1.1.1 Boronic acid affinity chromatography
Under alkaline conditions, boronic acids have a strong affinity towards cis-diol
containing compounds such as catechols, nucleic acids, some proteins and
carbohydrates (Frolov and Hoffmann 2008).
The Amadori product contains also cis-diol groups. m-Aminophenylboronic acid was
used, for example, to estimate glycated hemoglobin in normal and diabetic persons
in the early 1980 (Yue, McLennan et al. 1982). Furthermore, m-aminophenylboronic
acid was used to isolate glycated protein and peptides from human serum prior to
the analysis by LC-MS-MS (Zhang, Tang et al. 2007). Recently, Frolov et al have
optimized a method to enrich glycated peptides from bovine serum albumin followed
by the analysis by LC-ESI-MS-MS and MALDI-MS (Frolov and Hoffmann 2008).
In the current experiment, m-aminophenylboronic acid was used to purify the
Amadori product LacLys. The elution solutions were D-sorbitol and acetic acid. The
signal intensity of LacLys, which was purified by affinity chromatography, was
CHAPTER 2. RESULTS AND DISCUSSION 38
compared to the control which was not subjected to affinity chromatogaphy
(15µg/ml).
Figure 16: The interaction of m-aminophenylboronic acid with lactulosyllysine
Using of D-sorbitol as a specific eluent, the intensity of LacLys increased about 50%
compared to the control. By the use of acetic acid, no real improvement was
observed. In contrast, Frololov et al have found that the elution of glycated peptide
with acetic acid was as efficient as with D-sorbitol based on the peak areas in RP-
HPLC (Frolov and Hoffmann 2008). Boronic acid has high affinity towards cis-diols,
which inteacts with LacLys and lactose. The addition of sorbitol leads to a
competition between LacLys, lactose and sorbitol to bind to boronic acid.
This led to an enrichment of LacLys, but probably also of lactose. Another drawback
of the method is that after affinity purification, LacLys is present in a sorbitol solution
which needs to be removed by a further purification procedure. Therefore, a new
attempt was carried out to enrich FMOC-LacLys from the reaction mixture using a
ZIC-HILIC column
2.2.1.2 Hydrophilic interaction liquid chromatograp hy (HILIC)
Most of compounds formed during the initial stage of the Maillard reaction are
relatively polar. Therefore, they are poorly retained on conventional reverse phase
columns (Hao, Lu et al. 2007).
CHAPTER 2. RESULTS AND DISCUSSION 39
HILIC is a mode of chromatography which is similar to normal phase liquid
chromatography (NPLC) because it also uses a hydrophilic stationary phase. HILIC
separation employs different polar stationary phases such as unbound silica, amine
bonded phases, amide bonded phases and zwitterion bonded phases (like ZIC-
HILIC) (Guo and Gaiki 2005). In contrast to classic NPLC, the hydrophobic organic
mobile phase is replaced by an aqueous-miscible solvent such as acetonitrile or
methanol. The combination of high-organic solvent (more than 70 %) with a low
aqueous-solvent enables HILIC to be ideal for the compound ionization by ESI as
well as to provide a good retention for polar analytes (Schettgen, Tings et al. 2007).
The analyte retention on HILIC columns is proportional to its polarity and inversely
proportional to the mobile phase polarity. Several retention mechanisms are involved
in HILIC columns, such as hydrophilic interaction and ion exchange (Grumbach,
Wagrowski-Diehl et al. 2004; Schettgen, Tings et al. 2007).
Early HILIC application focused on the separation of peptides and carbohydrate
(Alpert 1990; Alpert, Shukla et al. 1994). Recently, the application of HILIC was
extended to include the analysis of amino acids (Hao, Lu et al. 2007), advanced
glycation end products (AGEs) (Schettgen, Tings et al. 2007) as well as drugs
(Grumbach, Diehl et al. 2003) and food like wheat gluten (Schlichtherle-Cerny,
Affolter et al. 2003).
2.2.1.2.1 ZIC-HILIC column
ZIC-HILIC columns share the main properties with the other HILIC columns. The
ZIC-HILIC column has a permanent and hydrophilic zwitterionic stationary phase
attached to porous silica. The weak electrostatic interaction between the charged
analyte and the neutral zwitterionic stationary phase results in a unique selectivity
especially for polar analytes which are poorly retained on reverse phase columns.
The zwitterionic stationary phase is stable and, according to the manufacture, it can
be operated in pH range 3-8 which allows changing the polarity of the analyte and
affecting the selectivity without changing the stability of the column.
CHAPTER 2. RESULTS AND DISCUSSION 40
Figure 17: Functional group packed in ZIC-HILIC columns
By the use of a ZIC-HILIC column, FMOC-LacLys eluted after 5.1 min. Unreacted
FMOC-Lys eluted after 3.93 min as shown in figure 15.
Figure 18: Chromatogram of the reaction mixture of FMOC-Lys and lactose separated on a ZIC-HILIC column
The appearing compound between FMOC-lysine and FMOC-LacLys showed the
molecular ion [M+1]+ of m/z 531.1 corresponding to protonated FMOC-fructosyllysine
and suggesting that lactose was hydrolysed during the synthesis.
After fractionation, one peak only appeared after 5.1 min. This peak showed the
molecular ion [M+1]+ of m/z 693 which indicates the presence of FMOC-LacLys, as
shown in figure 19. The synthesis of LacLys is illustrated in figure 20.
CHAPTER 2. RESULTS AND DISCUSSION 41
Figure 19: Chromatogram and mass spectrum of FMOC-LacLys obtained after fractionation of the reaction mixture of FMOC-Lys and lactose on a ZIC-HILIC column.
Figure 20: Synthesis of LacLys
CHAPTER 2. RESULTS AND DISCUSSION 42
2.2.1.3 Identification of LacLys by 1H-NMR
After the removal of the blockage group by morpholine, 1 mg of the synthesized
LacLys was dissolved in 1ml of D2O and then the structure of this product was
analyzed by 1H-NMR.
The synthesized LacLys showed the following 1H-NMR spectral data (600 MHz,
D2O) : δ=3.41 (t, 1H, αCH-Lys, J=5.8), δ =3.252 (s, 2H, Lys-CH2-lac), δ=3.01 (t, 2H,
εCH-Lys, J=7.5 ) δ=1.76 (m, 2H, βCH-Lys J not resolved), δ=1.7 (m, 2H, δCH-Lys,
J=7.6), δ=1.41 (m, 2H, γCH-Lys, J=6.6 ). δ=4.4 (d, 1H, CH-Gal (C1), J=3.4), δ=3.17
(t, 1H, CH-Glu (C4), J=6.3), δ=3.51 (t, 1H, CH-Gal (C4), J=5.5), δ=4.05-4.136 (m,
1H, CH-Gal (C5); 1H, CH-Glu (C5)), δ=3.53-3.85 (many H signals belonging to CH-
Glu (C3, C6);CH-Gal (C2, C3, C6); and all OH of glucose and galcatose)
The interpretation of the 1H-NMR spectrum of LacLys was achieved by comparison
to the 1H-NMR spectra of lysine and lactose, which were recorded separately.
Additionally, the interpretation was based on 1H-NMR spectral data of lactose and
lysine as well as fructosyllysine which were predicted by ChemDrawUltra11.01.
Furthermore, the spectral data of LacLys were compared to those of fructosyllysine
reported in literature (Smith and Thornalley 1992; Vinale, Fogliano et al. 1999).
It is expected that the resonance signal, which is assigned to the protons of the bond
between lactose and lysine, should appear as a singlet in 1H-NMR spectrum
because both neighboring atoms to this bond are not bound to protons (n = 0). Thus,
the multiplicity of the bond between lysine and lactose is 1. The multiplicity of the
bond is determined by the number (n) of magnetically equivalent protons on the
neighboring atoms and is given by the quantity n + 1 (Skoog 2005). This singlet
signal appeared between the proton signals of α- and ε-carbons of lysine as shown
in figure 21.
CHAPTER 2. RESULTS AND DISCUSSION 43
Figure 21: 1H-NMR spectrum of the synthesized LacLys shows the signal of α-C and ε-C of lysine as well as the expected signal of the bond between lactose and lysine
2.2.1.4 Determination of the purity of LacLys by 1H-NMR
The synthesized LacLys was measured quantitatively by 1H-NMR using DMF
(99.8%) as a standard to estimate the purity and acetone as a standard for axis
calibration.The 1H-NMR spectrum of lysine in D2O exhibited one triplet at δ=3.41
assigned to the proton at α-carbon of lysine (figure 21). The resonance signal of this
proton did not interfere with the other signals observed in the spectrum; therefore,
this signal was adopted to estimate the purity of LacLys by comparing its area to that
one of the aldehyde proton of DMF (δ=7.926). It might be better to estimate the
purity of LacLys depending on the resonance signal of the protons in the bond
position between lactose and lysine, but this signal was not completely resolved. The
purity of LacLys was 6.13%. Despite of this low purity, the synthesized product can
be used for quantification of LacLys in milk products, since the absolute content of
the standard could be determined by quantitative 1H-NMR. Thus, for first time
lactulosyllysine was synthesized and its purity was determined by quantitative 1H-
NMR. The main impurity was attributed to morpholine used to remove the FMOC-
group. Morpholine showed 3 signals at δ of 3.132, 3.294 and 3.95 as (figure 21).
CHAPTER 2. RESULTS AND DISCUSSION 44
Figure 22: Signal of the aldehyde proton of DMF which appeared at 7.926 ppm
2.2.2 Synthesis of CML
CML was prepared as reported in the literature using iodoacetic acid as alkylating
agent (Huber and Pischetsrieder 1998) with some modifications (figure 24). In order
to block the α-amino group and thus, to prevent the formation of a carboxymethyl
group on the α-amino group of lysine, Nα-carbobezyloxy-L-lysine (Cbz-lysine) was
used for the synthesis. Under the applied chromatographic conditions, the product
Nα-Cbz-CML eluted after 17 min as shown in figure 23.
Figure 23: Chromatogram of the reaction mixture of Cbz-lysine and iodoacetic acid shows the formation of Cbz-CML
The limited participation of Cbz-lysine in the reaction as well as the double alkylation
on the primary amino group (formation of α-Cbz-bis-CML) necessitated fractionation
by preparative HPLC to isolate α-Cbz-CML. After the removal of the blockage group
Cbz by hydrogenation, the synthesized product was analyzed by MS-MS. The mass
CHAPTER 2. RESULTS AND DISCUSSION 45
spectrum showed the molecular ion [M+1]+ of m/z 205.1 corresponding to CML.
Additionally, the mass spectrum showed the fragments m/z 205.1→ 84, m/z 205.1→
130 and m/z 205.1→ 56 which are identical to those reported in the literature (Yalcin
and Harrison 1996; Teerlink, Barto et al. 2004; Delatour, Hegele et al. 2009).
However, the synthesized CML was not completely pure; therefore, a CML standard
was purchased to validate the method as well as to quantify CML in milk products.
Figure 24: Synthesis of CML
2.2.3 Synthesis of MeSO
Methionine sulfoxide was synthesized using H2O2 as an oxidizing agent as reported
in literature (Brot, Werth et al. 1982; Fliss, Weissbach et al. 1983). The method was
further optimized to improve the yield.
The residue of the reaction mixture of methionine and H2O2 was loaded on a ZIC-
HILIC column. As it shown in figure 25, most of methionine (Me) was oxidized to
methionine sulfoxide (MeSO), which eluted after 6.7 min. A slight formation of
methionine sulfone was observed (MeSOO, retention time 6 min). The peak of
unreacted methionine appeared after 5.4 min.
CHAPTER 2. RESULTS AND DISCUSSION 46
Figure 25: Chromatogram of the reaction mixture of methionine and H2O2 which was incubated at 21 °C. The chromatogram shows the form ation of MeSO and MeSOO.
In order to improve the yield of MeSO, many trials were carried out in which different
amounts of methionine, H2O2 and HCl were incubated for 1-2 hours at 21 °C. The
trials are summarized in table 4.
trial methionine
0.25 M (µl)
HCl 0.5 M
(µl)
30% H2O2
(µl)
incubation time
(hour)
1 400 200 10 1
2 400 100 5 1
3 400 100 5 2
4 400 100 0 1
5 400 0 5 1
6 200 100 0 1
7 200 0 5 1
8 200 0 10 1
Table 4: Amount of methionine (0.25 M), HCl and H2O2 (30%) used to form MeSO
The best yield of MeSO was obtained by the incubation of 200 µl of 0.25 M
methionine and 10 µl of 30% H2O2 for 1 hour at 21°C. Methionine was totally
oxidized to methionine sulfoxide without the formation of methionine sulfone. The
synthesized product was then analyzed by LC-MS-MS showing the molecular ion
[M+1]+ of m/z 166.1 equivalent to the molecular ion of MeSO. In order to confirm the
CHAPTER 2. RESULTS AND DISCUSSION 47
product identity of the synthesized MeSO, high resolution electron impact mass
spectrometry (HREIMS) was carried out which showed that the synthesized product
had the same mass as calculated for MeSO (165.0460 Da). Additionally, the
synthesized MeSO revealed the same 1H-NMR spectral data as an authentic
standard. However, the synthesized MeSO had a lower purity than a commercial
standard (about 85%). This purity was estimated by the peak areas of both product
standards. Therefore, a commercial MeSO standard was used for method validation
as well as for quantitative analysis.
2.2.4 Trial to synthesize lysine aldehyde
Synthesis of lysine aldehyde was attempted using egg shell membrane (ESM) which
is an easily accessible source for LOX. LOX was isolated from ESM according to a
method reported in literature using potassium phosphate buffer containing NaCl and
urea (Kagan, Sullivan et al. 1979). The LOX extract was then incubated with Cbz-
lysine in the presence of urea at 37 °C to catalyse deamination of the ε-amino group.
The reaction mixture was analyzed by LC-MS.
Using a RP-18 column, unreacted Cbz-lysine eluted after 7.76 min. In the solution
which contained the LOX extract, an unknown compound appeared after 10.38 min,
as shown in figure 26. The unknown compound was detected first after 3 days. Its
yield increased gradually till the 9-th day. Additional formation was not observed in
the next 5 days. At the same time, Cbz-lysine decreased gradually till the 9-th day
when 18% had reacted. The molecular ion [M+1]+ of Cbz-lysine and the unknown
compound were 281.2 and 280.2 m/z, respectively, indicating a possible formation of
Cbz-lysine aldehyde.
CHAPTER 2. RESULTS AND DISCUSSION 48
Figure 26: Chromatogram and mass spectrum of the reaction mixture of Cbz-lysine and urea extract of ESM shows unreacted Cbz-lysine as well as the formation of unknown compound. The unknown compound showed m/z 280.2 corresponding to the protonated Cbz-lysine aldehyde with a mass difference of -1Da compared to Cbz-lysine.
The resulting reaction mixture contained additionally to the target compound, urea,
the enzyme extract as well as unreacted Cbz-lysine. Therefore, it was important to
isolate it before removal of the blockage group.
The isolation was achieved by HPLC fractionation using similar conditions to those
which were used previously for the detection. The 1H-NMR spectrum of this product
did not reveal any signal of an aldehyde proton, which appears generally about 10
ppm, although the analysis by mass spectrometry showed the molecular ion m/z
280.2. Furthermore, the molecular ion m/z 146.2 corresponding to protonated lysine
aldehyde was not detected after the removal of the blocking group. It is likely that
Cbz-allysine was formed, but it was not stable under the conditions of synthesis and
purification, probably because of the high reactivity of the aldehyde group.
2.2.5 Trial to synthesize Cys-SOH
The synthesis was attempted using Fenton`s reagent which is a mixture of hydrogen
peroxide and Fe2+, typically FeSO4. Fenton`s reagent is used to generate highly
reactive hydroxyl radicals which can react with the sulfhydryl group of the cysteine
residue to give a variety of oxidation products. This method has been applied on
some peptides of macrophage enzymes and resulted in a variety of oxidation
products which were identified by LC-MS-MS (Shetty, Spellman et al. 2007).
Cysteine was incubated with H2O2 at different temperatures as well as for different
time periods. In order to stop the oxidation of cysteine, methionine was added since
CHAPTER 2. RESULTS AND DISCUSSION 49
it is more susceptible to oxidation. A precursor ion scan of the reaction mixtures of
cysteine and hydrogen peroxide did not show the ions m/z 138, 154 and 172, which
are corresponding to protonated cysteine sulfenic acid, cysteine sufinic acid and
cysteine sulfonic acid, respectively. The reaction led to the formation of a new
product that eluted after 3.87 min and showed the molecular ion [M+1]+ of m/z 241.2
(figure 27). The product showed maximum absorption at 260-270 nm which is the
optimal wavelength for monitoring the disulfide bond (Ashby and Nagy 2007). These
data indicate the formation of cystine as the main product of cysteine oxidation (MW
240.3). Cysteine sulfenic acid was either not formed or was an unstable intermediate
which underwent further reactions.
Figure 27: Chromatogram of the reaction mixture of cysteine and H2O2 (recorded at 250 nm) shows the scavenger methionine and a new product with m/z 241.2 which was assigned to cystine
2.2.6 OH-Trp
OH-Trp was not synthesized. The compound 5-OH-Trp, which is commercially
available, was used as a standard compound.
2.2.7 Discussion
The use of tandem mass spectrometry to detect oxidation and glycation products in
milk protein requires first the preparation of standard compounds for identification
and quantification of the products in the samples. LacLys was synthesized by the
reaction of FMOC-lysine and lactose under nitrogen atmosphere to avoid the
oxidative cleavage of FMOC-LacLys to FMOC-CML. Due to the low reactivity of
CHAPTER 2. RESULTS AND DISCUSSION 50
lactose, only 10-14 % of FMOC-lysine underwent the reaction. Additionally, lactose
was hydrolysed during the reaction to give glucose and galactose. Both
monosaccharides have higher glycation activity compared to lactose (Chevalier,
Chobert et al. 2001). Consequently, glucose and galactose promote the formation of
other Amadori products thus reducing the yield of FMOC-LacLys. This low yield was
not sufficient for further synthesis; therefore FMOC-LacLys was isolated from the
reaction mixture by fractionation on a ZIC-HILIC column followed by the removal of
the blocking group. The identity of the resulting product was confirmed by MS-MS.
The content of the standard was determined by quantitative 1H-NMR using DMF as
an internal standard. DMF was chosen as a standard for quantitation because it is
not volatile. Additionally, the signal of the aldehyde proton did not interfere with the
other resonance signals in the 1H-NMR spectrum. The purity of LacLys was 6.13%.
The main impurity was identified as morpholine, which was used to remove the
blockage group (FMOC-group) and could not be removed from the mixture, because
both LacLys and morpholine showed similar solubility in water. Since the absolute
content of the standard could be determined by quantitative 1H-NMR, the
synthesized product could be used for quantification of LacLys in milk products.
The synthesis of CML and MeSO has been performed using an alkylating agent and
an oxidizing agent, respectively. The structures of the synthesized compounds were
confirmed by 1H-NMR and MS. For quantification however, commercial CML and
MeSO standards were applied, which were available in higher purity.
Both, lysine aldehyde and cysteine sulfenic acid could not be synthesized. Cys-SOH
is particular stable in the absence of other sulfhydryl groups, limited solvent access
and in association with apolar elements in the protein structure (Claiborne, Miller et
al. 1993). Since these conditions were not given during the synthesis, Cys-SOH can
undergo further reactions. For example, there is evidence for the transitional
formation of Cys-SOH in protein, where it rapidly react with thiols to form mixed
disulfides (Giles, Tasker et al. 2001).
In this part of work, LacLys, CML and MeSO as marker compounds for protein
modification in milk were succssefuly synthesized. Additionally, 5-OH-Trp is
commercially available. Therefore, it was decided to develop a method for the
detection and quantification of these compounds in milk products by LC-ESI-MS-MS.
CHAPTER 2. RESULTS AND DISCUSSION 51
2.3 Method development for the analysis of
glycation and oxidation products in milk by LC-ESI-
MS-MS
2.3.1 Introduction
During heat treatment, which is applied to improve food safety and sensory quality
as well as to increase the shelf life of milk products, the quality and the nutritional
value of milk proteins can be adversely affected by oxidative damage and Maillard
reaction. For example, the essential amino acid lysine is blocked by lactose during
the Maillard reaction leading to the formation of lactulosyllysine which is further
degraded to give different products like CML (figure 28). LacLys and CML are used
as indicators for the early and advanced stages of the Maillard reaction in milk,
respectively (van Boekel 1998). Milk proteins are also sensitive to oxidative
modifications which affect particularly methionine, lysine and tryptophan (figure 29).
As a result, the digestibility of milk protein and the bioavailability of these essential
amino acids are decreased (Mauron 1990).
The aim of the following part of work was to develop a method to detect the oxidation
products 5-OH-Trp and MeSO as well as the glycation products LacLys and CML,
which are formed during the heat treatment of α-lactalbumin in a milk resembling
system by LC-ESI-MS-MS.
Figure 28: Reaction of lysine with lactose during heating of whey protein
CHAPTER 2. RESULTS AND DISCUSSION 52
Figure 29: Some protein oxidation products
2.3.2 ESI-MS-MS conditions
Analysis was performed using the mass spectrometer Sciex 4000 QTRAP equipped
with electrospray ionization in positive mode by multi-reaction monitoring (MRM).
The mass analyzer was coupled to a Dionex UltiMate 3000 liquid chromatography.
The three most intense mass transitions of each compound were monitored. The
parameters of the mass analyzer which are dependent on the LC flow rate as well as
those depending on the compounds themselves were optimized to achieve the
highest signal intensity for the analytes. The data acquisition was performed with a
scan time of 200 ms.
CHAPTER 2. RESULTS AND DISCUSSION 53
2.3.2.1 Flow-dependent MS parameters
Source-dependent parameters are generally optimized at the flow rate of liquid
chromatography to get the highest intensities. These parameters are:
• GS1 (Gas 1) controls the nebulizer gas which helps to generate small droplets
from the sample flow.
• GS2 (Gas 2) controls the turbo gas which helps to evaporate the spray
droplets and prevent the solvent from entering the instrument.
• Temp (Temperature) controls the temperature of the turbo gas. Therefore it
helps to evaporate the solvent and to produce sample ions in the gas phase.
• CUR (Curtain gas flow): This parameter controls the curtain gas interface,
which controls the flow of gas between curtain plate and the orifice. The
curtain gas interface prevents the solvent`s droplets from entering and
contaminating the ion optic.
• IS (ion spray voltage) controls the voltage applied on the needle to ionize the
sample.
• CAD (Collision activated dissociation gas) controls the pressure of the
collision gas in the collision cell, which, for MS/MS scan, collides with the
precursor ions for fragmentation.
• Ihe (Interface Heater) switches the interface heater on and off. Heating the
interface helps to maximize the ion signal and prevents the contamination of
the ion optic (Manual user API 4000 QTRAP, 2008).
The optimized parameters are displayed in table 5.
2.3.2.2 Compound-dependent MS parameters
Compound-dependent parameters vary depending on the compound to be analyzed.
These parameters are:
• DP (Declustering potential) controls the potential difference between Q0 and
the orifice plate. It is used to minimize the solvent cluster ions which may
attach to the sample. Thus DP prevents the solvent from entering and
contaminating the ion optic.
• EP (Entrance potential) controls the entrance potential, which guides and
focuses the ion through Q0 region.
CHAPTER 2. RESULTS AND DISCUSSION 54
• CE (Collision energy) controls the collision energy received by the precursor
ion during the acceleration into the collision cell, where they collide with the
CAD gas and are fragmented.
• CXP (Collision Cell Exit Potential) controls the collision cell exit potential and
is used to focus and accelerate the ions out of Q2 towards Q3 (Manual user
API 4000 QTRAP, 2008).
The optimized parameters are displayed in table 6.
Figure 30 : Ion optic in the vacuum chamber of an MS-MS triple quadrupole system (Manual user API 4000 QTRAP, 2008). Q1: first quadrupole; Q2: collision cell; Q3: third quadrupole; ST: stubby lens between Q0 and Q1; ST2: stubby lens between Q1 and Q2; ST3: stubby lens between Q2 and Q3; RF: radio frequency; CEM: channel electron multiplier
CUR 30 (psi) IS 5500 (V)
Temp 500 °C GS1 60 (psi) GS2 75 (psi) CAD medium ihe on
Table 5: Optimized MS-parameters dependent on the LC-flow
CHAPTER 2. RESULTS AND DISCUSSION 55
compound precursor ion
(m/z) product ion
(m/z) DP (V) EP (V) CE (V) CXP (V)
LacLys 471.2 225.1 106 10 33 14 471.2 128.1 106 10 39 10 471.2 84.1 106 10 77 14
5-OH-Trp 221.1 162.1 116 8 25 6 221.1 134 116 8 35 4 221.1 77.1 116 10 73 6
CML 205.1 84.1 116 8 29 2 205.1 130.1 116 8 59 4 205.1 56.1 116 8 17 4
MeSO 166.1 74 46 8 33 8 166.1 56 46 10 19 4 166.1 102.1 46 8 11 4
Table 6: Monitored transitions of LacLys, 5-OH-Trp, CML and MeSO and the optimized MS-parameters dependent on these compounds
2.3.3 MS2 fragmentation of the investigated analyte s
The fragments of CML, MeSO, LacLyc and 5-OH-Trp were determined by product
ion scan, and then the three most intense fragments of each compound were chosen
for identification. The fragment which has the highest intensity was used as quantifier
fragment, as seen in table 6 where the quantifier fragments are printed in bold. The
other two fragments were used as qualifier fragments. However, since CML is
formed only in little amount in milk proteins, it was identified during the investigation
of glycation in milk samples by the quantifier fragment in addition to one of the
qualifier fragments (m/z 205.1→ 84.1 and m/z 205.1→ 130.1.1, respectively).
Otherwise, high concentrations of milk samples have to be used to detect the second
qualifier fragment (m/z 205.1→ 56.1). The predicted structures of these fragments
are shown in the figures 31, 32, 33 and 34.
The quantifier and qualifier fragments of CML (m/z 205.1→84.1 and m/z
205.1→130.1.1, respectively) were identical to those reported in literature (Delatour,
Fenaille et al. 2006; Schettgen, Tings et al. 2007). The qualifier fragments of MeSO
(m/z 166.1→56) (Sochaski, Jenkins et al. 2001) and (m/z 166.1→102) (Thornalley,
Battah et al. 2003) were reported in literature. The quantifier fragment of MeSO (m/z
CHAPTER 2. RESULTS AND DISCUSSION 56
166.1→74) has not been reported in literature. The quantifier fragment (m/z
471.2→225.1) and the qualifier fragments (m/z 471.2→128.1, m/z 471.2→84.1) of
LacLys were analogous to those of the Amadori product fructosyllysine (figure 14)
(Hegele, Buetler et al. 2008; Hegele, Parisod et al. 2008). The fragmentation of 5-
OH-Trp has not been reported in literature.
Figure 31: Product ion mass spectrum of CML and the corresponding fragments
Figure 32: Product ion mass spectrum of MeSO and the corresponding fragments
CHAPTER 2. RESULTS AND DISCUSSION 57
Figure 33: Product ion mass spectrum of LacLys and the corresponding fragments
Figure 34: Product ion mass spectrum of 5-OH-Trp and the corresponding fragments
2.3.4 Chromatographic seperation of CML, LacLys, Me SO
and 5-OH-Trp using a C18 column
In order to detect the compounds MeSO, 5-OH-Trp, LacLys and CML in milk
proteins, it was important to determine the retention time of these compounds.
Therefore, a mixture of the standard compounds MeSO, 5-OH-Trp, LacLys and CML
was analyzed under the optimized parameters of mass spectrometry (table 5 and 6).
The compounds were separated on a C18 column 150*2.1mm, particle size 5 µm,
CHAPTER 2. RESULTS AND DISCUSSION 58
protected with a pre-column 20*2.1mm. The column was housed in the oven at 20
°C. The flow rate and injection volume were set at 200 µl/min and 5 µl, respectively.
Since CML and LacLys are very polar compounds, they have only little retention on
RP-columns. To overcome this problem, ion pair reagents like nonafluoropentanoic
acid (NFPA) are used. NFPA was chosen due to its high compatibility with MS-MS
compared to other ion pair reagents like trifluoroacetic acid (TFA). Mobile phases
were acetonitrle (solvent B) and 5 mM NFPA in water (solvent A). The gradient was
optimized to achieve best separation of the four analytes. The gradient started with
10% acetonitrile, which increased in 3 min to 40% and in the next 3 min to 50%.
Acetonitrile was kept for 4 min at 50%, and then increased rapidly in 2 min to 100%
where it was kept for further 2 min. Acetonitrile decreased then in 6 min to the initial
conditions (10%) followed by the equilibration for 5 min before injecting the next
sample. All samples were dissolved in a mixture of water/acetonitrile (50/50%).
Methionine sulfoxide eluted after 4.1 min separately from the other compounds.
LacLys, 5-OH-Trp and CML eluted almost together after 7.2 to 7.6 min (figure 35). In
order to separate LacLys, 5-OH-Trp and CML from each other, the gradient was
systematically optimized but the separation of the compounds on the RP-18 column
could not be achieved. However, the method can be still applied because the
compounds LacLys, 5-OH-Trp and CML are separated by their specific mass
transitions. The detection of the three fragments of each compound depended on the
compound concentration. Thus, only one fragment was detected by the analysis of
low concentrations of the standard compounds. Therefore, high protein
concentrations were required for the investigation of oxidation and glycation products
in whey proteins as well as in milk products, since the identification of a modified
amino acid by MS-MS needs at least the detection of two fragments.
CHAPTER 2. RESULTS AND DISCUSSION 59
Figure 35: LC-MS-MS chromatogram of the standards mixture, obtained in MRM mode, on a C18 column. The standard mixture contained MeSO, 5-OH-Trp, LacLys and CML
An unknown peak appeared after 8.08 min which showed the quantifier fragment of
LacLys (m/z 471.2→225.1) but no qualifier fragment was detected. This peak
appeared during the analysis of standards, a mixture of water and acetonitrile
(50/50%), water alone and also during the analysis of milk samples. Additionally, it
appeared when the elution gradient was run on the C18 column without loading any
sample indicating that the presence of this peak is related to the mobile phases
themselves. Since the used acetonitrile was of MS grade while the used ion pair
reagent had a purity of 97%, it is expected that this peak belongs to a fragment of an
unknown compound present with the ion pair reagent.
2.3.5 Glycation of α-LA in a milk model mixture
α-Lactalbumin (α-LA) is the second major whey protein after β-LG in milk. It
represents about 30% of the total whey proteins and it is an important source for the
essential amino acid tryptophan. In order to study glycation in milk protein, a milk
model containing lactose and α-LA in the relevant concentrations diluted in a milk
salt matrix were heated at 60 °C. Three controls we re prepared: α-LA and lactose
without heat treatment, native α-LA as well as α-LA heated alone at 60 °C. After the
heat treatment, lactose and minerals were removed by dialysis to avoid artificial
modification during sample preparation. The lyophilized residue was then subjected
CHAPTER 2. RESULTS AND DISCUSSION 60
to multi-enzyme digestion to liberate the modified amino acids prior to the analysis
by LC-MS-MS. For qualitative experiments, the enzymatic digestion has been
applied as described in literature (Hasenkopf, Ronner et al. 2002) using three
enzymes for three days at 37 °C. For quantification , enzymatic protein hydrolysis has
been further optimized (see 2.8).
Pepsin, pronase and aminopeptidase were added after each other in 24 hours
intervals. Pepsin is an enzyme which cleaves the peptide bond preferably at the
carboxylic side of phenylalanine, tryptophan and leucine and methionine. Pepsin,
according to the supplier, has the optimum activity at a pH range of 1.8-2.2; therefore
the protein was dissolved in 0.02 M HCl (pH 2). Pronase is a mixture of non-specific
proteases, which digest proteins into individual amino acids. The molecular weight of
the various enzymes present in pronase ranges between 20-60 kDa and the
optimum pH value is 6-7.5. Aminopeptidase is an enzyme which cleaves the terminal
amino acids of peptides and proteins. The optimal pH value of aminopeptidase
activity, according to the supplier, varies between 7-7.5 up to 9; therefore it was
necessary to adjust the pH of the protein solution to 7.5 before adding pronase and
aminopeptidase. As a blank for the enzymatic hydrolysis, the enzymes and buffers
were added to 1ml of HCl 0.02 M without protein. The blank was treated and
analyzed exactly like the protein samples.
The hydrolyzate was separated from the enzymes by ultrafiltration using centrifugal
concentrator devices with a MWCO of 10 kDa (figure 36), since all used enzymes
have molecular weight higher than 20 kDa.
After lyophilization, the residue was reconstituted in a water/acetonitrile mixture
(50/50%). The concentration of 25µg/ml was enough for the detection of the modified
amino acids by MS-MS (25 µg of the sample before enzymatic hydrolysis).
Figure 36 : A centrifugal concentrator device which was used for the clean-up of the enzymatic hydrolyzate
CHAPTER 2. RESULTS AND DISCUSSION 61
Many modifications were detected after the incubation of α-LA and lactose in a milk
model mixture at 60 °C. The incubation temperature was chosen to provide a
sufficient extent of modification and to prevent at the same time the denaturation of
α-LA which occurs at temperatures higher than 65 °C (Czerwenka, Maier et al.
2006). After 3 days of incubation of α-LA with lactose at 60 °C, both MeSO and
LacLys were clearly detected by their three characteristic fragments. Because of the
low amount of CML in the samples, only the quantifier fragment (m/z 84.1) was
detected. By increasing the concentration of the sample, the second fragment of
CML was additionally detected (m/z 130.1). Moreover, the signal of CML increased
with increasing incubation time, where the highest signal was detected after 14 days
incubation. The highest signals of MeSO and LacLys, in contrast to CML, were
detected after 7 days and then decreased after 14 days of incubation. 5-OH-Trp was
not detected in any sample.
The peak of MeSO appeared after 3.81 min, which is 0.31 min earlier than the
standard MeSO. This early elution could be caused by other components of the
sample matrix which co-eluted at the same time. CML and LacLys eluted at the
same retention time as the standards (7.2 min and 7.6 min, respectively).
In the three controls neither LacLys nor CML were detected. This is expected since
no lactose was present in the native or the heated α-LA (the second and third
control). Additionally, this result confirms that lactose could be sufficiently removed
during dialysis from the unheated α-LA/lactose mixtures to prevent artificial formation
of LacLys during enzymatic hydrolysis.
Methionine sulfoxide was detected in all controls samples (heated and native α-LA).
The detection of methionine sulfoxide in native α-LA can be attributed either to
previous formation of methionine sulfoxide during the isolation of α-LA or to new
oxidation of methionine during sample preparation.
In order to test the latter hypothesis, 5 µg of methionine was subjected to the
conditions of the enzymatic hydrolysis, cleaned up and analyzed exactly like the
samples of milk model mixture, with one exception: The pepsin solution was
replaced by 50 µl of HCl 0.02 M, to avoid the autolysis of pepsin under the conditions
of enzymatic hydrolysis and thus, to avoid the overestimation of methionine
sulfoxide. The chromatograms showed only a minor formation of MeSO during
CHAPTER 2. RESULTS AND DISCUSSION 62
enzymatic hydrolysis suggesting that the artificial formation of MeSO during sample
preparation can be neglected. Compared to the heated control, the signal of MeSO
has clearly increased in the presence of lactose in the samples. This result leads to
the assumption that the presence of lactose can enhance the oxidation of methionine
during the heat treatment of α-LA. MeSO concentration decreased after 14 days of
incubation, whereas in the heated control MeSO was continuously formed during the
entire incubation process (figure 38).
Figure 37: Extracted ion chromatogram (XIC) of glycated α-LA shows the formation of CML (right). LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of LacLys (left). α-LA and lactose were heated in a milk matrix at 60 °C for 3 (A), 7 (B) and 14 (C) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. CML formation increased during the whole incubation period, while the highest signal of LacLys was obtained after 7 days of incubation.
CHAPTER 2. RESULTS AND DISCUSSION 63
Figure 38: LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of MeSO. α-LA and lactose were heated in a milk matrix at 60 °C for 3 (1 a), 7 (2 a) and 14 (3 a) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. MeSO was detected in native α-LA (standard) and in α-LA heated without lactose for 3 (1 b), 7 (2 b) and 14 (3 b) days
CHAPTER 2. RESULTS AND DISCUSSION 64
2.3.6 Oxidation of α-LA in a milk model mixture
The aim of this experiment was to investigate the formation of protein oxidation
products. Superoxide radicals, which are converted into hydrogen peroxide, may be
generated from glycated proteins such as the Amadori product (Mossine, Linetsky et
al. 1999). Thus, the superoxide radicals generated from glycated proteins in milk can
enhance the oxidation of methionine as well as tryptophan. Therefore, this
experiment aimed to study the formation of MeSO and 5-OH-Trp during the heat
treatment of the milk model under oxidative conditions.
In order to induce oxidation, α-LA was heated in the presence of H2O2 at 120 °C.
Native α-LA as well as α-LA heated without H2O2 served as controls.
5-OH-Trp was not detected neither in oxidized α-LA nor in the control. Methionine
sulfoxide was found in all samples, even in the control (native α-LA and α-LA heated
without H2O2). The signal intensity of MeSO increased gradually with increasing the
heat treatment in the presence of H2O2 compared to the control samples. This result
indicats that H2O2, as a relatively mild oxidant, promotes the oxidation of methionine.
Moreover, the heat treatment of α-LA at 120 °C without the addition of H 2O2 did not
enhance the oxidation of methionine; no real difference in the intensity was found
between the heated control samples (for 10, 20 and 30 min at 120 °C) and the native
α-LA (standard), as seen in figure 39.
CHAPTER 2. RESULTS AND DISCUSSION 65
Figure 39: LC-MS-MS chromatogram of oxidized α-LA obtained in MRM mode shows the formation of MeSO. 5-OH-Trp was not detected. α-LA and H2O2 were heated in a milk matrix for 10, 20 and 30 min at 120 °C (1a, 2a and 3a, respectively) and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. LC-MS-MS spectra obtained in MRM mode of native α-LA (standard) and α-LA heated for 10, 20 and 30 min at 120 °C (1b, 2b and 3 b, respectively) which were used as control
CHAPTER 2. RESULTS AND DISCUSSION 66
2.3.7 Discussion
The heat treatment of α-LA with lactose at 60 °C induced oxidation and gly cation
reactions and led to the loss of both, lysine and methionine. Most of LacLys was
formed during the first three days of incubation, and then the concentrations
increased slightly till the seventh day. During the next 7 days, a decrease of LacLys
was observed. Further studies are necessary to investigate if the degredation of
LacLys during advanced heat treatment is also relevant for milk processing.
LacLys has been identified as the most common modification in milk protein and has
been detected in various dairy products (Meltretter and Pischetsrieder 2008). The
decrease of LacLys during longer incubation in parallel to the increase of CML can
be explained by the oxidative degradation of LacLys to give CML as well as the
participation of degradation products of LacLys in the formation of CML (Ahmed,
Argirov et al. 2002). However, a decreased release of LacLys due to protein
insolubility and protease resistance, as a result of severe modification and cross-
linking, can not be fully excluded (Ahmed, Ahmed et al. 2005).
In general, heating of α-LA at 60 °C led to the formation of MeSO regardles s of the
presence of lactose which might result from oxidation by metal ions present in the
buffer. However, the signal of MeSO increased clearly in the presence of lactose.
This result indicates a role of lactose in oxidation reactions. The Amadori product
promotes the generation of the superoxide radical ion, which is converted into
hydrogen peroxide (Mossine, Linetsky et al. 1999). The latter then reacts with metal
ions to form the hydroxyl radicals which are highly reactive and attacks amino acids
residues leading to oxidation of proteins (Dean, Fu et al. 1997). Since it was shown
in the present study, that hydrogen peroxide leads readily to MeSO formation, this
mechanism could explain promotion of oxidation reaction by lactose. The decrease
of MeSO signal after 14 days of incubation at 60 °C could be caused by further
oxidation to methionine sulfone.
5-OH-Trp was not detected in glycated nor in oxidized α-LA. The absence of 5-OH-
Trp does not proof that tryptophan was resistant to oxidation in the samples. The
indole ring of tryptophan is oxidized in proteins in different ways leading to the
formation of various regioisomers of OH-Trp as well as to kynurenine and N-
formylkynurenine. These products were detected in actin of rats after exposure to
oxidative stress (Fedorova, Kuleva et al. 2010). Formation of tryptophan oxidation
CHAPTER 2. RESULTS AND DISCUSSION 67
products in milk protein is still not assured. The mass shift of 16 Da, which was
detected in some peptides of glycated whey proteins, suggested oxidation of
tryptophan residue. However, this assumption was equivocal since this mass shift
could also result from the oxidation of cysteine residue present at the same peptide
to give cysteine sulfenic acid. Furthermore, the mass shift of 32 Da detected at
another peptide was thought to be caused either by a single oxidation of both
tryptophan and methionine to give hydroxyltryptophan and methionine sulfoxide,
respectively, or a double oxidation of one of them leading to the formation of N-
formylkynurenine or methionine sulfone (Meltretter, Becker et al. 2008). Further
studies are required to determine if tryptophan was really oxidized in processed milk
or milk models.
CHAPTER 2. RESULTS AND DISCUSSION 68
2.4 Detection of glycation and oxidation products in
milk
Cow milk is processed to give different dairy products like butter, yogurt and cheese.
The modern industrial processes can produce more durable milk forms like UHT and
sterilized milk as well as easily transportable forms like milk powder. However, the
thermally treatment applied during the product manufacturing can induce different
modification for milk components similar to those, which have been detected in α-LA
in the previous experiments. The aim of this part of work was the detection of some
glycation and oxidation products in milk proteins formed during industrial processing.
The investigated milk samples were low fat UHT milk and sterilized milk as examples
for liquid long shelf life products.
Additionally, liquid and powdered hypo-allergenic infant formulas (HA-infant formula)
were investigated. Hypoallergenic infant formulas are hydrolyzed milk formulas
which are recommended for preventing or alleviating the atopy response of infants
with a familiar history of atopy. Hypoallergenic infant formulas are divided into
extensively and partially hydrolyzed formulas. Extensively hydrolyzed formulas are
mostly used in therapeutic application because they contain a mixture of amino acids
or low molecular weight peptides with a very low residual antigenicity. The partially
hydrolyzed formulas, which less extensively hydrolyzed, are used to prevent the
development of atopy response for infant with high risk for atopy (Nentwich,
Michkova et al. 2001).
For the analysis of glycation and oxidation products in the commercial milk samples,
protein preparation by dialysis, enzymatic hydrolysis and amino acids purification
were carried out as described for the glycated and oxidized α-LA samples.
Additionally, fat was separated from milk before the dialysis by centrifugation for one
hour at 4 °C. Finally, the samples were analyzed un der the same chromatographic
and MS-MS conditions which were used previously to detect the modification of α-
LA. For LC-MS-MS analysis, 25 µg of the enzymatically hydrolysed residue was
dissolved in 1 ml of a water/acetonitrile mixture (50/50%).
CHAPTER 2. RESULTS AND DISCUSSION 69
2.4.1 Analysis of MeSO and LacLys
The LC-MS-MS chromatograms of UHT milk, sterilized milk, liquid and powder HA-
infant formula have shown a clear formation of MeSO and LacLys (figure 40). As it
was observed for α-LA, MeSO eluted at 3.81 min, which is earlier than the standard
MeSO, while LacLys from samples eluted at the same retention time as the standard
(7.6 min).
Figure 40: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of MeSO and LacLys. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. LacLys and MeSO were identified by their specific mass transitions (LacLys: m/z 471.1→225.1, m/z 471.1→128.1, m/z 471.1→84.1; MeSO: m/z 166.1→74, m/z 166.1→56, m/z 166.1→102.1).
An unknown peak appeared at a retention time of 7 min and showed the ion
products m/z 166.1→74 and m/z 166.1→56, which are the characteristic fragments
of MeSO. To investigate the identity of this peak, MeSO (as standard) was added to
the protein hydrolyzate followed by LC-MS-MS analysis. The chromatograms
showed only an increase of the signal intensity of the peak with a retention time of
3.81 min, which is the peak of MeSO. For further investigation, UHT milk was heated
for 30 and 60 min at 120 °C and analyzed as describ ed before. Since the heat
treatment enhances glycation and oxidation reactions, the peak area of MeSO
increased gradually during the heat treatment. On the other hand, the area of the
unknown peak was almost stable. This means that the peak at RT of 7 min is not
derived from MeSO but is an interferent with lower polarity than MeSO. Later, by the
CHAPTER 2. RESULTS AND DISCUSSION 70
use of the ZIC-HILIC column, this unknown compound eluted before MeSO at the
retention time 4.5 min. This observation confirms its lower polarity than MeSO,
because the retention on a ZIC-HILIC column is proportional to the polarity
(Grumbach, Wagrowski-Diehl et al. 2004). The unknown compound is probably
generated from the milk matrix.
Figure 41: The increase of the MeSO signal and the stability of the unknown peak during heat treatment of milk
The peak areas calculated under the quantifier fragments of both, MeSO and LacLys
(m/z 166.1→74 and m/z 471.2→225.1, respectively), was higher in the samples of
HA-milk and sterilized milk compared to those of UHT milk indicating severe heat
damage in these products (figure 42). The high temperature used for sterilization
promoted the formation of LacLys compared to HA-milk treatment. In contrast, HA-
milk samples showed a higher signal of MeSO than sterilized milk. In order to draw
valid conclusions on the influence of industrial processes on product formation, the
development of a quantification method is necessary. Because of matrix effects on
ionization, the use of internal standard or standard addition for quantification is
required.
CHAPTER 2. RESULTS AND DISCUSSION 71
Figure 42 : Relative signal of MeSO and LacLys in different milk samples
2.4.1.1 Formation of MeSO during sample work-up
MeSO was detected in the blank in which enzymatic hydrolysis was carried out in the
absence of sample proteins. It could be excluded that the buffers and enzymes were
contaminated with MeSO. A reason for the MeSO background could be the autolysis
of the enzymes during enzymatic hydrolysis, especially of pepsin, under the alkaline
conditions.
In order to check this possibility, each of the three enzymes was removed from the
blank hydrolyzate after 1 day incubation and before the addition of the next enzyme.
The enzymes were removed by ultrafiltration using the centrifugal concentrator
devices used previously for the clean-up of protein hydrolyzate (MWCO of 10kDa).
This control was then analyzed by LC-MS-MS and the results were compared to
those of the blank containing the three enzymes untill the end of hydrolysis. The
chromatograms showed about 31% to 42% lower intensity of MeSO after the
removal of pepsin or both pepsin and pronase, respectively, indicating that the
hydrolysis of pepsin is the main source for MeSO in the blank (figure 43). The
presence of MeSO in the blank after the removal of pepsin is caused either by
inadequate removal of pepsin and thus further hydrolysis during the second and third
days, or by hydrolysis of the other enzymes. The intermediate removal of pepsin
during enzymatic hydrolysis of raw and UHT milk, led to the loss of some peptides
resulting in a 21-24 % lower signal intensity for MeSO and LacLys. Therefore, for
further investigation, the enzymes were not removed before the end of enzymatic
hydrolysis (3 days), but the overestimation of MeSO was determined quantitatively
and then subtracted from results of the milk samples.
CHAPTER 2. RESULTS AND DISCUSSION 72
Figure 43: Formation of MeSO during the different steps of enzymatic protein hydrolysis. A blank without sample protein was subsequently hydrolysed by pepsin, pronase and aminopeptidase. The enzymes remained in the samples throughout hydrolysis (A). Pepsin was removed before further hydrolysis (B). Pepsin and pronase were removed before further hydrolysis (C)
Figure 44: The decrease of MeSO and LacLys signal intensities after the removal of pepsin from milk hydrolysate after 24 hours of enzymatic hydrolysis
2.4.2 Analysis of 5-OH-Trp
5-OH-Trp was not detected in any of the samples, even if the sample concentration
was increased to 50 and 75 µg/ml.
CHAPTER 2. RESULTS AND DISCUSSION 73
2.4.3 Analysis of CML
CML was not detected in the samples of UHT milk; this could be related to the low
concentration of the analyzed sample (25µg/ml) and/or to the low modification during
the ultra-high temperature processing. In the other samples, a small peak appeared
after 7.35 min which showed the most intense product ion of CML (m/z 205.1→ 84.1)
indicating a possible formation of CML, as seen in figure 45.
Figure 45: LC-MS-MS chromatogram of sterilized milk obtained in MRM mode shows a clear formation of MeSO and LacLys and a possible formation of CML. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. MeSO and LacLys were identified by their specific mass transitions. Only one mass transition of CML was detected (m/z 205.1→ 84.1).
In order to ensure the identification of CML, it was necessary to increase the
sample’s concentration to the threshold at which two fragments of CML can be
detected. This could be problematic, especially for samples with low modification
rate like UHT and pasteurized milk, because the signals in concentrated samples are
more susceptible to matrix effects. Matrix effects, in particular signal suppression,
might lead to underestimation of the actual concentration and a higher limit of
detection (LOD) (Zimmer 2003). Additionally, the concentrated samples could be
damaging for the spryer needle of the LC-MS-MS apparat.
CML and LacLys are very hydrophilic analytes; therefore they have little retention on
reversed-phase columns. This problem was overcome by the use of the ion pair
CHAPTER 2. RESULTS AND DISCUSSION 74
reagent NFPA. The ion pair reagent is a large molecule which can act as a counter
ion to the analyte leading to the formation of a neutral pair. The neutral pair partitions
between the mobile and stationary phase, or the hydrophobic part of the ion pair
reagent adsorbs to the stationary phase to form an ion exchange surface. Thus, the
increase of the retention time on C18 column caused by the use of NFPA is
depending on the positive charge which is carried on CML and LacLys. Therefore it
will be difficult to separate CML from LacLys. Moreover, the co-elution of LacLys with
CML can cause matrix effects, especially ionization suppression which results in a
reduction of the signal intensity and a lower sensitivity. This effect can also result
from other polar and/or non-polar compounds in the matrix, which co-elute with CML,
even if these compounds are not defined in the MRM method. This loss of sensitivity
is more notable and negatively affected for CML detection due to its low formation
compared to LacLys.
A possibility to improve the method`s sensitivity for CML detection was to seperate
CML from LacLys and probably from other undefined matrix compounds, which have
similar retention time as CML. Since the separation of CML from LacLys could not be
achieved on the C 18 column despite of extended variation of the elution gradient, a
ZIC-HILIC column was used for further experiments.
2.4.4 Separation of 5-OH-Trp, MeSO, CML and LacLys
using a ZIC-HILIC column
HILIC is a mode of chromatography that uses a hydrophilic stationary phase with
organic water miscible solvent like acetonitrile and methanol. The use of an eluent
with a high portion of organic solvent (more than 70%) for HILIC chromatography is
ideal for the compound ionization by ESI-MS. Additionally, suitable retention times
for polar analytes are obtained (Schettgen, Tings et al. 2007). The analyte retention
on the HILIC columns is proportional to its polarity and inversely proportional to the
mobile phase polarity. Therefore, it is expected that CML can be separated form
LacLys by the use of HILIC columns such as ZIC-HILIC column. ZIC-HILIC columns
share the main properties with the other HILIC columns
MS-MS conditions were the same which were previously used for the analysis of 5-
OH-Trp, MeSO, CML and LacLys on the C18 column; however, the HPLC gradients
were changed to be compatible with the ZIC-HILIC column.
CHAPTER 2. RESULTS AND DISCUSSION 75
As it is seen in figure 46, a complete separation of the compounds 5-OH-Trp, MeSO,
CML and LacLys (mixture of standards) was achieved. These compounds eluted
after 4.9, 6.5, 10.5 and 25.6 min, respectively.
Figure 46: LC-MS-MS chromatogram obtained in MRM mode of 5-OH-Trp, MeSO, CML and LacLys (standard mixture) on a ZIC-HILIC column 150*2.1mm, 3.5 µm
2.4.5 Comparison of the signal intensities using C1 8 or
ZIC-HILIC columns
A standard solution containing 5-OH-Trp, MeSO, CML and LacLys was analyzed
once using a C18 column and then using a ZIC-HILIC column. The concentrations of
these compounds were 0.25, 0.008, 0.08 and 0.6µg/ml, respectively. The two trials
were carried out on two consecutive days. The chromatograms showed clear
differences in the intensities of the analytes between the two columns with a huge
advantage for the ZIC-HILIC column. The increase of the intensities using the ZIC-
HILIC column varied between 8, 17, 25, and 200 times for LacLys, MeSO, CML and
5-OH-Trp, respectively (figure 47). Thus, detection limits are considerably
decreased. This will be of special importance for CML which is present in the
samples in concentrations close to the detection limit.
Analysing UHT milk samples on the ZIC-HILIC column showed that the intensities for
CML as well as MeSO became only 3 times higher and for LacLys 5 times higher
compared to separation on the C18 column. Furthermore, the analytes intensities
CHAPTER 2. RESULTS AND DISCUSSION 76
showed a higher variation on the ZIC-HILIC column compared to the C18 column.
High variation was especially noted for LacLys in milk samples where the coefficient
of variation (CV) reached 40%. The reason for such imprecision could be the mass
spectrometer parameters of the ion source. They were optimized for an eluent ratio
of 50/50%, but LacLys eluted at ratio 20/80 of organic and aqueous solvents,
respectively.
Consequently, it was decided to analyze the compounds using two different methods
to achieve optimal results for quantification. The C18 column was chosen for further
investigation of LacLys. The ZIC-HILIC column was chosen for further investigation
of CML and 5-OH-Trp. Since MeSO is detected easily in milk samples and since the
variation of its signal intensity was similar on C18 and ZIC-HILIC columns, C18
column was chosen for further investigation of MeSO to avoid the long run time on
ZIC-HILIC (52 min).
Figure 47: Comparison of the signal intensities of the standards MeSO, CML, 5-OH-Trp and LacLys on C18 and ZIC-HILIC columns
2.4.6 Analysis of milk samples by LC-MS-MS using a ZIC-
HILIC column
UHT milk, sterilized milk and HA-infant formula were analyzed using a ZIC-HILIC
column. For this purpose, 50 µg of enzymaticlly hydrolyzed resdiue was dissolved in
1 ml of a water/acetonitrile mixture. Under these conditions, CML was detected
CHAPTER 2. RESULTS AND DISCUSSION 77
clearly in all milk samples at the retention time of 10.5 min. The formation of CML
was proportional to the thermal treatment. The highest signal was detected in the
sample of sterilized milk followed by the samples of HA infant formula with a
negligible difference between the liquid and powder forms. The mild heat treatment
of UHT milk resulted in the lowest signal for CML. Thus, the difficulty to detect the
low level of CML in UHT milk has been overcome by the replacement of the C18
column by a ZIC-HILIC stationary phase. However, an unknown peak appeared at a
retention time of 5 min, which showed the fragments of CML. In order to clarify the
origin of this peak, CML-standard was added to UHT milk samples before analysis.
The LC-MS-MS chromatograms showed an increase of the signal intensity of the
peak with a retention time of 10.5 min, which is the peak of CML. The signal of the
unknown peak, however, did not increase after spiking, indicating that the peak did
not originate from CML.
Figure 48: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of CML. Milk proteins were subjected to enzymatic hydrolysis prior to the LC-MS-MS analysis using a ZIC-HILIC column. An unknown peak with the same specific mass transitions (m/z 205.1→ 84.1, m/z 205.1→ 130.1) appeared after 5 min.
For further investigation of this peak, UHT milk was defatted and then heated for 30
and 60 min at 120 °C. CML was then analyzed as desc ribed before. Since the heat
treatment promotes the oxidation and glycation reaction, the peak of CML at 10.5
min increased gradually with increasing heat treatment. In contrast, the intensity of
the unknown peak was almost stable. This confirms that the compound at a retention
time of 5 min is not derived from CML and is not formed during the heat treatment.
CHAPTER 2. RESULTS AND DISCUSSION 78
More likely, the unknown compound is derived from the milk matrix. Despite the
higher sensitivity obtained by the use of ZIC-HILIC column compared to C18 column,
5-OH-Trp was not detected in any of the investigated samples.
Figure 49: The increase of CML signal and the stability of the signal of the unknown compound during heat treatment of milk
2.4.7 Discussion
The industrial production of dairy products requires thermal treatment in order to
ensure microbiological safety as well as to increase the shelf life of the products.
However, when exposed to heat, the amino acids in milk proteins may undergo
oxidation and glycation reactions leading to the formation of different compounds
such as lactulosyllysine, carboxymethyllysine, methionine sulfoxide,
hydroxytryptophan and others. In order to detect these modified amino acids in milk
proteins, a complete enzymatic hydrolysis has been applied using three enzymes
(pepsin, pronase and aminopeptidase) for 3 days at 37 °C. The release of the
modified amino acids was followed by the analysis by LC-MS-MS which represents a
powerful analytical technique to determine qualitatively and quantitatively low levels
of the target compounds with a high a degree of certainty.
By the use of LC-ESI-MS-MS, the analytes LacLys, CML and MeSO were detected
in UHT milk and sterilized milk as well as in hypoallergenic infant formula. 5-OH-Trp
was not detected in any of the investigated samples.
At present, different analytical approaches have been introduced to determine MeSO
in milk proteins. The partial enzymatic hydrolysis prior to the analysis by MALDI-
TOF-MS allowed for example the detection of MeSO in many peptides of whey
CHAPTER 2. RESULTS AND DISCUSSION 79
proteins (Meltretter and Pischetsrieder 2008). MeSO was also detected and
quantified in milk protein via reverse-phase HPLC system with UV detection after a
complete enzymatic hydrolysis for 20 hours at 37 °C (Baxter, Lai et al. 2007). In the
current study, MeSO was detected under similar chromatographic conditions after
complete enzymatic hydrolysis. However, MS-MS detection was used, which allows
product identification by three ion products (m/z 166.1→74, m/z 166.1→56 and m/z
166.1→102.1) additional to the retention time. Thus, higher certainty can be
achieved.
It is a common problem in the analysis of the polar compounds like CML and LacLys
that they have only little retention on reversed-phase columns due to their high
polarity. This problem has been overcome by the use of ion pair reagents such as
NFPA, which, in contrast to other ion pair reagent is volatile and better compatible
with ESI-MS-MS (Teerlink, Barto et al. 2004). The combination of C 18 column,
NFPA and MS-MS detection has been used successfully to detect advanced
glycation products in some human fluids (Teerlink, Barto et al. 2004; Gonzalez-
Reche, Kucharczyk et al. 2006) as well as detect early and advanced glycation
products in dairy products (Hegele, Buetler et al. 2008; Hegele, Parisod et al. 2008;
Delatour, Hegele et al. 2009).
The use of NFPA is more preferable for MS-MS, because it yields higher signal
intensity compared to other ion pair reagent like TFA (Assar, Moloney et al. 2009).
However, the possibility of ion suppression by NFPA can not be completely
excluded. Furthermore, the use of NFPA decreases the pH of the mobile phases
which accelerates the deterioration of RP-columns (Schettgen, Tings et al. 2007). In
the current study, moreover, the separation of CML and LacLys was not achieved on
the C18 column by the use of NFPA. Co-elution could weaken the signal intensity,
especially the signal intensity of CML, requiring the use of higher sample
concentrations. To avoid these problems, hydrophilic interaction liquid
chromatography was introduced as an alternative to reverse-phase chromatography.
The retention of the analytes on the ZIC-HILIC column was proportional to their
polarity enabling a complete separation of CML and LacLys (RT of 10.5 and 25.6
min, respectively). Additionally, the use of the ZIC-HILIC column enhanced clearly
the signal intensity of the analytes in the standard solutions as well as in milk
samples. However, a strong variation of the signal intensities of LacLys was
observed by the use of the ZIC-HILIC column. This imprecision could be caused by
CHAPTER 2. RESULTS AND DISCUSSION 80
the mass spectrometer parameters of the ion source, since these parameters were
optimized at an eluent ratio of 50/50 5mM ammonium acetate buffer and acetonitrile,
but the elution of LacLys was at ratio 80/20. Since the sensitivity of the method was
sufficient for LacLys and MeSO, further investigation of both analytes was performed
on a C18 column.
In general, sterilized milk showed a high modification rate which can be explained by
the high and prolonged heat treatment applied for sterilization (heating at 107-115 °C
for 20-40 s followed by heating in bottle at 120-130 °C for 8-12 min). This thermal
treatment enhanced glycation reactions and led to a high level of LacLys and CML.
The degradation of glycation products promotes oxidation reactions and explains the
high intensity of MeSO in sterilized milk. The level of MeSO in infant formula was
higher than the other investigated milk samples including sterilized milk.
However, since strong matrix effects like ion suppression are expected during the
analysis by ESI-MS-MS (Zimmer 2003), it is not recommended to quantify according
to peak areas. The quantification should be accomplished either by stable isotope-
labeled internal standard or by standard addition method.
5-OH-Trp was not detected in any of the investigated milk samples including infant
formulas. Infant formulas are generally supplemented with α-LA to resemble the
human milk as well as to compensate the deficiency of tryptophan in the whole
bovine milk proteins (Heine, Klein et al. 1991). The absence of 5-OH-Trp in infant
formula as well as in other milk samples is consistent with the results for glycated
and oxidized α-LA. This result does not mean, as discussed previously, the absence
of tryptophan oxidation products in the samples, because other OH-Trp regioisomers
and other oxidation products like kynurenine, N-formylkynurenine may be present.
However, the identification of these products requires further investigation.
CHAPTER 2. RESULTS AND DISCUSSION 81
2.5 Analysis of ornithine in milk proteins
2.5.1 Introduction
The formation of ornithine from arginine is well known. In vivo, ornithine is formed via
enzymatic catalysis by arginase in the urea cycle with concurrent production of urea.
Arginase is classified into arginase ו which is highly expressed in the liver and
arginase which is expressed in the kidney, prostate, brain and mamary glands
(Deignan, Livesay et al. 2007).
Ornithine is prepared conveniently from arginine under alkaline conditions.
Additionally, it can be gained from arginine in acidic solution at high temperature.
However, the latter reaction is very slow and leads to less than 1 % of ornithine
under the conditions usually used for the acidic hydrolysis of protein (HCl 6N, 24
hours at 110 °C) (Murray, Rasmussen et al. 1965)
Figure 50: Ornithine formation from arginine
Ornithine is not found in native proteins in vivo; however it has been detected in
long-live proteins like collagens and lens crystalline where the level of ornithine
increases during protein aging. The mechanism of ornithine formation is not known
but it may involve a conversion of the arginine residue into arginine-derived AGEs,
mainly argpyrimidine and pentosidine as well as Nδ-(5-hydro-4-imidazolon-2-yl)-
ornithine (G-H1) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1),
which then are degraded into ornithine (Sell 2004). In food products, ornithine was
detected in sausages (Villmann, Sandmeier et al. 2007). Since glycation is very
pronounced during processing of milk, the aim of the following part of the work was
to analyze if ornithine is formed in milk proteins during heating.
CHAPTER 2. RESULTS AND DISCUSSION 82
Figure 51: Some derivatives of imidazolone formed from the reaction of arginine and dicarbonyls
2.5.2 LC-ESI-MS-MS analysis of ornithine
The chromatographic conditions (C 18 column, mobile phases and gradient) as well
as the mass spectrometric flow-dependent parameters were the same which were
used previously to detect LacLys and MeSO. The mass spectrometric compound-
dependent parameters were optimized to get the highest signal intensity for
ornithine.
Only two fragments of ornithine (m/z 133.1) were determined, which were the
quantifier fragment (m/z 133.1→ 70.1) and the qualifier fragment (m/z 133.1→ 116)
(figure 52). These fragments were the same as reported in literature (Argirov, Leigh
et al. 2005).
Precursor ion (m/z)
Product ion (m/z)
DP (V) EP (V) CE (V) CXP (V)
133.1 70.1 46 10 25 4
133.1 116 46 10 11 8
Table 7: Mass spectrometric compound-dependent parameters of ornithine
CHAPTER 2. RESULTS AND DISCUSSION 83
Figure 52: Product ion scan of ornithine shows the fragments and their proposed structures
The standard of ornithine eluted after 8.4 min and showed its characteristic
fragments (m/z 133.1→ 70.1 and m/z 133.1→ 116). In the samples of raw and UHT
milk proteins (25 µg/ml), a peak appeared at 8.3 min which showed only the
quantifier fragment (m/z 133.1→ 70.1), as seen in figure 53. Furthermore, no
difference was observed between the peak areas of raw milk and those of UHT milk
as well as of UHT milk heated for 10, 20, 30 and 60 min at 120 °C.
CHAPTER 2. RESULTS AND DISCUSSION 84
Figure 53: LC-MS-MS chromatogram of the standard ornithine (A) and UHT milk proteins (B) obtained in MRM mode. Milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. Panel B shows a peak at the retention time of ornithine which showed only one of its characteristic fragments (m/z 133.1→ 70.1). The second fragment, which was characteristic for the standard (m/z 133.1→ 116), was not detected
Figure 54: Peak areas obtained for the quantifier fragment (m/z 133.1→ 70.1) of the compound detected at the retention time of ornithine
CHAPTER 2. RESULTS AND DISCUSSION 85
2.5.3 Discussion
In the present part of the work, the presence of ornithine in milk proteins was
investigated. In thermally heated milk proteins, a compound was detected which
eluted at the retention time of the standard ornithine and which showed one mass
transitions characteristic for the MS/MS fragmentation of ornithine (m/z 133.1→
70.1). The second important mass transition of ornithine (m/z 133.1→ 116),
however, was not detected. A single fragment is usually inadequate for the
identification of an analyte by MS-MS. When chromatography is involved, one
fragment in combination with the retention time may be considered adequate,
especially if the ions are generated by soft ionization technique. However, the use of
more than one fragment is still advisable (Ardrey 2003). In the current study, the
fragment m/z 70.1 was generated by the soft ionization technique (ESI) and it is
identical to the fragments reported in the literature. Additionally, the compound had
the same retention time as the standard ornithine. However, since two fragments of
ornithine (as standard) were detected under the same chromatographic and mass
spectrometric conditions, the detection of ornithine in milk proteins was questionable.
Furthermore, the fact that the concentration of the compound was independent from
the heat treatment of milk proteins indicates that it represents rather a matrix
compound of milk than ornithine.
It has been reported that the fragment m/z 133.1→ 70.1 can be generated from
arginine (Argirov, Leigh et al. 2005). In the current study, however, the compound
was not detected by the analysis of arginine standard or arginine that was subjected
to enzymatic hydrolysis. These results indicate that the detected fragment was not
generated from arginine. Further studies are therefore required to find out if the
compound indeed originates from milk matrix components or if the analytical
conditions prevented the detection of the second characteristic fragment of ornithine
in milk proteins.
CHAPTER 2. RESULTS AND DISCUSSION 86
2.6 Method optimization for the detection of MeSO,
CML and LacLys in proteins of processed milk
In the previous chapters, a method was established to detect MeSO, CML and
LacLys in milk proteins. A complete hydrolysis was accomplished by the application
of three proteases in the course of three days prior to analysis by LC-MS-MS. In the
present part of the work, the conditions of hydrolysis were optimized to obtain the
best release of the target analytes and, consequently, to achieve maximal sensitivity.
Moreover, the method was optimized to avoid overestimation or underestimation of
these analytes during the sample preparation.
The optimization process involved three steps which were the duration of the
enzymatic protein hydrolysis, the reduction of LacLys to prevent its conversion into
CML during protein hydrolysis and thus to avoid overestimation of CML. Additionally,
the optimization process involved purification of the hydrolyzate.
2.6.1 Optimization of protein hydrolysis
In order to determine the conditions which lead to a sufficient release of the modified
amino acids concurrently with minimal hydrolysis time, an another enzymatic
hydrolysis protocol was applied which takes 27 hours (Delatour, Hegele et al. 2009).
This new enzymatic hydrolysis method was compared to that one described by
Hasenkopf et al (72 hours). Additionally, some samples were subjected to acidic
hydrolysis and compared to the results of enzymatic hydrolysis. Acidic hydrolysis
requires reduction of LacLys prior to analysis to prevent its conversion into CML
during hydrolysis. The different hydrolysis methods were compared by the ninhydrin
assay, which measures the release of α-amino groups as an indicator for hydrolysis
efficiency. Additionally, the concentrations of the target analytes were monitored by
LC-MS-MS.
LacLys can be decomposed into CML by the oxidative cleavage of the carbohydrate
chain during the enzymatic digestion which results in an overestimation of CML
(Assar, Moloney et al. 2009; Delatour, Hegele et al. 2009). In order to prevent this
CHAPTER 2. RESULTS AND DISCUSSION 87
artificial formation of CML, LacLys was reduced in the present work by sodium
borohydride (NaBH4).
Figure 55: Reduction of lactulosyllysine with borohydride to form a product stable during acidic hydrolysis
The reduction of LacLys required the use of solid phase extraction (SPE) instead of
ultrafiltration to remove the reducing agents, which interfere with LC-MS-MS
analysis.
2.6.2 Results
The ninhydrin assay showed that the enzymatic hydrolysis for 72 hours of heated
UHT milk and sterilized milk resulted in a higher absorbance at 570 nm compared to
the samples hydrolyzed for 27 hours, even if higher concentrations were used (figure
56). These results were consistent with those obtained by MS-MS analysis where
longer hydrolysis duration resuted in higher signal intensities of MeSO, CML and
LacLys (figure 57). These findings indicate that the longer enzymatic hydrolysis
leads to higher release of the target analytes from proteins.
CHAPTER 2. RESULTS AND DISCUSSION 88
Figure 56: Protein samples hydrolyzed with a cocktail of enzymes for 72 hours or 27 hours were analyzed by ninhydrin assay. Enzymatic hydrolysis of sterilized milk and UHT milk heated for 10, 20, 30 and 60 min (120 °C) for 72 ho urs resulted in higher absorbance indicating a higher content of free amino acids
Figure 57: LC-MS-MS signal intensity of MeSO, CML and LacLys obtained after the enzymatic hydrolysis of sterilized milk sample for 72 hours or 27 hours
In order to investigate the influence of the purification step of the hydrolyzate on the
target analytes, SPE was introduced and compared to ultrafiltration. The first
experiment was carried out to investigate the target analytes recovery from the
stationary phase of the SPE column. A solution of the standards MeSO, LacLys and
CML was prepared and divided into two portions. The first one was loaded on a SPE
column. The eluate and the second part of the standard solution, which was used as
control, were lyophilized prior to LC-MS-MS analysis. The standards LacLys, CML
and MeSO which were loaded on a SPE column showed a decreased signal
CHAPTER 2. RESULTS AND DISCUSSION 89
intensity compared to the other part of the standards which was not loaded on the
SPE column. In particular, more than 50% of the signal intensity of LacLys was lost
during SPE (figure 58).
These results were consistent with those obtained by the analysis of milk samples
such as sterilized milk and UHT milk. Considerably lower peak intensities were
observed when samples were subjected to SPE after hydrolysis instead of
ultrafiltration. The observed loss was independent from the enzymatic hydrolysis
protocol applied.
Centrifugal vacuum concentrator (Speed-Vac) was used to concentrate some
samples after purification step instead of lyophilization. The typical application of
centrifugal vacuum concentrator is the removal of water and organic solvents such
as methanol, acetonitrile, chloroform or acetone. Additionally, centrifugal vacuum
concentrators are used to remove strong acids and bases from the samples. This is
of especial importance for acidic protein hydrolysis, since strong acids can not be
removed by lyophilization. A slight increase of CML was noted in the samples which
were dried by Speed-Vac after SPE compared to those dried bylyophilization. This
increase could be caused by LacLys degradation, since the drying by Speed-Vac
was carried out at 35 °C and lasted about 7 hours ( figure 59 and 60).
Figure 58: Decrease of the signal intensity of the standards MeSO, CML and LacLys after purification by SPE column compared to control. The samples were analyzed by LC-MS-MS. The highest loss was noted for LacLys
CHAPTER 2. RESULTS AND DISCUSSION 90
Figure 59: Signal intensity of MeSO, CML and LacLys detected by LC-MS-MS in UHT milk which was heated for 60 min at 120 °C. The sample w as purified either by ultrafiltration or by SPE, and then dried by Speed-Vac
Figure 60: Signal intensity of CML detected by LC-MS-MS in the samples of heated UHT milk which were dried either by lyophilization or Speed-Vac after purification
LacLys was reduced by 0.1 M NaBH4 prior to enzymatic protein hydrolysis to
investigate the artificial formation of CML during enzymatic hydrolysis. Some milk
samples, in which lactulosyllysine was reduced by NaBH4, showed a decrease of the
signal intensity of CML. This decrease did not exceed 25% compared to the signal
intensities obtained without reduction, independent of the enzymatic hydrolysis
protocol. Additionally, this decrease was not noted in all samples.
CHAPTER 2. RESULTS AND DISCUSSION 91
Figure 61: Decrease of the signal intensity of CML in heated UHT milk and sterilized milk in which LacLys was reduced by NaBH4 prior to enzymatic hydrolysis. The samples were analyzed by LC-MS-MS
In order to determine the conditions which lead to a sufficient release of the modified
amino acids, acidic hydrolysis was applied and compared to enzymatic hydrolysis
protocols by LC-MS-MS analysis as well as the ninhydrin assay. LacLys was not
detected in the acidic hydrolyzate of sterilized milk, heated UHT milk and HA-infant
powder formula. This result is expected, since LacLys was reduced by NaBH4 prior
to hydrolysis. However, the signal intensity of CML was about 400 % higher
compared to the samples subjected to enzymatic hydrolysis, independent if
enzymatic hydrolysis was carried out with or without the addition of NaBH4 (figure
62). The concentration of NaBH4 was increased to 2M in order to assure a complete
reduction of LacLys. However, the increase of NaBH4 concentration interfered with
analysis by LC-MS-MS so that the three analytes could not be detected anymore.
Remarkably lower MeSO intensities were found in the acidic hydrolyzates compared
to the enzymatic hydrolyzates. This decrease of intensities was observed when
NaBH4 was added prior to acidic hydrolysis. The analysis of a model mixture of
MeSO and NaBH4, which was heated under the conditions used previously for acidic
hydrolysis, showed a decrease of MeSO concentration, which was however not
accompanied by the formation of methionine. This result indicates that NaBH4 did
not reduce MeSO directly during acidic hydrolysis, but MeSO has undergone further
reaction. MeSO intensities in the enzymatic hydrolyzates did not decrease by the
addition of NaBH4.
CHAPTER 2. RESULTS AND DISCUSSION 92
Figure 62: LC-MS-MS signal intensity of the analytes MeSO, CML, and LacLys which were detected in (A) HA-infant powder formula and (B) sterilized milk. The diagrams show an increase of CML in the acidic hydrolyzate compared to both enzymatic hydrolyzates. LacLys was not detected after the reduction by NaBH4 in the acidic nor the enzymatic hydrolyzates. Clear decrease of MeSO signal was found in the acidic hydrolyzate
The enzymatic hydrolysis (72 hours) of heated UHT milk, sterilized milk, HA-infant
powder formula and lactalbumin resulted in a higher absorbance in the ninhydrin
assay compared to those which were acidicly hydrolyzed (figure 63). Before the
ninhydrin assay, acidic hydrolysates were buffered to a pH between 5 to 5.5, so that
an influence of the pH value on the color reaction can be excluded.
CHAPTER 2. RESULTS AND DISCUSSION 93
Figure 63: Protein samples of UHT milk heated for 10, 20, 30 and 60 min (120 °C), sterilized milk, HA-infant powder formula and lactalbumin. The samples were analyzed by ninhydrin assay after acidic or enzymatic hydrolysis for 72 hours. The absorbance at 570 nm of the enzymatic hydrolyzate (3µg protein) was higher compared to the acidic hydrolyzate (10µg protein)
2.6.3 Discussion
The experiments were carried out to determine the optimal conditions to release
MeSO, CML and LacLys from milk proteins as completely as possible. The analysis
by LC-MS-MS showed that a longer duration of enzymatic hydrolysis led to higher
signal intensities of these analytes: Signal intensities increased about 5 times after
enzymatic hydrolysis for 72 hours compared to 27 hours.
It may be hypothesized that the increase of LacLys is caused by an artificial
formation during the hydrolysis as a result of the reaction between the released
lysine residue and remaining lactose. It may be also assumed that the prolonged
hydrolysis for 72 hours can enhance the oxidative cleavage of LacLys and thus, will
increase the artificial formation of CML. Additionally, both, LacLys degradation and
the prolonged sample incubation, may enhance the oxidation of methionine.
However, during the sample preparation, lactose is removed by dialysis, so that
artificial lactosylation is less likely. Moreover, the reduction of LacLys prior to
enzymatic hydrolysis led only in some samples to a decrease of CML intensity,
which was always lower than 25% compared to the samples which were not
CHAPTER 2. RESULTS AND DISCUSSION 94
reduced. The results indicate that LacLys is relatively stable during enzymatic
hydrolysis so that the artificial formation of CML during prolonged enzymatic
hydrolysis should play only a minor role. The stability of LacLys under enzymatic
hydrolysis reduces the possibility of methionine oxidation, since methionine is
oxidized by hydroxyl radicals generated from LacLys decomposition (Mossine,
Linetsky et al. 1999). Furthermore, the incubation of methionine under the conditions
of enzymatic hydrolysis did not result in methionine oxidation, as it was seen
previously (see 2.3.5), so that the artificial formation of MeSO can be excluded.
These results in addition to those of the ninhydrin assay, which showed a higher
absorbance after enzymatic protein hydrolysis for 72 hours compared to 27 hours,
refer to a higher hydrolysis efficacy. Consequently, these results support a higher
release of the modified amino acids rather than their artificial formation.
The analysis of the modified amino acids by LC-MS-MS showed that the method
which is used to purify the samples affect the recovery of the target analytes. Lower
signal intensity was found after the purification by solid phase extraction compared to
samples purified by ultrafiltration. The lower recovery can be caused by the
adsorption of the analytes on the stationary phase. This is particularly the case for
LacLys, which showed about 50% loss during SPE. LacLys may also be degraded
during drying by the centrifugal vacuum concentrator. This step was carried out at 35
°C and lasted 7 hours which explains the slight inc rease of CML signal compared to
the samples dried by lyophilization.
Additionally, acidic hydrolysis was used to release the modified amino acids prior to
sample cleanup by SPE and LC-MS-MS analysis. Several authors report that the
Amadori product is not stable and can undergo spontaneous oxidative cleavage
during hydrolysis in 6 M HCl at elevated temperature. However, these conditions are
required to completely release amino acids from the proteins (Guy and Fenaille
2006; Hegele, Buetler et al. 2008).
To ascertain that CML was not formed artificially from LacLys, milk protein samples
were reduced by NaBH4 prior to acidic hydrolysis. It is well known that acidic
hydrolysis yields a better protein cleavage (20-25% more) than enzymatic hydrolysis
(Sell 2004; Delatour, Hegele et al. 2009). Therefore, an increase of CML intensity
was expected using acidic hydrolysis. However, the analysis by LC-MS-MS showed
that CML intensity in the acidic hydrolyzate was 4 times higher instead of only 25%
higher compared to the enzymatic hydrolyzate. This result was also in contrast to a
CHAPTER 2. RESULTS AND DISCUSSION 95
previous study which reported that the enzymatic hydrolysis resulted in an artificial
formation of CML, where CML concentrations in the enzymatic hydrolyzate were 3
times higher compared to the acidic hydrolyzate (Delatour, Hegele et al. 2009).
Another study did not observe any overestimation of CML when the proteins were
subjected to acidic hydrolysis without prior reduction by NaBH4 (Charissou, Ait-
Ameur et al. 2007).
The high level of CML in the acidic hydrolyzate led to doubt the sufficiency of the
reduction step and indicated that the absence of LacLys has resulted from the
reduction by NaBH4 as well as from the oxidative cleavage of LacLys. This
degradation of LacLys might lead then to an artificial formation of CML and explain
the increase of the signal intensity of CML.
The results of the ninhydrin assay could not completely confirm or falsify those
obtained by LC-MS-MS analysis. All samples of the acidic hydrolyzates developed
less color compared to the enzymatic hydrolyzates. Two factors are important to
develop the maximum color in the ninhydrin assay. The first is a sufficient reducing
agent to convert ninhydrin to the reduced hydantoin which is essential to develop the
color. The other factor is the temperature which must approach the boiling point over
the heating period. In the current experiment, a concentration of stannous chloride
was applied, which was described as sufficient to provide an adequate reducing
capacity in the range of 1-10 µg of protein hydrolyzate (Starcher 2001). Additionally,
a boiling water bath was used to heat the reaction mixture and it was found that 5
min was enough to provide the heating necessary to develop the color for all
samples. However, the color development in the samples of the acidic hydrolyzate
was less than the enzymatic hydrolyzate, independent of the duration of enzymatic
hydrolysis. This result could be caused by insufficient buffer capacity since the pH of
the acidic and enzymatic hydrolyzates was 5 and 5.5, respectively.
Interestingly, the intensity of MeSO decreased in the samples of the acidic
hydrolyzate compared to the enzymatic hydrolyzate. A previous study reported that
up to 59 % of methionine can be destroyed during acidic hydrolysis (Jennings. D. M
1969), while others reported that MeSO is reduced to methionine during acidic
hydrolysis (Guy and Fenaille 2006; Brock, Ames et al. 2007).
In the current study, the decrease of MeSO during acidic hydrolysis was observed
when NaBH4 was used. On the other hand, a considerable increase of MeSO was
found in the samples, which were subjected to acidic hydrolysis without NaBH4. This
CHAPTER 2. RESULTS AND DISCUSSION 96
refers to a possible influence of NaBH4 on MeSO intensity. However, NaBH4 mainly
reduces aldehyde and ketone to yield alcohols (da Costa, Pais et al. 2006).
Moreover, the model mixture of MeSO, NaBH4 and HCl (6 N), which was heated at
110 °C, did not show the conversion of MeSO into me thionin despite the decrease of
MeSO signal intensity. This result suggests that MeSO was not reduced to
methionine, but has undergone further reaction. No effect of NaBH4 on methionine
sulfoxide was reported in literature.
It was decided for several reasons to use enzymatic hydrolysis without the reduction
step to release the target analytes. 1)- The analysis by LC-MS-MS revealed
overestimation of CML as well as underestimation of MeSO in the acidic hydrolyzate
compared to the enzymatic hydrolyzate. 2)- The decrease of CML in the enzymatic
hydrolyzate, which was caused by the reduction of LacLys, was not observed in all
reduced samples and was always lower than 25%. Thus, the effect of NaBH4 on the
artificial formation of CML is still doubtful. 3)- The reduction of LacLys by NaBH4
required the use of SPE as a cleanup tool, which resulted in the adsorption of the
analytes on the stationary phase of the SPE column. 4)- The reduction step would
prevent the detection and quantification of LacLys in parallel with CML and MeSO.
On the basis of these experimental data, it was decided for further investigation to
adopt enzymatic hydrolysis for 72 hours without applying a reduction step.
Additionally, it was decided to use ultrafiltration as a cleanup tool. Finally, the use of
ZIC-HILIC column is also considered an improvement, because it resulted in a better
sensitivity for the detection of CML.
CHAPTER 2. RESULTS AND DISCUSSION 97
2.7 Method validation for the quantification of
LacLys, CML and MeSO in milk proteins
2.7.1 Introduction
Is the last chapters, a method was established to detect some glycation and
oxidation products in milk proteins by LC-ESI-MS-MS. Additionally, the conditions of
protein hydrolysis were optimized to release the target analytes as complete as
possible as well as to avoid artificial formation of the analytes during sample
preparation. In the present chapter, the validation of the method will be described.
The method was validated for each analyte by evaluation the within-day repeatability
(intraday repeatability), the between-days repeatability (inter-day repeatability), the
recovery as well as the limit of detection (LOD) and the limit of quantitation (LOQ).
The selectivity of the method was achieved by monitoring two mass transitions of
each compound. The most intense transition was used as the quantifier fragment
and the second one as the qualifier fragment.
analyte quantifier fragment qualifier fragment
LacLys m/z 471.2→ 225.1 m/z 471.2→ 128.1 MeSO m/z 166.1→ 74 m/z 166.1→ 56
CML m/z 205.1 → 84.1 m/z 205.1→ 130.1
Table 8: The quantifier and qualifier fragments of LacLys, MeSO and CML
UHT milk was chosen to investigate the repeatability of the method in mildly heated
milk proteins, while UHT milk heated for 1 hour at 120 °C was chosen as an example
for severely heated milk proteins.
To investigate the within-day repeatability, 7 to 8 samples of UHT milk or heated
UHT milk protein were enzymatically hydrolyzed for 3 days, worked up at the same
day, and then analyzed by LC-MS-MS. Since the standard addition method was
used to quantify the three analytes, which is rather time consuming, it was difficult to
analyze all samples at one day. Therefore, each group of samples, UHT milk or
heated UHT milk, required two days for anaylsis.
CHAPTER 2. RESULTS AND DISCUSSION 98
To investigate the between-day repeatability, 4 to 6 samples of UHT milk or heated
UHT milk were enzymatically hydrolyzed in consecutive days, worked up at the
same day, and were then analyzed by LC-MS-MS in consecutive days.
In order to investigate the recoveries of the analytes, UHT milk proteins were spiked
with three different and known amounts of LacLys, MeSO and CML before
enzymatic hydrolysis.
LOD was defined as the concentration of the analyte for which the signal to noise
ratio of the qualifier fragment is 3, while LOQ was defined as the concentration of the
analyte for which the signal to noise ratio of the quantifier fragment is 10. However,
the noise of the qualifier fragment of MeSO (m/z166.1→ 56) was high, resulting in a
LOD of MeSO similar to the LOQ. In order to avoid this problem, both, LOD and LOQ
of MeSO, were determined based on the signal to noise ratio of the quantifier
fragment. Moreover, since there were no milk samples which did not contain any
MeSO, LOQ and LOD of MeSO were determined by the dilution of three samples of
UHT milk to the extent at which the signal to noise ratio of the quantifier fragment
was 10 and 3, respectively.
LOQ and LOD of LacLys were determined by the dilution of three samples of UHT
milk protein hydrolyzate to the extent at which the signal to noise ratio of the
quantifier (m/z 471.2→ 225.1) and qualifier (m/z 471.2→ 128.1) fragments were 10
and 3, respectively.
Since CML is not detectable in the applied concentration of raw milk, it was used in a
low concentration as a blank matrix to determine LOQ and LOD of CML. Different
amounts of CML were added to this matrix, and then each sample was analyzed four
times by LC-MS-MS. The average of signal to noise ratios of these 4 analyses was
calculated to determine LOQ and LOD.
During the validation of the method, each analyte was quantitatively measured by
standard addition.
2.7.2 Quantitative analysis by standard addition
It is well known that electrospray ionization suffers signal suppression when
polar/ionic compounds coelute with the analyte of interest. The common approaches
to overcome this problem are the use of standard addition or the use of stable
isotope labeled internal standards (Ardrey 2003). Since the isotopic labelled MeSO
CHAPTER 2. RESULTS AND DISCUSSION 99
and LacLys are not available as internal standards, the method of standard addition
was adopted for quantitative measurement.
For this method, increasing known amounts of each standard compound are added
to the sample which contains an unknown amount of the analyte. The signal of the
analyte of interest is related to the amount of the added standard as well as to the
amount of the analyte originally present in the sample (Ardrey 2003).
The signal intensity was expressed as the peak area under the quantifier fragment of
each analyte in the unknown sample as well as in those to which the standard
compounds were added. The peaks areas (y axis) were then related to the
concentrations of the added analyte (x axis). The following equation is used to
determine the unknown concentration of the analyte:
y= m*x+b
y: the signal of the analyte
m: the slope of the calibration curve
x: the concentration of the analyte
b: y-intercept of calibration curve
In the case of standard addition, y is the signal of the unknown analyte and the
added standard, while the y-intercept is equivalent to the signal of the unknown
analyte. The extension of calibration curve will intercept the x axis at the point
corresponding to the unknown concentration of the analyte. At this point, y is 0,
which means that x=m/b
CHAPTER 2. RESULTS AND DISCUSSION 100
Figure 64: Schematic illustration shows the method of standard addition. (http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/3/anc/croma/kalibrierung.vlu/Page/vsc/de/ch/3/anc/croma/datenauswertung/quantitativ/standardaddition/standardadditionm80ht0801.vscml.html, date 9/2/2011)
2.7.3 Results
2.7.3.1 Within-day repeatability
0.59 ± 0.038 µg of MeSO with a coefficient of variation (CV) of 6.4 % was detected in
1 mg of UHT milk protein residue as an average of 8 samples, which were prepared
simultaneously for analysis. The heat treatment of UHT milk resulted in a higher level
of MeSO. 0.69 ± 0.068 µg was detected analysing 7 samples with a CV of 9.96 %.
0.86 ± 0.074 µg and 1.13 ± 0.09 µg of LacLys were found in UHT milk and heated
UHT milk protein, respectively, as an average of 8 samples of each group with
similar coefficients of variation (8.2 and 8.6%). These values of LacLys were
calculated taking into consideration the purity of the synthesized LacLys (6.13%).
The variation within day for CML was good. 0.0279 ± 0.002 µg with a CV of 7.11%
and 0.0486 ± 0.0046 µg with a CV of 9.6% were detected in UHT and heated UHT
milk, respectively.
CHAPTER 2. RESULTS AND DISCUSSION 101
MeSO
n=8 LacLys
n=8 CML n=7
UHT-milk µg/mg
0.59 ± 0.038 0.86 ± 0.074 0.0279 ±0.002
coefficient of variation % 6.4 8.6 7.1
MeSO
7=8 LacLys
n=8 CML n=8
heated UHT-milk 60min, 120 °C
µg/mg 0.69 ± 0.068 1.13 ± 0.09 0.0486 ±0.0046
coefficient of variation %
9.96 8.2 9.6
Table 9: Within-day repeatability of the method for MeSO, LacLys and CML
2.7.3.2 Between-day repeatability
The between-day repeatability of the method for LacLys and MeSO was calculated
by analysing 6 samples of UHT milk as well as heated UHT milk proteins which were
prepared and analyzed in sequence with a time difference of 24 hours. 4 samples of
each UHT and heated UHT milk proteins were analyzed to investigate the between-
day repeatability for CML. As seen in table 10, 0.56 ± 0.054 to 0.68 ± 0.059 µg of
MeSO with CV of 9.6 and 8.7 % were detected in UHT and heated UHT milk,
respectively. The between-days repeatability of the method for LacLys in UHT was
good (CV 7.9%). In heat treated UHT milk, the CV was slightly higher (9.96%). The
best coefficients of variation were found in for CML, which were between 5.8 and
7.4%.
CHAPTER 2. RESULTS AND DISCUSSION 102
MeSO n=6
LacLys n=6
CML n=4
UHT-milk µg/mg
0.56 ± 0.054 1.01 ± 0.08 0.019 ± 0.0014
coefficient of variation % 9.6 7.9 7.4
heated UHT-milk 60min, 120 °C
µg/mg 0.68 ± 0.059 1.49 ± 0.15 0.031±0.0017
coefficient of variation %
8.7 9.96 5.8
Table 10: Between-day repeatability of the method for MeSO, LacLys and CML
2.7.3.3 Recovery
Three different amounts of each of the synthesized LacLys, MeSO and CML were
added to 4 samples of UHT milk proteins before applying enzymatic hydrolysis. The
contents of these analytes in the unspiked UHT milk (control) as well as in the spiked
UHT milk samples were determined by standard addition.
The recovery of each analyte was calculated as the ratio of the measured to the
expected concentration. The measured amount is the content of the analyte in the
sample after spiking (spiking 1, spiking 2 or spiking 3). The expected value of the
analyte is theoretically the sum of the spiked standard compound and the analyte
already present in the control. The average of the recoveries of four samples was
calculated and considered as the final recovery of the analyte. As seen in table 11,
the recovery of LacLys ranged between 110.5 and 111.6 % with a tendency for a
higher standard deviation at a higher amount of the spiked standard.
The recoveries of CML were optimal for the first and the second additions (0.012 and
0.024µg/mg) which were almost 100 % ± 5.5. By increasing the amount of spiked
CML to 0.048µg/mg, the standard deviation increased to 9.4, however the recovery
was still very good (96.6 % ± 9.4). In general, MeSO showed a high recovery which
reached 113.1 % as well as a high standard deviation which ranged between 10.7
and 14.3 %.
The first three experiments were simultaneously performed, while a fourth
experiment was carried out after 2 months. The fourth experiment showed generally,
CHAPTER 2. RESULTS AND DISCUSSION 103
independent of the spiked amounts of the analytes, lower contents of LacLys and
MeSO compared to the other experiments. Such differences in the analyte content
might result from the use of different standard solutions for standard addition.
However, the recoveries of LacLys and MeSO in this experiment were consistent
with the other experiments.
Unspiked sample (control)
Spiking 1 (0.357 µg added to 1 mg protein)
LacLys µg/mg
measured LacLys µg/mg
expected LacLys µg/mg recovery %
Experiment 1 1.55 2.3 1.91 120.4
Experiment 2 1.52 1.84 1.88 97.9
Experiment 3 1.75 2.50 2.1 119
Experiment 4 0.73 1.14 1.09 104.6
mean 110.5±9.6
Unspiked sample (control)
Spiking 2 (0.715 µg added to 1 mg protein)
LacLys µg/mg
measured LacLys µg/mg
expected LacLys µg/mg recovery %
Experiment 1 1.55 2.66 2.27 117.4
Experiment 2 1.52 2.38 2.23 106.5
Experiment 3 1.75 2.4 2.46 97.4
Experiment 4 0.73 1.81 1.45 124.3
mean 111.6±10.6
Unspiked sample (control)
Spiking 3 (1.43 µg added to 1 mg protein)
LacLys µg/mg
measured LacLys µg/mg
expected LacLys µg/mg
recovery %
Experiment 1 1.55 3.61 2.98 121.1
Experiment 2 1.52 3.72 2.95 126.1
Experiment 3 1.75 2.79 3.18 87.7
Experiment 4 0.73 2.33 2.16 107.9
mean 110.7±17
Table 11: Recovery of LacLys
CHAPTER 2. RESULTS AND DISCUSSION 104
Unspiked sample (control)
Spiking 1 (0.16 µg added to 1 mg protein)
MeSO µg/mg
measured MeSO µg/mg
expected MeSO µg/mg recovery %
Experiment 1 1.86 2.3 2.02 113.9
Experiment 2 1.9 1.87 2.06 90.8
Experiment 3 1.7 2 1.86 107.5
Experiment 4 1.06 1.45 1.22 118.9
mean 107.8 ±10.6
Unspiked sample (control)
Spiking 2 (0.32 µg added to 1 mg protein)
MeSO µg/mg
measured MeSO µg/mg
expected MeSO µg/mg recovery %
Experiment 1 1.86 2.25 2.18 103.2
Experiment 2 1.9 2.91 2.22 131.1
Experiment 3 1.7 1.85 2.02 91.6
Experiment 4 1.06 1.51 1.38 109.4
mean 108.8 ±14.3
Unspiked sample (control)
Spiking 3 (0.64 µg added to 1 mg protein)
MeSO µg/mg
measured MeSO µg/mg
expected MeSO µg/mg
recovery %
Experiment 1 1.86 2.96 2.5 118.4
Experiment 2 1.9 3.05 2.54 120.1
Experiment 3 1.7 2.21 2.34 94.4
Experiment 4 1.06 2.03 1.7 119.4
mean 113.1±10.8
Table 12: Recovery of MeSO
CHAPTER 2. RESULTS AND DISCUSSION 105
Unspiked sample (control)
Spiking 1 (0.012 µg added to 1 mg protein)
CML µg/mg
measured CML µg/mg
expected CML µg/mg
recovery %
Experiment 1 0.076 0.086 0.088 97.7
Experiment 2 0.042 0.049 0.054 90.7
Experiment 3 0.053 0.07 0.065 107.7
Experiment 4 0.047 0.057 0.059 96.6
mean 98.2 ±6.1
Unspiked sample
(control)) Spiking 2 (0.024 µg added to 1 mg protein)
CML
µg/mg measured CML
µg/mg expected CML
µg/mg recovery %
Experiment 1 0.076 0.106 0.10 106.0
Experiment 2 0.042 0.07 0.066 106.2
Experiment 3 0.053 0.076 0.077 98.3
Experiment 4 0.047 0.066 0.071 93.4
mean 101 ±5.4
Unspiked sample (control)
Spiking 3 (0.048 µg added to 1 mg protein)
CML µg/mg
measured CML µg/mg
expected CML µg/mg
recovery %
Experiment 1 0.076 0.132 0.124 106.5
Experiment 2 0.042 0.095 0.09 105.6
Experiment 3 0.053 0.088 0.101 87.1
Experiment 4 0.047 0.083 0.095 87.4
mean 96.6 ±9.4
Table 13: Recovery of CML
2.7.3.4 Limit of detection and limit of quantitatio n
LOQ and LOD of MeSO were determined based on the quantifier fragment (m/z
166.1→ 74). LOQ and LOD of LacLys were determined based on the quantifier
fragment (m/z 471.2→ 225.1) and the qualifier fragment (m/z 471.2→ 128.1). LOQ
and LOD of MeSO and LacLys were dependent on the injection volume. LOQ and
CHAPTER 2. RESULTS AND DISCUSSION 106
LOD were 2 and 1 pg of MeSO on column, respectively, equivalent to 400 and 200
pg MeSO pro 1 ml of sample solution (injection volume is 5 µl).
For the determination of LOQ of LacLys, the unknown peak, which showed the
quantifier fragment of LacLys (m/z 471.2→ 225.1) and which appeared directly after
LacLys (figure 65), was not considerd as noise. Otherwise, LOQ would increase
more than 20 fold.
Figure 65: An unknown peak appeared at 8.08 min and showed the quantifier fragment of LacLys
LOQ and LOD were 6.5 and 4 pg of LacLys on column, which is equivalent to 1.3
and 0.75 ng LacLys pro 1 ml of sample solution, respectively.
These values were calculated taking into consideration the purity of the synthesized
LacLys (6.13%) and the injection volume (5µl). Additionally, the unknown peak,
which appeared directly after LacLys (8.08 min), was not considerd as noise.
LOQ and LOD of CML were 8 and 4 ng, respectively, spiked in 25 µg of raw milk
proteins. These values are equivalent to 40 and 20 pg of CML on column (injection
volume is 5 µl).
2.7.4 Discussion
The coefficients of variation of within-day and between-day repeatability of the entire
method to detect the analytes LacLys, CML and MeSO were determined. These
values did not exceed 10 % at low as well as at high level of modifications. The
coefficients of variation reflect the variation in both, enzymatic hydrolysis and the
instrumental analysis. The results for CML are consistent with those obtained by
Assar at al. However, in this study the hydrolyzates were analyzed only on two
CHAPTER 2. RESULTS AND DISCUSSION 107
different days to estimate the between-day repeatability, and only twice on the same
day to exam the intra-day repeatability (Assar, Moloney et al. 2009).
In lactose free milk, UHT milk and raw milk samples, 0.01 µg CML was detected in
65, 210 and 700 µg protein, respectively, with S/N ratio of about 10. Additionally,
0.005 µg CML was detected in 35 µg protein of infant powder formula and HA infant
powder formula with S/N ratio, based on the qualifier fragment (m/z 205.1→ 130.1)
of about 3. The protein content in these five samples was determined by Kjeldahl.
This result indicates that the ion suppression is very low up to 1 mg of analyzed milk
proteins. During validation, LOQ and LOD of 8 and 4 ng in 25 µg protein residue,
were determined. These values are equivalent to 40 and 20 pg CML on column,
respectively. Since protein matrix up to 1 mg did not interfere with signal intensity, a
LOQ and LOD of 8 and 4 ng pro 1mg protein were defined.
The value of LOQ and LOD of CML differs from those reported in literature. One
study reported, for example, that LOQ and LOD of CML are 27 and 8 ng per mg
protein, respectively. However, these values were estimated by extrapolation and not
by experimental determination (Delatour, Hegele et al. 2009). Another study reported
that LOQ and LOD of CML were 0.03 and 0.009 mmol per mol lysine, respectively.
The content of lysine in this study was not measured directly, but estimated
depending on milk lysine content reported in literature. Additionally, LOD and LOQ
were calculated based on the quantifier fragment of CML only (Assar, Moloney et al.
2009). In both studies, CML was analyzed by LC-ESI-MS-MS using a stable isotope
labeled standard. It is important to mention that LOD and LOQ of the analytes CML
as well as MeSO and LacLys is dependent on the cleanness of the sprayer needle
and the mass analyzer.
So far, a LC-MS-MS method for the analysis of MeSO and LacLys in milk proteins
has not been reported. The repeatability of the analysis of MeSO and LacLys was
good, since the coefficient of variation did not exceed 10 %. In the current study,
LOQ and LOD were 6.5 and 4 pg of LacLys on column, respectively, and 2 and 1 pg
of MeSO on column, respectively. The detected amount of LacLys and MeSO in
UHT milk, as an example for a mildly heat treated milk product, was much higher
than the LOD and LOQ. Thus, it can be concluded that the method is sensitive
enough to detect these two analytes in all milk products.
The high recovery of LacLys might be caused by the added LacLys standard, which
may contain impurities of lactose. Lactose could then react with the released lysine
CHAPTER 2. RESULTS AND DISCUSSION 108
during hydrolysis leading to the formation of LacLys. Consequently, the content of
LacLys may enhance the oxidation of methionine leading to a higher recovery of
MeSO.
The validation results indicate that the method is precise and sensitive enough to
quantify lactulosyllysine, carboxymethyllysine and methionine sulfoxide in various
dairy products.
CHAPTER 2. RESULTS AND DISCUSSION 109
2.8 Quantitation
2.8.1 Introduction
After validation, the method was employed for the analysis of LacLys, CML and
MeSO in several dairy products. The analysis involved raw milk and mildly heat-
treated milk like pasteurized milk, UHT milk as well as lactose free milk and severely
heat-treated milk such as condensed milk and sterilized milk. Additionally, different
infant formulas were analyzed which varied between regular and hydrolyzed
formulas, liquid and powder formulas as well as "from birth" and toddler liquid
formulas. Two samples of each milk product were purchased from two different
manufactures for quantification and then subjected separately to analysis.
The sample preparation involved defatting, dialysis, enzymatic hydrolysis for 72
hours of 1 mg of the lyophilized residue, and then cleanup by ultrafiltration. The
protein content was determined by Kjeldahl which was applied to the milk residues
obtained after defatting and dialysis. The analytes were quantified by standard
addition. The content of CML was expressed as ng per mg protein, while the
contents of both, LacLys and MeSO, were expressed as µg per mg protein.
2.8.2 Results
The contents of MeSO, LacLys and CML in the analyzed samples are summarized in
table 14.
CHAPTER 2. RESULTS AND DISCUSSION 110
sample MeSO µg per mg protein
LacLys µg per mg protein
CML ng per mg protein
raw milk 1 1.13 0.043 15.10
raw milk 2 0.79 0.087 14.40
pasteurized milk 1 0.91 0.29 17.20
pasteurized milk 2 0.40 0.30 22.10
UHT milk 1 0.85 1.05 60.60
UHT milk 2 0.44 1.03 40.40
lactose free milk 0.49 1.24 142.60
sterilized milk 1 2.38 3.60 454.10
sterilized milk 2 2.14 3.45 547.50
vanilla drink 1 3.05 4.83 322.40
vanilla drink 2 1.97 4.27 451.40
condensed milk 4% , 1 1.52 7.48 532.40
condensed milk 7.5 % 1.79 7.23 538.20
condensed milk 4% , 2 3.09 6.05 899.00
condensed milk 4%, 3 1.06 8.70 586.70
sugared condensed milk 7.5 %, 1 1.41 8.52 624.70
sugared condensed milk 7.5 %, 2 2.20 11.48 832.50
powdered infant formula 1 (from birth) 2.85 4.08 254.40
powdered infant formula 2 (from birth) 7.61 1.50 154.10
powdered infant HA formula 1 (from birth)
3.86 4.26 420.20
powdered infant HA formula 2 (from birth) 11.31 2.23 250.00
liquid toddler formula 1 (from 12 month) 3.46 3.42 321.6
liquid toddler formula 1 (from 12 month) 2.99 2.80 245.74
Table 14: MeSO, LacLys and CML contents in some dairy products determined by LC-ESI-MS-MS
CHAPTER 2. RESULTS AND DISCUSSION 111
2.8.3 Discussion
The analytes LacLys and CML were quantified in different milk products where they
increased generally with increasing heat treatment. The highest contents were found
in condensed and sterilized milk, while mild heat treatment like pasteurization and
UHT processing induced a low level of modification. Additionally, LacLys and CML
as well as MeSO were detected in important amounts in infant formulas. Some of the
present results are consistent with those reported in literature. For example, Baxter
et al quantified MeSO in milk products by HPLC/UV and found 3 and 11.3 mg in 100
g of pasteurized milk and sugared condensed milk, respectively (Baxter, Lai et al.
2007). These results are in good accordance with the current results, where 0.4-0.91
µg and 1.41-2.2 µg of MeSO per mg proteins were detected in pasteurized milk and
sugared condensed milk proteins (equivalent to 1.29-2.93 mg and 10.7-16.6 mg per
100 g of product, respectively). The latter values were calculated assuming a milk
density of 1.025 g/ml and a protein content of 3.3 % in pasteurized milk. Protein
concentration of sugared condensed milk was taken from the information given by
the supplier (7.6 g protein in 100 g of the product).
However, some results differed completely from literature data. For example, 2.08 ng
of CML per mg protein were detected in raw milk (Ahmed, Mirshekar-Syahkal et al.
2005). In the current study, 14.4-15 ng of CML per mg protein was detected. Such
differences may result from different instrumental methods as well as different
hydrolysis protocols and different methods used for the determination of the protein
content. Additionally, the conditions applied in the production process may vary
between different manufactures.
The content of CML in lactose free milk was 3 folds higher than in UHT milk despite
of the same heat treatment and the same content of LacLys. For the production of
lactose free milk, lactose is enzymaticallly hydrolysed to glucose and galactose prior
to heat treatment. The higher content of CML may result from the participation of
glucose and galactose in glycation. It was found that glucose and galactose can
induce a higher level of glycation in milk proteins compared to lactose (Chevalier,
Chobert et al. 2001). CML levels in hydrolyzed infant formulas were higher than in
the regular formulas from the same manufacture (e.g. powdered infant formula 1
versus powdered infant HA formula 1). This result is consistent with literature data
(Delatour, Hegele et al. 2009). Furthermore, Delatour et al and Birlouez et al
CHAPTER 2. RESULTS AND DISCUSSION 112
reported that liquid infant formulas show in general higher CML levels than powdered
formulas (Birlouez-Aragon, Pischetsrieder et al. 2004; Delatour, Hegele et al. 2009).
In the current study, toddler liquid formulas showed similar or slightly higher CML
contents compared to powdered regular formulas.
Higher levels of LacLys, CML and MeSO were detected in infant formulas and
toddler formulas compared to processed cow milk (UHT and pasteurized milk). A
combination of factors can lead to this result beside the heat treatment. These
factors include the high content of lactose (8% of lactose) (Assar, Moloney et al.
2009) and the high level of lysine in infant formula caused by the addition of α-LA to
resemble mother milk (Heine, Klein et al. 1991). Additionally, it was shown that the
supplementation of infant formulas with ascorbic acid and iron enhances glycation of
milk proteins and leads to higher levels of CML and furosine (Leclere, Birlouez-
Aragon et al. 2002). This supplementation produces also hydroxyl radicals and thus
promotes oxidation reactions (Almaas, Rootwelt et al. 1997). Furthermore, iron is
chelated by glycated proteins and can generate oxygen species which react with
neighbouring amino acids (Leclere, Birlouez-Aragon et al. 2002). These factors
explain the high level of CML and LacLys as well as the highest level of MeSO in
infant formulas compared to the other analyzed samples. The high content of LacLys
and CML in sterilized milk results from the high temperature applied to ensure
microbiological safety. Subsequently, the degradation of the Amadori product
promotes the generation of hydroxyl radicals (Mossine, Linetsky et al. 1999) and
thus explains the high levels of MeSO in these samples. The detection of MeSO in
raw milk was unexpected, especially since the content was higher than in the mildly
heat-treated milk products. This could result from hydrogen peroxide which is widely
used as bacteriostatic agent to clean milk containers. It has been shown that the
addition of H2O2 to whey protein solutions at bacteriostatic level led to a significant
increase of methionine sulfoxide (Baxter, Lai et al. 2007). High levels of
modifications were found in vanilla drinks which were obtained from two different
suppliers. Unfortunately, additional details were not reported on the product`s label
which might help to interpret the results.
CHAPTER 2. RESULTS AND DISCUSSION 113
sample MeSO analytical method reference
pasteurized milk 3mg/100g product HPLC/UV Baxter et al (2007)
pasteurized milk 1.29-2.93mg/100g product LC-ESI-MS-MS current study
sugared condensed milk 11.3mg /100g product HPLC/UV Baxter et al (2007)
sugared condensed milk 10.7-16.6mg/100g product LC-ESI-MS-MS current study
Table 15: MeSO content in different milk products reported in literature
sample CML analytical method reference
raw milk 9.3ng/mg protein LC-ESI-MS-MS Assar et al (2009)
raw milk 337nM (2.08ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)
raw milk 1.19-2.35ng/mg protein LC-ESI-MS-MS Delatour et al (2009)
raw milk 14.4-15.05ng/mg protein LC-ESI-MS-MS current study
pasteurized milk 10.4-16.2ng /mg protein LC-ESI-MS-MS Assar et al (2009)
pasteurized milk 877nM (5.42ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)
pasteurized milk 0.79-1.81ng/mg protein LC-ESI-MS-MS Delatour et al (2009)
pasteurized milk 16.3mg/mg protein LC-ESI-MS-MS Fenaille et al (2006)
pasteurized milk 17.2-22 ng per mg protein LC-ESI-MS-MS current study
UHT milk 8.88ng/mg protein LC-ESI-MS-MS Delatour et al (2009)
UHT milk 29.2-46.4ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)
UHT milk 40-60.55ng/mg protein LC-ESI-MS-MS current study
sterilized milk 12.77nM
(12.77ng/mg protein) LC-ESI-MS-MS Ahmad et al (2005)
sterilized milk 454-547ng/mg protein LC-ESI-MS-MS current study
condensed milk 205ng/mg protein LC-ESI-MS-MS Delatour et al (2009)
condensed milk 533-899ng/mg protein LC-ESI-MS-MS current study
powdered infant formula 1.2ng /mg protein ELISA Birlouez-Aragon (2004)
powdered infant formula 54ng / mg protein GC-MS Chrissou et al (2007)
powdered infant formula 25-140ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)
powdered infant formula 154-254.44ng /mg protein LC-ESI-MS-MS current study
powdered infant HA formula 184ng/mg protein LC-ESI-MS-MS Delatour et al (2009)
powdered infant HA formula
134-322ng/mg protein LC-ESI-MS-MS Fenaille et al (2006)
powdered infant HA formula 250-420ng/mg protein LC-ESI-MS-MS current study
Table 16: CML content in different milk products reported in literatu
CHAPTER 3. MATERIALS AND METHODS 114
3 Materials and methods
3.1 Materials and apparatus
3.1.1 General materials
Tubes 15 and 50 ml (Sarstedt)
Syringe filter 0.22µm, PVDF (Roth)
Tubes 0.5, 1.5 and 2 ml (Eppendorf)
Pipette tips (Eppendorf)
3.1.2 Apparatus
Analytical balance (Sartorius)
pH-Meter (Metrohm)
Pipette (Brand, Eppendorf)
Ultrasonic bath (Bandelin SONOREX)
Magnetic stirrer (Heidolph)
Thermo mixer (Eppendorf)
Rotary evaporator (Büchi, Heidolph)
Centrifuge (Hettich universal 32R)
Shaking water bath SW22 (Julabo)
Freeze-dryer (Novalyphe-NL150, Savant)
Speed-Vac concentrator (Thermo Electron corporation)
NMR-spectrometry analyzer (Bruker Avance 360 MHz)
High resolution impact mass spectrometry (JMS-GC-Mate JEOL)
Kjeldahl apparatus consists of kjeldahl apparatus IR600, exhauster and water vapour
distillatory (Omnilab)
CHAPTER 3. MATERIALS AND METHODS 115
LC-ESI-MS-MS consists of API 4000 QTRAP (AB SCIEX) coupled to liquid
chromatography Ultimate 3000 RS (Dionex)
Analytic HPLC:
AS-1555, Intelligent Sampler, Jasco
DG-980-50, 3-Line Degasser Jasc
MD-1510, Multiwavelength Detector, Jasco
LG-980-02S, Ternary Gradient Unit; Jasco
PU-1580, Intelligent pump, Jasco
Preparative HPLC:
AS-2057Plus, Intelligent Sampler, Jasco
UV-2077Plus, 4- Intelligent UV/VIS Detector, Jasco
CHF122SB, Fraction Collector, Advantec
PU-2087Plus, Intelligent Prep. Pump, Jasco
CO-200, oven, Jasco
3.1.3 Materials
Synthesis
Nα-FMOC-Lys-HCl (Fluka)
Lactose anhydrous (Fluka)
Triethylamine (Acros Organics)
Morpholine (Fluka)
Dimethylformamide (Sigma-Aldrich)
Diethylether (Fisher scientific)
Dimethylformamide 99.8% (Acros-organics )
Cbz-Lys-OH (Fluka)
Na2HPO4*2H2O (Fluka)
NaH2PO4-2H2O (Fluka)
Iodoacetic acid (Fluka)
Palladium black (Fluka)
Spiritus ethanol (BFB-Nürnberg)
K2HPO4 (Fluka)
KH2PO4 (Fluka)
NaCL (Prolabo)
CHAPTER 3. MATERIALS AND METHODS 116
Urea (Merck)
Methionine (Sigma-Aldrich)
Cysteine (Fluka)
H2O2 30% (Merck)
FeSO4 heptahydrate (Fluka)
Hydrolysis
Pepsin (Roche)
Pronase (Roche)
Leucine aminopeptidase (Sigma-Aldrich)
Ammonium acetate (Acros Organics)
Ammonium carbonate (Acros Organics)
Ammonia (Grüssing)
Sodium tetraborate (Acros Organics)
Sodium hydroxide (Grüssing)
Sodium borohydride (Fluka)
Tris (Acros Organics)
Concentrated hydrochloric acid (Merck)
Ninhydrin reagent
Sodium acetate (Fluka)
Stannous chloride dehydrate (Merck)
Ninhydrin (Sigma-Aldrich)
Ethelyne glycol (Acros Organics)
Materials and equipments for liquid chromatography and mass
spectrometry
RP-thin layer (Merck)
1ml polypropylene column (Qiagen GmbH)
m-Aminophenylboronic acid-agarose (Sigma-Aldrich)
Magnesium chloride hexahydrate (Merck)
D-sorbitol (Fluka)
Concentrated acetic acid (Merck)
ZIC-HILIC column: 150*2.1mm, 3.5 µm (Sequant)
CHAPTER 3. MATERIALS AND METHODS 117
ZIC-HILIC column: 150*4.6 mm, 5 µm (Sequant)
Supelcosil C18 column: 150*2.1mm, 5µm (Supelco)
C18-100 column: 250*4 mm, 5µm (Nucleosil)
C18-100 column: 250*21 mm, 7µm (Nucleosil)
Strata C18-E (phenomenex)
NFPA (Sigma-Aldrich)
Formic acid 99% (Acros Organics)
Acetonitrile MS grade (Fisher scientific)
Ammonium acetate MS grade (Fluka)
Methanol HPLC grade (Prolabo)
Acetonitrile HPLC grade (Prolabo)
5-Hydroxytryptophan (Sigma-Alrich)
Methionine sulfoxide (Sigma-Adrich)
Methionine sulfone (Sigma-Aldrich)
Carboxymethyllysine (NeoMPS GROUPE SNPE)
Oxidation and glycation in milk models
α-lactalbumin typ (Sigma-Aldrich)
Lactose anhydrous (Fluka)
H2O2 30% (Merck)
NaCL (Prolabo)
Na2HPO4*2H2O (Fluka)
NaH2PO4-2H2O (Fluka)
Dialysis membranes MWCO of 4-6 and 8-10kDa (Roth)
Nanosep centrifugal devices 10K (Pall)
3.2 Buffers and solutions
Milk resembling Phosphate Buffer Saline (PBS), pH 6 .8
1000 ml of doubly distilled water
2.26 mM Na2HPO4*2H2O (0.466g)
7.38 mM NaH2PO4-2H2O (1.152g)
CHAPTER 3. MATERIALS AND METHODS 118
8 mM NaCL (0.467g)
Solutions for enzymatic hydrolysis (Hasenkopf et al )
• Pepsin solution: 2 mg pepsin in 1 ml HCl 0.02M
• Pronase solution: 2 mg pronase in 1 ml of ammonium acetate 2M, pH 8.2
buffer.
• Ammonia 5%
• Ammonium acetate buffer 2M, pH 8.2
100 ml of doubly distilled water
15.616 g of ammonium acetate
The pH was adjusted to 8.2 with ammonia 25%
Solutions for enzymatic hydrolysis (Delatour et al)
• Pepsin solution: 1 mg pepsin in 1 ml HCl 0.02M
• Pronase solution: 1 mg pronase in 1 ml of tris buffer 2M, pH 8.2
• Sodium borate 0.2 M, pH 9.5
10 ml doubly distilled water
0.7627 g of NA2[B4O5(OH)].8H2O
• Sodium hydroxide 0.1M
20 ml doubly distilled water
0.0799 g of NaOH
• Sodium borohydride 1M
10 ml of 0.1 M NaOH
0.3783 g of NaBH4
• Ammonium carbonate 1M, pH 10.5
10 ml doubly distilled water
0.9609 g of (NH4)2CO3
• 25% Ammonia to adjust the pH to 10.5
• Tris buffer 2M, pH 8.2
10 ml doubly distilled water
2.4228 g of (HOCH2)3CNH2
35% HCl to adjust pH to 8.2
CHAPTER 3. MATERIALS AND METHODS 119
Solutions for the ninhydrin assay
• Sodium acetate buffer 4N, pH 5.5
10.8 mg of sodium acetate (water free),
2 ml of glacial acetic acid (acetic acid 99%)
Water to complete the volume to 10 ml
pH was adjusted to 5.5 using NaOH 2M (about 10 ml NaOH)
• Stannous chloride dihydrate (MW 225 g)
1 ml ethylene glycol
118 mg of SnCL2
• Ninhydrin reagent
200 mg ninhydrin
7.5 ml of ethelyne glycol
2.5 ml of sodium acetate buffer 4N
250 µl of SnCL2 suspension
Solutions to isolate lysyl oxidase
• Potassium phosphate buffer 16 mM/NaCL 120 mM
1000 ml of doubly distilled water
12 mM K2HPO4 (2.090g)
3.8 mM KH2PO4 (0.517g)
7.013 g of NaCl
pH was adjusted to 7.7 by NaOH 2N (80 g NaOH dissolved in 1000 ml of doubly
distilled water)
• Potassium phosphate buffer 16 mM/NaCl 120 mM/urea 4N
250 ml of potassium phosphate buffer 16 mM/NaCL 120 mM
60.06 g of Urea
CHAPTER 3. MATERIALS AND METHODS 120
Standard`s concentration to determine the fragments of
LacLys, CML, MeSO and 5-OH-Trp
4.2 µM of the synthesized LacLys, 1µM of CML, 1.2 µM of MeSO and 0.9 µM of 5-
OH-Trp were dissolved in a mixture of formic acid (0.1%) in a water/acetonitrile
mixture (50/50%).
Aqueous mobile phases for liquid chromatography
• 5 mM CH3COONH4, pH 6.8
1000 ml of doubly distilled water
0.385 g of CH3COONH4
• 10 mM CH3COONH4, pH 6.8
1000 ml of doubly distilled water
0.770 g of CH3COONH4
• 5 mM NFPA
1000 ml doubly distilled water
0.766 µl of NFPA 97%
Solutions for affinity chromatography
• Washing buffer: CH3COONH4 250 mM/MgCl2 50 mM, pH 8
100 ml doubly distilled water
1.927g of CH3COONH4
1.0165g of MgCL2* H2O
• Elution buffer: D-sorbitol 200 mM
100 ml of washing buffer
3.644g D-sorbitol
• Elution buffer: acetic acid 250 mM
98.569 ml doubly distilled water
1.431 ml of concentrated acetic acid 99%, d=1.049
Ammoniac 25% to adjust the pH to 2.83
CHAPTER 3. MATERIALS AND METHODS 121
3.3 Methods
3.3.1 Lactulosyllysine synthesis
3.3.1.1. Trial to synthesize lactulosyllysine
A solution of Nα-FMOC-Lys-HCl (0.493 mmol, 200 mg) and triethylamine (0.5 mmol,
69 µl) in 8 ml of methanol was stirred at room temperature for 20 min, and then 80
mg of lactose anhydrous was added.
The reaction mixture was refluxed at 64 °C for 3 ho urs under nitrogen atmosphere.
After the removal of solvents by rotary evaporator, the residue was reconstituted in
water and purified on a column (200*20mm) filled with RP-18 silica (LiChroprep 25-
40µm). Five mixtures of water and methanol were run on the column and then
collected. These fractions were: fraction A (50 ml, 100% water), fraction B (70 ml,
80:20%, water/MeOH), fraction C (50 ml, 50:50%, water/MeOH), fraction D (50 ml,
20:80%, water/MeOH) and fraction E (100 ml, 100% MeOH).
In order to determine the fractions which contain the target compound, an aliquot of
each fraction (20 µl) was loaded on a RP-thin layer chromatography. Mobile phases
were a mixture of water and methanol (20:80%). After 3-4 hours the plate was
sprayed by ninhydrin solution (5% in ethanol) to detect the target compound FMOC-
LacLys. Pink spots appeared in the fractions C and D indicating the presence of
FMOC-LacLys. After the removal of methanol by rotation evaporator, the rest of the
fractions C and D were lyophilized.
3.3.1.2 Chromatographic conditions for the separati on of FMOC-
LacLys
The lyophilized residues (from the fractions C and D) were dissolved in acetonitrile
(1mg/ml) and then were loaded on a Nucleusil C18 column, 250*4, 5 µm using
mobile phases of formic acid 0.5% in water (solvent A) and acetonitrile (solvent B).
Acetonitrile was 10 % at the beginning of run, it increased then in 10 min to 50%,
and then to 100% during the next 5 min. Acetonitrile was kept at 100% for 5 min. The
flow rate was 0.8 ml/min.
CHAPTER 3. MATERIALS AND METHODS 122
3.3.1.3 Removal of the blocking group of FMOC-LacLy s
242 mg of the lyophilized residue (from the fractions C and D) were dissolved in a
mixture of DMF/MeOH (9:1, 1.6 ml) followed by the addition of 0.52 ml of
morpholine. The mixture was stirred for 2 hours at 25 °C. After the removal of
volatiles, the residue was suspended in 100 ml diethyl ether and filtered. The
synthesized product was obtained as a yellowish powder on the filter paper.
3.3.1.4 Trial to purifiy LacLys using m-Aminoboroni c acid
m-Aminophenylboronic acid attached to insoluble agarose matrix was suspended in
washing buffer (ammonium acetate 250 mM, pH 8 containing 50 mM MgCL2) and
then packed into polypropylene column (1ml, 85* 7mm). In order to keep the reactive
boronate form [RB(OH)3], the column was equilibrated with washing buffer. 200 µl of
the synthesized LacLys (1mg dissolved in ml of washing buffer) were loaded on the
column and kept at 4 °C to retain LacLys. After 1 h our, 10 ml of the washing buffer
was added to remove the unbounded substances and to maintain the medium an
alkaline pH. Finally, LacLys eluted specifically using the elution buffer D-sorbitol (200
Mm), which competes with LacLys to bind to boronate. Alternatively, LacLys eluted
non specifically by acidification of the boronate complex by 250 mM acetic acid pH
2.8. The eluates were collected in 5 ml fractions, and then lyophilized and
reconstituted in water (15 µg of the lyophilized residue per ml water). As control, a
solution of the synthesized LacLys, which was not subjected to affinity
chromatogaphy, was prepared (15µg/ml). The lyophilized residues and the control
were loaded on a C18 column, 150*2.1mm, particle size 5 µm, followed by MS-MS
analysis under the chromatographic and spectrometric conditions used to separate
LacLys on the C18 column.
3.3.1.5 Synthesis of FMOC-LacLys
A solution of Nα-FMOC-Lys-HCl (0.493 mmol, 200 mg) and triethylamine (0.5mmol,
69 µl) in 20 ml MeOH was stirred at room temperature for 20 min, and then a
solution of lactose anhydrous (0.584 mmol, 200mg) dissolved in 1 ml water was
added. The reaction mixture was stirred at 64 °C fo r 3-5 hours under nitrogen
atmosphere then filtered. After the removal of solvents by rotary evaporator, the
residue was reconstituted in water and then lyophilized. The lyophilized rest was
CHAPTER 3. MATERIALS AND METHODS 123
taken up in MeOH, and then filtered to remove the non-reacted lactose. MeOH was
then removed by rotary evaporator; the residue was taken up in water and
lyophilized.
3.3.1.5.1 Fractionation of FMOC-LacLys on a ZIC-HILIC column
30 mg of the lyophilized residue, which resulted from refluxing lactose and FMOC-
Lys, was dissolved in 1 ml of a water/acetonitrile mixture (50/50%). The solution was
loaded on a ZIC-HILIC column (150*4.6 mm, 5µm) to separate FMOC-LacLys from
other reaction products.
The separation was achieved using acetonitrile (solvent B) and ammonium acetate
buffer (10 mM). The flow rate was 800 µl per min and the injection volume was 20 µl.
At the beginning of run Solvent B was 80 % then decreased in 4 min to 50% and
then in the next 4 min to 20%. The fractions between 4.8 and 5.8 min were collected.
Acetonitrile was removed by rotary evaporator followed by freeze-drying to remove
water. In order to be sure that the other the compounds were removed, 1 mg of the
freeze-dried residue was reconstituted in 1 ml of a water/acetonitrile mixture
(20/80%). 20 µl was then loaded on the ZIC-column under the chromatographic
conditions which were mentioned. Only the peak of FMOC-LacLys appeared. Finally,
the blockage group (FMOC) was removed by the use of morpholine (See 5.3.1.3).
3.3.1.5.2 Determination of the purity of LacLys by 1H-NMR
2.4 mg of synthesized LacLys (5.1µmol) were dissolved in 1.3 ml D2O. 0.393 µl of
DMF was added (5.1µmol). DMF (99.8%) was used as standard to estimate the
purity of LacLys. The mixture was measured quantitatively by 1H-NMR, whereby the
relaxation time (D1) was 20 sec. Acetone has been added as a standard for axis
calibration and has appeared as a single signal (S, 2.22 ppm).
3.3.2 Synthesis of Cbz-CML
280 mg of Nα-carbobezyloxy-L-lysine (Nα-Cbz-L-lysine) and 186 mg of iodoacetic
acid were dissolved in 10 ml of 2M phosphate buffer. The pH of the reaction mixture
was adjusted to 10 by 2N NaOH, and then the solution was stirred at room
temperature for 2 days. 2 millilitres of ammonia (25%) were added and the solution
was stirred overnight at room temperature. The product Nα-Cbz-CML was isolated
CHAPTER 3. MATERIALS AND METHODS 124
by preparative HPLC on a Necleusil column, C18-100, 7µm, 250*21 mm using the
mobile phases methanol and 5 mM ammonium format buffer, pH 7. The flow rate
and the injection volume were 8 ml/min and 1 ml, respectively. The total run time
was 40 min. Methanol increased in 22 min from 0% to 65%, and then in 4 min to
100%. Methanol was kept at 100% till the end of run. The fractions between 17 and
21 min were collected followed by the removal of methanol by rotation evaporator. At
last, the fractions were lyophilized.
Removal of the blocking group of Cbz-CML
200 mg of the lyophilized residue, which was obtained after fractionation of the
reaction mixture of Cbz-Lysine and iodoacetic acid, was dissolved in 10 ml dry
ethanol. Palladium black was added as catalyser and the solution was hydrogenated
for one day. The solution was then filtered on diatomite (celite 535) and washed first
with water and then with spirituous ethanol. The obtaind aqueous solution was
lyophilized. CML was obtained as a white powder.
3.3.3 Synthesis of methionine sulfoxide (MeSO)
200 µl of 0.25 M methionine was mixed with 100 µl of 0.5M HCl and 5 µl of 30%
H2O2. The reaction mixture was incubated for 1 hour at 21°C then lyophilized. The
lyophiliyed residue was dissolved in a water/acetonitrile (20/80%) mixture then
MeSO was isolated by fractionation from the reaction mixture. The separation of
MeSO was performed on a ZIC-HILIC column (150*4.6 mm, 5µm) using mobile
phases of acetonitrile (solvent B) and 10 mM ammonium acetate buffer. The
gradients were (time in min/B %): (0/70) (4/50) (12/50). The fractions between 6.5
and 7.5 min were collected and lyophilized.
3.3.4 Trial to synthesize lysine aldehyde
LOX was isolated as reported in literature with some modifications (Kagan, Sullivan
et al. 1979). Shell membranes of 10 fresh chicken eggs were washed with doubly
distilled water, and then were ground and filtered to remove water. 20 ml of
potassium phosphate buffer (16 mM pH 7.7) containing 0.12M NaCl (buffer A) was
added to 8 g of the wet ground membranes. The solution was stirred at +4 °C for 3
hours followed by centrifugation at +4 °C for 30 mi n and RPM of 5000. The
CHAPTER 3. MATERIALS AND METHODS 125
centrifugation process was repeated. The two salt extracts were collected, filtered,
and then dialysed against buffer A for 24 hours at +4 °C (molecular weight cutoff of
4-6 kDa).
The pellet was further extracted by potassium phosphate buffer/NaCL containing
urea 4M (buffer B). 50 ml of buffer B was added to the pellet, and then the solution
was stirred at +4 °C for 16 hours followed by centr ifugation at +4 °C for 30 min and
RPM of 5000. The urea extract was collected and the pellet was stirred again using
the same buffer for 8 hours followed by centrifugation. The two urea extracts were
filtered and dialysed against buffer A for 24 hours at +4 °C. At last, both salt extract
and urea extract were lyophilized.
After obtaining the lysyl oxidase extracts, 100 mg of the lyophilized extract, which
contained urea, were dissolved in 35 ml of 0.1M phosphate buffer, pH 7.4. Another
solution was prepared containing 100 mg of the lyophilized salt extract (without
urea). 5 g of urea and 350 mg of Cbz-lysine were then added to each solution. And
then, both solutions were incubated in a shaking water bath at 37 °C. The aliquots
were taken after 2, 3, 7, 9 and 14 days followed by lyophilization. The lyophilized
residues were reconstituted in water. The samples were then loaded on a C18
column 250*4 mm, 5 µm. Acetonitrile and formic acid 0.5% in water were used as
mobile phases. The flow rate was 800µl/min and the injection volume was 20 µl. At
run beginning, acetonitrile was 10% then increased in 10 min to 50%, and then
increased in the next 10 min to 100%. Acetonitrile was then kept at 100% for 3 min.
Mass spectrometry and UV (280nm) were used for detection. The unknown product
was detected mainly in the presence of LOX urea extract, while its formation in the
presence of LOX salt extract was negligible.
The isolation of the predicted Cbz-Lys was achieved by fractionation using the same
chromatographic conditions used for detection. The only difference was the
replacement of acetonitrile with methanol. This replacement has led to higher
pressure; therefore the flow rate was reduced to 700µl/min. These changes
increased the retention time of the target compound to 15.9 min. The fractions
between 15 and 18 min were collected to isolate the target compound, evaporated
by rotation evaporator to remove methanol, and then lyophilized. Finally the blocking
group (Cbz) was removed by hydrogenation, as described previously to synthesize
CML (see 5.3.2).
CHAPTER 3. MATERIALS AND METHODS 126
3.3.5 Trial to synthesize cysteine sulfenic acid (C ys-SOH)
Briefely, 60 ml of 1mM cysteine, 0.5 mM of FeSO4 heptahydrate and 0.2 mM H2O2
were incubated at 37 °C.
Aliquots of 19 ml were taken after 10, 20 and 30 min. 1 ml of methionine 20 mM was
added to each aliqouts to stop the oxidation of cysteine. The aliquots were
lyophilized, reconstituted in water and then loaded on a C18 column, 250*4 mm,
particle size 5 µm. The mobile phases were acetonitrile and formic acid 0.5% in
water. At run beginning, acetonitrile was 10%, and then it increased in 10 min to
50%. UV detection and mass spectrometry were used for the detection. Later, many
attemps were carried out to synthesize Cys-SOH, in which FeSO4 was not used.
Additionally, different temperature (25, 37 and 120 °C) and/or different incubation
times (5, 10, 20, 30, 60, 100 and 120 min) were applied. The aliquots were
lyophilized then treated as menthioned previously.
3.3.6 The conditions of liquid chromatography for t he
seperation of CML, MeSO, LacLys and 5-OH-Trp on a C 18
column
The compounds CML, MeSO, LacLys and 5-OH-Trp were separated on a C18
column from Supelco, 150*2.1mm, particle size 5 µm, protected with a pre-column
20*2.1mm. The column was housed in the oven at 20 °C. The flow rate and injection
volume were set at 200µl/min and 5 µl, respectively. Mobile phases were acetonitrle
(solvent B) and 5 mM NFPA in water (solvent A). The gradient was optimized as
possible to achieve the better separation between the four analytes. At run
beginning, acetonitrile was 10% then it increased in 3 min to 40%, and then
increased in the next 3 min to 50% where it continued for 4 min at 50%. Acetonitrile
increased then in two min to 100% and continued for 2 min at 100% (until 14 min).
Acetonitrile decreased then in 6 min to the initial conditions (10%) followed by
equilibration for 5 min before taking the next sample. All samples were dissolved in a
mixture of water/acetonitrile (50/50%). The same MS-MS conditions were used for
these analytes on both C18 and ZIC-HILIC columns.
CHAPTER 3. MATERIALS AND METHODS 127
3.3.7 The conditions of liquid chromatography for t he
seperation of CML, MeSO, LacLys and 5-OH-Trp on a Z IC-
HILIC column
The separation was performed on a ZIC-HILIC column from Sequant, 150*2.1 mm
and 3.5 µm particle sizes. The column was protected with a pre-column (Sequant)
20*2.1mm, particle size 5 µm and housed in oven at 20 °C. The mobile phases were
acetonitrile (solvent B) and 5mM ammonium acetate buffer, pH 6.8. The flow rate
was constant during run time at 200µl/min. The injection volume was 5 µl. The initial
composition of acetonitrile was 70%, and then it decreased gradually in 18 min to
25% and then decreased in the next 2 min to 20%. Acetonitrile was kept for 10 min
at 20 %. Acetonitrile increased then to the initial percentage between 30 and 45 min
(70 %) followed by the equilibration for 7 min before taking the next sample. All
samples were dissolved in a mixture of water/acetonitrile (50/50%).
3.3.8 Stimulation of glycation in a milk model mixt ure
13 mg of α-LA and 493 mg of lactose, equivalent to 92 and 144.4 mM, respectively,
were dissolved in 10 ml of milk-resembling phosphate buffer saline (10mM of PBS
with 8mM NaCL). One aliquot was taken before heating and was used as the first
control. The protein solution was heated in a shaking water bath at 60 °C. Aliquots of
2.5 ml were taken after 3, 7 and 14 days followed by cooling on ice to stop the
reaction, dialysis against water (MWCO 8-10 kDa) and hydrolysis with enzymes
according to Hasenkopf et al. At last, the samples were cleaned up by ultrafiltration
(Nanosep centrifugal devices)
As a second control, a solution of α-LA without lactose in the same buffer was
heated for 3, 7 and 14 days and treated like that one with lactose. The third control
was native α-LA.
3.3.9 Stimulation of oxidation in a milk model mixt ure
A solution containing 13 mg of α-LA and 8.5 µl of 30% H2O2 (d=1.1) in 10 ml of milk-
resembling PBS was heated at 120 °C (equivalent to 92 µM and 8.4 mM of α-LA and
H2O2 , respectively). Aliquots of 2.5 ml were taken after 10, 20 and 30 min. As a
CHAPTER 3. MATERIALS AND METHODS 128
control, a solution of α-LA without H2O2 in the same buffer was heated for 10, 20 and
30 min at 120 °C. The second control was native α-LA (standard). The aliquots were
cooled immediately on ice and treated like the samples of glycated α-LA (see 5.3.8).
3.3.10 Preparation of milk samples for LC-MS-MS ana lysis
3.3.10.1 Milk defatting
5 ml of liquid milk or reconstituted powderd milk were defatted by centrifugation at 4
°C and 3850 RPM. Centrifugation lasted for 1-3 hour s depending on the fat amount.
Centrifugation lasted 1 hour in low fat UHT milk and 3 hours in condensed milk. Fat
layer was then removed with laboratory spatula.
3.3.10.2 Removal of lactose and minerals from milk and milk model
mixture
Lactose and minerals were removed from milk samples (5ml) as well as from milk
models (2.5ml) by dialysis against doubly-distilled water for 24 hours at +4 °C
(molecular weight cut-off of 8-10 kDa). Water was changed after 12 hours. The
dialysis was followed by freeze-drying.
3.3.10.3 Enzymatic protein hydrolysis (Hasenkopf et al)
1mg of the freeze-dried residue, which was obtained after dialysis, was dissolved in
1 ml HCl 0.02M. An aliquot of 50 µl of pepsin solution (2mg/ml in HCl 0.02M) was
added to the protein solution followed by incubation at 37 °C in a thermo mixer. After
24 hours of incubation, the protein solution was buffered with 250 µl of 2M
ammonium acetate buffer pH 8.2. 50 µl of pronase solution was then added (2mg/ml
in 2M ammonium acetate buffer, pH 8.2). The pH was adjusted to 7.5 by ammonia
solution (5%). The sample was then incubated for 24 hours at 37 °C. The protein
solution was then supplemented with 4.1 µl of aminopeptidase and further incubated
for 24 hours at 37 °C.
3.3.10.4 Purification of protein hydrolyzate by ult rafiltration
After enzymatic hydrolysis, the enzymes were separated from the protein
hydrolyzates by ultrafiltration. 411 µl of the protein hydrolyzate (equivalent to 300 µg
CHAPTER 3. MATERIALS AND METHODS 129
of the residue which was enzymatically hydrolyzed) were put in a centrifugal
concentrator device (Nanosep device with molecular weight cutoff of 10 kDa), and
were then centrifuged for 10 min at 4 °C and RPM of 14400. The centrifugation was
followed by lyophilization. The dry residue was reconstituted in 1ml of a water/
acetonitrile mixture (50/50%), and then diluted with the same mixture to the
concentration to be analyzed.
3.3.10.5 Enzymatic protein hydrolysis (Delatour et al)
0.5 mg of the lyophilized residue, which was obtained after milk dialysis, was
dissolved in 90 µl of water, and then 10 µl of 1M ammonium carbonate pH 10.5 and
100 µl of 1M sodium borohydride were added. The protein solution was incubated for
1 hour at 37 °C, lyophilized and then reconstituted in 1 ml of HCl 0.02 M. An aliquot
of 18 µl of pepsin solution (1mg/ml in HCl 0.02M) was added. The protein solution
was incubated for 1 hour at 37 °C in a thermo mixer . This step was repeated two
times. The protein solution was then buffered with 250 µl of 2M tris buffer; pH 8.2
followed by the addition of 15 µl of pronase (1mg/ml in tris buffer 2M). The protein
solution was then incubated for two times at 37 °C (each incubation period lasted for
30 min). After 1 hour, the sample was further supplemented with 4.1 µl of
aminopeptidase and the incubation continued for 24 hours at 37 °C. Finally, 15 µl of
pronase was added to the sample followed by incubation at 37 °C for one hour. This
step was repeated two times. Samples were cleaned up by solid phase extraction.
Some samples were hydrolyzed according to this method without the addition of
sodium borohydride. Thus, 0.5 mg of protein residue was directly dissolved in 1 ml of
HCl followed by the addition of 18 µl of pepsin.
3.3.10.6 Acidic protein hydrolysis (Delatour et al)
0.5 mg of the lyophilized residue, which was obtained after milk dialysis, was
dissolved in 50 µl water, and then was diluted with 1.5 ml of 0.2 M sodium borate pH
9.5. 1ml of 1M sodium borohydride was added and the solution was incubated for 4
hours at 21 °C. 2.75 ml of HCl 35% was then added a nd the protein sample was
hydrolysed at 110 °C for 24 hours.
CHAPTER 3. MATERIALS AND METHODS 130
3.3.10.7 Purification of protein hydrolyzate by sol id phase
extraction
The acidic hydrolyzates were dried by Speed-Vac followed by reconstitution in 500 µl
of water. This means 0.5 mg of the lyophilized residue, which was obtained after
dialysis and was then acidic hydrolysed, was reconstituted in 500 µl of water.
The cartridges (Sartra C18-E) were inserted into the chamber. Tubes were set under
the cartridge to collect the eluates. A vacuum port with a gauge was used to control
the vacuum applied on the chamber. The column (Sartra C18-E) was first
equilibrated with 100 µl of NFPA in 1 ml water. An aliquot of the enzymatic or acidic
hydrolyzate (equivalent to 200 µg of the residue which was previously hydrolyzed)
was diluted with water till 1000 µl. 100 µl of 100 mM NFPA was added, and then the
sample was loaded on the SPE column. 2 ml of 10mM NFPA in water were run on
the column for two consecutive times (this was fraction A about 5 ml). Then, 2 ml of
methanol/10mM NFPA in water (5/95%) were run on the column for two consecutive
times (this was the fraction B about 4 ml). At last, 2 ml of methanol/10mM NFPA in
water (50/50%) were run on the column for two times (fraction C). The three fractions
were collected and dried. The analytes were detected mainly in the first fraction.
Lyophilization was mainly used for drying; however, during the investigation of the
role of drying method, some samples were dried by Speed-Vac at 35 °C.
Figure 66: The chamber for solid phase extraction with the cartridges
CHAPTER 3. MATERIALS AND METHODS 131
3.3.10.7.1 The standards MeSO, LacLys and CML on a SPE column
A solution of the standards MeSO, LacLys and CML was prepared (25 µg of each
standard per 1ml water) and divided into 2 portions. The first one was loaded on a
Sartra C18-E column and treated like the samples of enzymatic and acidic
hydrolyzate (see 5.3.10.7). However, to avoid any loss of the standards in the
fractions B and C, the three fractions were collected. The collected fractions as well
as the second portion of standards were lyophilized, reconstituted in water and then
analyzed by LC-MS-MS on a C18 column, 150*2.1, 5 µm.
3.3.11 Trial to detect ornithine in milk protein
Milk samples were prepared as mentioned previously for the detection of LacLys,
CML, MeSO and 5-OH-Trp. The preparation involved milk defatting, dialysis,
enzymatic hydrolysis (for 72 hours) and cleanup by ultrafiltration. In order to detect
ornithine in heated milk, some samples of UHT milk were heated at 120 °C after fat
removal. The aliquots were taken after 10, 20, 30 and 60 min, cooled on ice.
Enzymatic hydrolysis as well as the other preparation steps was then carried out
(see 5.3.10.2-5.3.10.4). The concentration of the analyzed sample was 25 µg per 1
ml of a water/acetonitrile mixture (25 µg of the residue which was enzymatically
hydrolysed).
To investigate the artificial formation of ornithine from arginine during enzymatic
hydrolysis, 25 µg of arginine were subjected to the conditions of enzymatic
hydrolysis. As a standard, two solutions of arginine and ornithine (0.25µg/ml) were
prepared. The samples of milk as well as the standards were analyzed by LC-MS-
MS on a C 18 column 150*2.1, 5 µm.
3.3.12 Investigation the effect of sodium borohydri de on
MeSO
1.5 ml of sodium borate 0.2 M pH 9.5, 1 ml of 1M sodium borohydride and 2.75 ml of
HCl 35% were added to 1 mg of MeSO. The reaction mixture was heated at 110 °C
for 24 hours. An aliquot, which was corresponding to 200 µg MeSO, was cleaned up
by SPE (Sartra C18-E). The first fraction was then lyophilized and analyzed by LC-
MS-MS on the C18 column (150*2.1, 5µm). Furthermore, in order to investigate the
CHAPTER 3. MATERIALS AND METHODS 132
conversion of MeSO into methionine, the first fraction was analyzed by HPLC/UV on
the ZIC-HILIC column (150*4.6, 5µm) under the chromatographic conditions which
were used previously for the fractionation of MeSO (see 5.3.3).
3.3.13 Ninhydrin assay
An aliquot of the hydrolyzate was diluted with water till 50 µl. The aliquot was
equivalent either to 3 µg of the residue which was enzymatically hydrolyzed
according to Hasenkopf et al, or was equivalent to 10 µg of the residue which was
acidic or enzymatically hydrolyzed according to Delatour et al. And then, 100 µl of
ninhydrin reagent was added. The pH of the samples was in range 5-5.5. The
samples were heated in a boiling water bath for 3-5 min, and then were left in a
water bath at room temperature for 5 min. 250 µl of n-buthanol was added to each
sample then the samples were left at room temperature for 15 min. Finally, 250µl of
each sample was taken and the absorbance was measured at 570 nm.
3.3.14 Method validation
The amounts of LacLys during method validation were calculated taking into account
the purity of the synthesized standard (6.13%)
3.3.14.1 Within-day repeatability (intra-day variat ion)
A solution of the standards was prepared which contained 2.94, 1.28 and 0.096 µg of
the synthesized LacLys, MeSO and CML, respectively, dissolved in 10 ml of a
water/acetonitrile mixture (50/50%). This standard solution was used as a stock
solution for standard addition during the investigation of within-day repeatability
where it was diluted many times (1 to 2, 1 to 4 and 1 to 8).
8 mg of UHT milk proteins or heated UHT milk proteins (heated for 60 min at 120 °C)
were dissolved in 8 ml of 0.02M HCl. These protein residues were obtained after fat
removal, dialysis and lyophilization. The protein solution was divided into 8 aliquots
(1mg per 1 ml) followed by enzymatic hydrolysis according to Hasenkopf et al.
Enzymatic hydrolysis was applied for eight samples at the same time using the same
enzymes and buffers solutions. After 3 days of hydrolysis, 411 µl of the enzymatic
hydrolyzate (corresponding to 300 µg of residue which was enzymatically
hydrolyzed) were cleaned up, lyophilized then reconstituted in 1 ml of a
CHAPTER 3. MATERIALS AND METHODS 133
water/acetonitrile mixture (50/50%). The protein samples were then diluted with
water/acetonitrile mixture to 100µg/ml. An aliquot of 300 µl was then diluted either
with 300 µl of water/acetonitrile mixture or with 300 µl of one of the standards
solutions. This means the concentration of the unknown sample became 50µg/ml.
The analysis by LC-ESI-MS-MS lasted 2 days to investigate the within-day
repeatability of the method for LacLys and MeSO on the C18 column (150*2.1, 5
µm). 4 known amounts of LacLys and MeSO were added to the unknown sample.
The analysis lasted 2 days to investigate the within-day repeatability of the method
for CML on the ZIC-HILIC column (150*2.1, 3.5 µm). However, 3 known amounts of
CML were added to the unknown sample. The addition 0.0048 µg/ml was abdicated
by the analysis of UHT milk, while the addition 0.0006µg/ml was abdicated by the
analysis of heated UHT milk.
sample concentration
added LacLys added MeSO added CML
50 µg/ml 0 0 0
50 µg/ml 0.0183 µg/ml 0.008 µg/ml 0.0006 µg/ml
50 µg/ml 0.0367 µg/ml 0.016 µg/ml 0.0012 µg/ml
50 µg/ml 0.0735 µg/ml 0.032 µg/ml 0.0024 µg/ml
50 µg/ml 0.147 µg/ml 0.064 µg/ml 0.0048 µg/ml
Table 17 : Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the within-day repeatability for UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 50 µg/ml
3.3.14.2 Between-day repeatability (inter-day varia tion)
The concentrations of LacLys and MeSO in the standard solutions were the same
which were mentioned previously to investigate the within-day repeatability.
Addaitionally, the samples were prepared as mentioned previously (5.3.14.1).
However, higher concentrations of CML were used. Additionally, enzymatic
hydrolysis was carried out in consecutive days where the eight samples were
completely hydrolyzed after 11 days. The concentrations of the samples increased to
75 µg/ml. 6 samples were then analyzed in a consecutive days to investigate the
between-day repeatability for LacLys and MeSO on the C18 column (150*2.1, 5µm).
4 samples were analyzed to investigate the between-day repeatability for CML
because the used ZIC-HILIC column (150*2.1, 3.5µm) was damaged. A new ZIC-
CHAPTER 3. MATERIALS AND METHODS 134
HILIC column (150*2.1, 3.5µm) was purchased; however, the samples for the
between-day repeatability were not further investigated.
sample concentration added LacLys added MeSO added CML
75 µg/ml 0 0 0
75 µg/ml 0.0183 µg/ml 0.008 µg/ml 0.001 µg/ml
75 µg/ml 0.0367 µg/ml 0.016 µg/ml 0.002 µg/ml
75 µg/ml 0.0735 µg/ml 0.032 µg/ml 0.004 µg/ml
75 µg/ml 1.147 µg/ml 0.064 µg/ml 0.008 µg/ml
Table 18: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the between-day repeatability in the samples of UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 75 µg/ml
3.3.14.3 Recovery
A solution of standards was prepared containing 21.45, 9.6 and 0.72 µg of the
synthesized LacLys, MeSO and CML, respectively. The standard compounds were
dissolved in 450 µl of water (this solution was used for addition 3). Two aliquots of
the standards solution, each one was 150 µl, were diluted with water to 300 µl or to
600 µl. These two standard solutions were used for addition 2 and addition 3,
respectively.
16 mg of UHT milk proteins, which were obtained after fat removal, dialysis and
lyophilization, were dissolved in 16 ml of HCl 0.02M. The protein solution was then
divided into 16 aliquots (1mg per 1 ml). An aliquot of each standards solution (30 µl)
was added to 4 aliquots of the protein solution followed by enzymatic hydrolysis
according to Hasenkopf et al. 4 aliquots of the protein solution were enzymatically
hydrolyzed without spiking any analyte. 425 µl of the enzymatic hydrolyzate
(corresponding to 300 µg of the residue which was enzymatically hydrolyzed) were
cleaned up, lyophilized, and reconstituted in 1 ml of a water/acetonitrile mixture
(50/50%). The samples were then diluted with the latter mixture either to 50µg/ml, to
determine the recovery of MeSO and LacLys, or were diluted to 300µg/ml to
determine the recovery of CML.
70 µl of the diluted samples (50 or 300µg/ml) were mixed either with 70 µl of
water/acetonitrile mixture or with 70 µl of one of the standards solutions. Thus, the
CHAPTER 3. MATERIALS AND METHODS 135
concentrations of the unknown samples as well as the concentration of the spiked
analytes decreased to 50% (table 20).
protein dissolved
in 1 ml HCl
0.02M
volume of added water (µl)
volume of added
standards solution
(µl)
LacLys (µg/30µl)
CML (µg/30µl)
MeSO (µg/30µl)
Protein (mg/µl)
without analyte addition
1 mg 30 0 0 0 0 1/1030
addition 3 1 mg 0 30 1.43 0.048 0.64 1/1030
addition 2 1 mg 0 30 0.715 0.024 0.32 1/1030
addition 1 1 mg 0 30 0.357 0.012 0.16 1/1030
Table 19 : The amounts of LacLys, CML and MeSO in 30 µl of the standards solutions which were spiked to each protein sample before enzymatic hydrolysis
sample concentration added LacLys added MeSO sample
concentration added CML
25 µg/ml 0 0 150 µg/ml 0
25 µg/ml 0.0459 µg/ml 0.025 µg/ml 150 µg/ml 0.003 µg/ml
25 µg/ml 0.0919 µg/ml 0.05 µg/ml 150 µg/ml 0.006 µg/ml
25 µg/ml 0.1839 µg/ml 0.1 µg/ml 150 µg/ml 0.012 µg/ml
25 µg/ml 0.3678 µg/ml 0.2 µg/ml
Table 20: The concentrations which were used for standard addition during the determination of the recovery of of LacLys, MeSO and CML. The concentration of the sample in which CML should be quantified was 150µg/ml and it was 25µg/ml to quantify LacLys and MeSO
3.3.14.4 LOD and LOQ
LOD and LOQ of LacLys and MeSO
In order to determine LOD and LOQ of LacLys, the content of LacLys was
determined in three samples of UHT milk by standard addition. Each sample
contained 25 µg per ml of a water/acetonitrile mixture (50/50). The analyte`s
concentrations for standard addition were mentioned previously for the within-day
repeatability (see 5.3.14.1). In order to determine LOQ und LOD, the three samples
were diluted many times (1:20, 1:25, 1:35, 1:40 and 1:50) with the water/acetonitrile
mixture to the extent at which the signal to noise ratio of the quantifier fragment (m/z
CHAPTER 3. MATERIALS AND METHODS 136
471.2→225.1) and qualifier fragment (m/z 471.2→128.1) were 10 and 3,
respectively. LOQ or LOD were then calculated by dividing the content of LacLys in
25µg of the hydrolyzed residue into the dilution factor. The calculated value
represents LOQ or LOD in 1 ml of the sample solution, and then it was divided into
200 to calculate the content in 5µl, which was the injection volume
In order to determine LOQ and LOD of MeSO, similar steps were carried out;
however, the determination of LOQ and LOD of MeSO was based only on the
quantifier fragment (m/z166.1→ 74). Additionally, the samples were diluted in the
range 1:35 to 1:150.
LOD and LOQ of CML
A solution of CML was prepared (5 µg/ml), and then was diluted to 0.312µg/ml. The
latter solution of CML was diluted many times as the following: 1:2.5, 1:5, 1:10, 1:20,
1:40, and 1:80. A water/acetonitrile mixture (50/50%) was used to dissolve and to
dilute the samples.
In order to determine LOD and LOQ of CML, an aliquot of raw milk hydrolyzate
(50µg/ml: 50 µg of the residue which was enzymatically hydrolyzed) was mixed
either with the same volume of water/acetonitrile mixture to give the sample 1, or it
was mixed with one of the diluted solutions of CML to give the samples 2, 3, 4, 5 and
6. Each sample was then analyzed 4 times by LC-ESI-MS-MS using the ZIC-HILIC
column (150*2.1, 3.5µm).
sample name
raw milk residue spiked CML
sample 1 25 µg/ml 0 µg/ml
sample 2 25 µg/ml 0.0624 µg/ml
sample 3 25 µg/ml 0.0312 µg/ml
sample 4 25 µg/ml 0.0156 µg/ml
sample 5 25 µg/ml 0.0078 µg/ml
sample 6 25 µg/ml 0.0039 µg/ml
sample 7 25 µg/ml 0.00195 µg/ml
Table 21: Amounts of CML which were spiked into 25 µg of raw milk protein hydrolyzate in order to determine LOD and LOQ
CHAPTER 3. MATERIALS AND METHODS 137
3.3.15 Qantitation
The concentrations of the samples to be analyzed varied between 25 and 900µg/ml,
as seen in table 22. The sample concentrations were 25µg/ml in most milk samples
to quantify MeSO or LacLys. In order to quantify LacLys in raw and pasteurized milk,
the concentrations increased to 200µg/ml. This means 25 or 200 µg of the residue,
which was defatted and dialysed prior to enzymatic hyrolysis, was dissolved in 1 ml
of a water/acetonitril mixture (50/50%). In the case of CML, the concentrations were
higher and reached 900µg/ml. However, one should keep in mind that the sample`s
concentration can be changed according the cleanness of the sprayer needle and
the mass analyzer, since the draggle will decrease the sensitivily and thus, will
require the use of higher concentrations to reach S/N of 10 (LOQ). By standard
addition, the concentrations of the added analytes were chosen depending on the
expected values of the analytes in milk products. For example, the concentrations of
added MeSO were 0.025, 0.05, 0.1 and 0.2µg/ml for all milk samples. The
concentrations of added LacLys were, taking into account the purity of LacLys
(6.13%), 0.046, 0.092, 0.184 and 0.368µg/ml. Only three amounts of CML were
added to milk samples, so that the concentrations were 0.05, 0.1 and 0.2µg/ml. In
the case of raw and pasteurized milk, lower amounts of CML were added, so that the
concentrations were 0.02, 0.04 and 0.08 µg CML per 1 ml.
sample name sample
concentration (CML)
sample concentration
(LacLys)
sample concentration
(MeSO)
sterilized milk 75 µg/ml 25 µg/ml 25 µg/ml
condensed milk 75 µg/ml 25 µg/ml 25 µg/ml
vanilla drink 75 µg/ml 25 µg/ml 25 µg/ml
lactose free milk 100 µg/ml 25 µg/ml 25 µg/ml
infant formulas 125 µg/ml 25 µg/ml 25 µg/ml
UHT milk 300 µg/ml 25 µg/ml 25 µg/ml
pasteurized milk 800 µg/ml 200 µg/ml 25 µg/ml
raw milk 900 µg/ml 200 µg/ml 25 µg/ml
Table 22: Concentrations of milk samples which were quantified. The concentration varied between 25µg/ml to quantifiy both MeSO and LacLys to 900µg/ml to quantify CML in raw milk proteins
CHAPTER 3. MATERIALS AND METHODS 138
3.3.15.1 Determination protein content by Kjeldahl
100 mg of the residue, which was obtained after defatting and dialysis, was put in a
kjeldahl flask, and then 20 ml of concentrated sulfuric acidic (98%) and a tablet of
catalyser were added.
The mixture was heated for 30 min at 80% energy level of the apparatus, and then
heated at 70% energy level till the disappearance of the black colour. The solution
was then heated at 60 % energy level for 1 hour followed by cooling for 30 min. A
blank, which contained sulfuric acid and catalyser without protein, was prepared and
treated like milk protein samples.
80ml of water was carefully added to the flask. The addition of NaOH 30% was
programmed for 5 seconds followed by distillation for 5 min. The distillate was
received in Erlenmeyer flask containing 90 ml of boric acid (2%) pH 4.3 and taschiro
indicator (a mixture of methyl red and methylene blue dissolved in ethanol). After the
end of distillation the colour of boric acid solution converted into light green. HCl
0.1N was then titrated on the previous solution untill the colour converted into pink.
HCl 0.1N, which was needed for the blank sample, was substracted from HCl which
was needed for the samples with proteins. The conversion factor was 6.25. Protein
content (%) was calculated as the following:
0.1N HCl(ml)*6.25*0.0014*100/ sample weight (g)
CHAPTER 4. SUMMARY 139
4 Summary
The Maillard reaction comprises a series of chemical reactions between amino
groups and carbonyl compounds leading to the formation of a variety of Maillard
reaction products (MRPs). Controlled browning is often used to develop desirable
flavor, odor or color in food like coffee, bakery products and roasted meat. However,
on the other hand, the Maillard reaction can lead to alteration of protein structure and
function as well as to the production of toxic compounds. The Maillard reaction takes
place also in the body leading to the formation of advanced glycation end products
(AGEs). The accumulation of AGEs in different tissues has been implicated in the
aging process and in the pathogenesis of complications associated with diabetes
mellitus such as nephropathy, retinopathy and neuropathy. Other diseases like
Alzheimer‘s disease, cataract and atherosclerosis were also related to the formation
of AGEs. Some of the consumed MRPs can survive the digestive process and can
be transported into the circulation resulting in elevation of AGE-serum concentration.
Due to the structural similarities between MRPs and AGEs, it is discussed that the
consumption of MRP-rich diet may participate in the development of many diseases
especially in patients with kidney diseases. Additionally, food-derived Maillard
products which cannot pass into the circulation may affect the intestinal immune
function and participate in the pathogenesis of inflammatory bowel diseases.
The presence of lactose and proteins as well as the neutral pH value render milk
as a suitable medium for the Maillard reaction during processing and storage. This
leads to the formation of various products like lactulosyllysine (LacLys) and
carboxymethyllysine (CML). In parallel to glycation, milk proteins are subjected to
various oxidation reactions where tryptophan, methionine and cysteine are
particularly sensitive to oxidative modifications. Glycation and oxidation lead to the
degradation of essential amino acids as well as to a decrease of the protein
digestibility. Consequently, these processes reduce the nutritional value of milk and
CHAPTER 4. SUMMARY 140
can alter the functional properties of milk whey proteins which are widely used in
food industry.
This work aimed to detect and quantify several glycation and oxidation products in
milk by liquid chromatography connected to electrospray ionization tandem mass
spectrometry (LC-ESI-MS-MS). LC-ESI-MS-MS represents a powerful analytical
technique for the qualitative determination since the analytes can be unequivocally
identified by their product ion spectrum. MS-MS is considerd also as a very selective
and sensitive detector for quantitation, even in complex matrices like food.
In the course of this work, experiments were carried out to synthesize reference
compounds of the major glycation and oxidation products which has been identified
before in milk products: LacLys, CML in addition to methionine sulfoxide (MeSO),
lysine aldehyde and cysteine sulfenic acid. The synthesis of LacLys could be
achieved by the reaction of FMOC-Lys and lactose under nitrogen atmosphere.
FMOC-LacLys was then isolated from the reaction mixture by fractionation on a ZIC-
HILIC column followed by the removal of the blocking group. The purity of the
standard was determined by quantitative 1H-NMR (6.13%). Thus, for the first time, a
standard with a defined content of LacLys could be synthesized. CML and MeSO
were synthesized using an alkylating agent (iodoacetic acid) or an oxidizing agent
(H2O2), respectively. For further analysis however, commercial standards of CML
and MeSO were used. Lysine aldehyde and cysteine sulfenic acid could not be
synthesized in sufficient purity, because their high reactivity led to degradation during
purification. Standards of 5-hydroxytryptophan (5-OH-Trp) and ornithine were
commercially available.
A method was then developed to detect the oxidation and glycation products, for
which standards were available, in α-lactalbumin (α-LA), which was heated in a milk
model as well as in processed and stored milk products. After removal of fat and
lactose, the target analytes were released from proteins by a complete enzymatic or
acidic hydrolysis prior to the analysis by LC-ESI-MS-MS. The tandem mass
spectrometry parameters which are dependent on the liquid chromatography flow
rate as well as the parameters which are dependent on the compounds were
optimized to achieve the highest intensity for LacLys, CML, MeSO, 5-OH-Trp and
ornithine.
LacLys and CML have little retention on C18 columns due to their high polarity. The
high polarity requires therefore the use of nonafluropentanoic acid as ion pair
CHAPTER 4. SUMMARY 141
reagent to increase the retention. Both, 5-OH-Trp and ornithine could neither be
detected in milk resembling model of lactose and α-LA nor in milk samples. Even
after the oxidation of α-LA by hydrogen peroxide at 120 °C, only MeSO was
detected. The analytes LacLys, CML and MeSO were detected clearly in milk
models which were heated at 60 °C for 14 days.
However, no separation could be achieved between CML and LacLys on the C18
column. Additionally, it was difficult to detect CML in milk samples, especially in
mildly heat-treated products like UHT milk, since CML is formed only in small
amounts. In order to improve the separation as well as to increase the sensitivity of
the method and thus, to avoid loading of high amounts of milk protein on the column,
a ZIC-HILIC column was introduced as alternative to the C18 column. The use of this
hydrophilic stationary phase allowed complete separation of CML and LacLys.
Additionally, a 3-5 fold increased signal intensity was achieved. However, the signal
intensity of LacLys in milk samples showed a higher variation on the ZIC-HILIC
column compared to the C18 column. Additionally, MeSO is detected easily in milk
samples and the variation of its signal intensity was similar on C18 and ZIC-HILIC
columns. Thus, the ZIC-HILIC column was chosen for further investigation of CML,
while the C18 column was used for further investigation of LacLys and MeSO.. Next, protein hydrolysis was studied and optimized to achieve maximal release of
MeSO, CML and LacLys from milk protein as well as to avoid at the same time
artificial formation. It was shown by LC-ESI-MS-MS analysis of the target analytes
and by ninhydrin assay that longer enzymatic hydrolysis increased the release of the
analytes (hydrolysis for 72 hours versus 27 hours). At the same time, the reduction
of LacLys to the unreactive alcohol by sodium borohydride did not lead to an
important decrease of the CML values. The result indicated that artificial formation of
CML resulting from the oxidative cleavage of LacLys during enzymatic hydrolysis
can be neglected.
MeSO was detected in the blank of the enzymatic hydrolysis due to autohydrolysis of
the enzymes, especially of pepsin. This blank value was taken later into
consideration for the quantitative measurements to avoid overestimation of MeSO.
The use of solid phase extraction (SPE) as a tool to remove the enzymes and purify
the hydrolyzate after enzymatic hydrolysis resulted in a loss of the analytes,
especially of LacLys. Therefore, ultrafiltration was used instead of SPE.
CHAPTER 4. SUMMARY 142
Prior to acidic hydrolysis, LacLys must be reduced by sodium borohydride to avoid
artificial formation of CML during acid treatment. As a consequence, reduction
prevents the detection and quantification of LacLys in parallel to CML and MeSO.
Additionally, sodium borohydride could not completely convert LacLys to the reduced
form. Consequently, it could not prevent the conversion of LacLys to CML resulting
in an overestimation of CML. Therefore, acidic hydrolysis was not adopted.
Furthermore, a decrease of MeSO was observed when NaBH4 was used prior to
acidic hydrolysis. MeSO might undergo further reaction in the presence of sodium
borohydride.
LacLys, CML and MeSO were quantified based on standard addition, since stable
isotope-labeled standards of LacLys and MeSO are not available to be used as
internal standards. The method was validated by evaluating the within-day and
between-day repeatability at low and high modification levels. In addition, the
recovery as well as detection limit and quantitation limit were determined for each
analyte. A high selectivity of the method was achieved by identifiying each analyte by
two characteristic mass transitions. The LC-ESI-MS-MS method showed good
precision. The coefficients of variation for within-day and between-day repetition of 4-
8 samples containing the analytes LacLys, CML and MeSO did not exceed 10%. In
order to investigate the recovery, three different amounts of each analyte were
spiked into UHT milk proteins prior to enzymatic hydrolysis. Some samples showed
a very good recovery. The recoveries of CML, for example, were optimal and ranged
between 96.6±9.4 % to 101±5.4 %. The recoveries of LacLys ranged between
110.5±9.6 % to 111.6±10.6 %, while the recoveriers of MeSO ranged between
107.8±10.6 % to 113.1±10.8 %. Since the recoveries did not exceed 115 %, they
were considered as acceptable.
The limit of detection (LOD) and the limit of quantitation (LOQ) for LacLys were
calculated by the dilution of UHT milk proteins to the extent at which the signal to
noise ratio of the qualifier and quantifier fragments were 3 and 10, respectively. LOD
and LOQ were 4 and 6.5 pg of LacLys on column and 1 and 2 pg of MeSO on
column. LOD and LOQ for MeSO were calculated based on the quantifier fragment
only (m/z 166.1 →74). LOD and LOQ for CML were determined by spiking different
amounts of CML to 25 µg of raw milk protein. LOD and LOQ of 4 and 8 ng CML per
mg protein were defined. These values were equivalent to 20 and 40 pg of CML on
column. Thus, a selective and validated method was developed to detect the major
CHAPTER 4. SUMMARY 143
glycation and oxidation products LacLys, CML and MeSO in dairy products. This
sensitive method allows the detection and the quantitation even of low levels of
modifications as present in mildly processed milk.
In the last part of this work, the method was employed for the quantitative
measurement of LacLys, CML and MeSO in different dairy products. The analysis
involved raw milk, mildly heat-treated milk like pasteurized milk and severely heat-
treated milk like sterilized milk. Additionally, different infant formulas were analyzed.
The concentrations of the analytes in cow milk were clearly dependent on the
applied heat treatment. The lowest levels of modifications were detected in raw milk
and the highest levels were found in sterilized as well as in condensed milk. One
exception was MeSO because its content in raw milk was higher than in mildly heat-
treated milk. High modification rates, especially of oxidation were found in infant
formulas with a tendency to higher values in hypoallergenic formulas compared to
regular formulas.
High modification rates of methionine and lysine block their physiological use as
essential amino acids and decrease the protein digestibility. Monitoring of LacLys,
CML and MeSO by the newly developed LC-ESI-MS-MS methods in dairy products
thus, allows evaluation of their nutritional value and assessment of the production
parameters.
CHAPTER 5. ZUSAMMENFASSUNG 144
5 Zusammenfassung
Die Maillard Reaktion umfasst eine Reihe chemischer Reaktionen zwischen
Aminogruppen und Carbonylverbindungen, die zur Bildung von vielfältigen Maillard-
Reaktionprodukten (MRPs) führen. Diese so genannte nicht-enzymatische Bräunung
ist häufig erwünscht und verleiht vielen Lebensmitteln, wie Kaffee, Backwaren und
gebratenem Fleisch ihren charakteristischen Geruch und Geschmack sowie eine
braune Farbe. Andererseits kann die Maillard-Reaktion zur Veränderung der
Proteinstruktur und der Proteinfunktion sowie zur Entstehung von toxischen
Verbindungen führen. Die Maillard-Reaktion läuft auch im menschlichen Körper ab
und führt zur Bildung von so gennanten „Advanced Glycation End-products (AGEs)“.
Die Akkumulation von AGEs in verschiedenen Geweben wird mit dem
Alterungsprozess und verschiedenen Komplikationen von Diabetes mellitus wie z.B.
Nephropathie, Retinopathie und Neuropathie in Zusammenhang gebracht.
Auch andere Krankheiten wie Alzheimer, Katarakt und Atherosklerose stehen in
Verbindung mit der Bildung von AGEs. Über die Nahrung aufgenommene MRPs
können während des Verdauungsprozesses resorbiert werden, was zu einer
Erhöhung der Konzentrationen von AGEs im Serum führt. Aufgrund der strukturellen
Ähnlichkeiten zwischen MRPs und AGEs wird diskutiert, dass besonders bei
Patienten mit Nierenkrankheiten der Konsum von MRP-reicher Nahrung an der
Entwicklung von verschiedenen Komplikationen beteiligt sein könnte. Zusätzlich
können die von Lebensmitteln stammenden Maillard-Produkte, die nicht resorbiert
werden, die intestinale Immunreaktionen beeinflussen und an der Pathogenese der
chronisch-entzündlichen Darmerkrankung beteiligt sein.
Durch die Anwesenheit von Laktose und Proteinen sowie den neutralen pH-Wert
stellt Milch bei der Verarbeitung und der Lagerung ein ideales Medium für die
Maillard-Reaktion dar, so dass es zur Bildung von verschiedenen Produkten wie
Laktulosyllysin (LacLys) und Carboxymethyllysin (CML) kommt. Parallel zur
CHAPTER 5. ZUSAMMENFASSUNG 145
Glykierung, unterliegen Milchproteine auch verschiedenen Oxidationsreaktionen,
wobei die Aminosäuren Tryptophan, Methionin und Cystein besonders betroffen
sind. Glykierung und Oxidation führen zum Abbau der essentiellen Aminosäuren und
zur Reduzierung der Proteinverdaubarkeit, wodurch diese Prozesse den Nährwert
der Milch mindern. Auch die funktionellen Eigenschaften der Molkenproteine, welche
in der Lebensmittelindustrie weit eingesetzt werden, können durch Glykierung und
Oxidation geändert werden.
Das Ziel dieser Arbeit war die Detektion und die Quantifizierung von verschiedenen
Glykierungs- und Oxidationsprodukten in Milch mittels
Hochleistungsflüssigchromatographie gekoppelt mit Elektrospray-Ionisation und
Tandem-Massenspektrometrie (LC-ESI-MS-MS). LC-ESI-MS-MS ist ein
leistungstarkes Analyzenverfahren für die Qualifizierung, da die Analyten über ihre
Produktionenspektren eindeutig identifiziert werden können. Weiterhin, stellt die
Tandem-Massenspektrometrie einen selektiven und empfindlichen Detektor zur
Quantifizierung dar, selbst wenn der Analyt in einer komplexen Matrix, wie
beispielweise einem Lebensmittel, zu untersuchen ist.
Zunächst wurde versucht, die Referenzesubstanzen von den wichtigsten
Glykierungs- und Oxidationprodukten zu synthesiziern, die in Milchprodukten vorher
identifiziert worden waren: LacLys, CML, Methioninsulfoxid (MeSO), Lysin-Aldehyd
und Cysteinsulfensäure (CysSOH). Die Synthese von LacLys erfolgte durch die
Reaktion von FMOC-Lys und Lactose unter Stickstoffatmosphäre. Anschließend
wurde FMOC-LacLys mit Hilfe einer ZIC-HILIC Säule aus der Reaktionsmischung
isoliert und die Schutzgruppe entfernt. Die Reinheit von LacLys wurde durch
qualitative 1H-NMR (6.13 %) bestimmt. Auf diese Weise konnte zum ersten Mal ein
Standard mit einem definierten Gehalt von LacLys synthetisiert werden. CML und
MeSO wurden mittels Alkylierungs- (Iodessigsäure) und Oxidationsreagenzien
(H2O2) synthetisiert. Allerdings wurden für die weiteren Untersuchungen
kommerzielle Standards von CML und MeSO verwendet. Lysin-Aldehyd und
CysSOH konnten nicht in ausreichender Reinheit synthetisiert werden, weil ihre
hohe Reaktivität zum Abbau während die Aufreinigung geführt hat. Die Standards
von 5-Hydroxytryptophan (5-OH-Trp) und Ornithin waren kommerziell erhältlich.
Anschließend wurde eine Methode entwickelt, um Glykierungs- und
Oxidationsprodukte, für die ein Standard zur Verfügung stand, zu detektieren. Der
Nachweis erfolgte in α-Lactalbumin, das in einem Milchmodell erhitzt worden war
CHAPTER 5. ZUSAMMENFASSUNG 146
und in kommerziell erhältlichen Milchprodukten. Nach der Entfettung und Entfernung
von Laktose wurden die Analyten aus den Milchproteinen durch eine komplette
enzymatische oder saure Hydrolyse freigesetzt und anschließend mittels LC-ESI-
MS-MS bestimmt. Die Parameter des Massenspektrometers, welche vom Fluss bei
der Flüssigchromatographie abhängen und diejenigen, welche von den
Verbindungen abhängig sind, wurden optimiert, um die maximale Intensitäten für
LacLys, CML, MeSO, 5-OH-Trp und Ornithin zu erreichen.
Aufgrund der hohen Polarität von LacLys and CML, weisen beide Verbindungen
kaum Retention an C18 Säulen auf. Deshalb wurde Nonafluropentansäure als
Ionenpaar-Reagenz verwendet, um die Retention der beiden Substanzen zu
erhöhen.
5-OH-Trp und Ornithin konnten weder im Milchmodell noch in Milchproben detektiert
werden. Auch nach der Oxidation von α-LA mittels Wasserstoffperoxid bei 120 °C
wurde nur MeSO detektiert. Die Analyten LacLys, CML and MeSO wurden in den
Milchmodellen detektiert, die bei 60 °C für 14 Tage n erhitzt wurden.
Allerdings konnten CML und LacLys auf einer C18 Säule nicht getrennt werden.
Auch die Detektion von CML in Milchproben, die mild erhitzt wurden, wie
beispielweise UHT-Milch, gestaltete sich als schwierig, da CML nur in geringen
Mengen gebildet wird. Um die Trennung zu verbessern und die Sensitivität zu
steigern, wurde statt einer C18 Säule eine ZIC-HILIC Säule benutzt. Durch eine
verbesserte Sensitivität kann weiterhin die Menge an Milchproteinen, die auf die
Säule geladen wird, reduziert werden. Die Anwendung dieser hydrophilen
stationären Phase hat eine vollständige Trennung zwischen CML und LacLys
ermöglicht. Weiterhin wurde die Signalintensität um das 3-5-fache gesteigert.
Allerdings zeigte die Signalintensität von LacLys in den Milchproben hohe Variation
an der ZIC-HILIC Säule im Vergleich zu der C18 Säule. Weiterhin wurde MeSO
lediglich in den Milchproben detektiert und die Variation ihre Signalintensität war an
der C18 und ZIC-HILIC Säule gleichartig. Aus diesem Grund wurde die ZIC-HILIC
Säule für weitere Untersuchungen von CML und die C18 Säule für weitere
Untersuchungen von LacLys and MeSO gewählt.
Als nächstes wurde die Proteinhydrolyse untersucht und optimiert, um die
maximale Freisetzung der Analyten MeSO, CML und LacLys von Milchproteinen zu
erreichen und, gleichzeitig, eine artifizielle Bildung zu vermeiden. Die Analyze der
Analyten mittels LC-ESI-MS-MS und Ninhydrin-Assay haben gezeigt, dass die
CHAPTER 5. ZUSAMMENFASSUNG 147
Zunahme der Hydrolysedauer die Freisetzung der Analyten erhöht (72 h gegenüber
27 h Hydrolyse). Gleichzeitig führte die Reduktion von LacLys mit Natriumborhydrid
zum unreaktiven Alkohol zu keiner bedeutenden Abnahme des CML-Werts. Diese
Ergebnisse zeigen, dass die artifizielle Bildung von CML durch oxidative Spaltung
des LacLys während der enzymatischen Hydrolyse vernachlässigt werden kann.
Bedingt durch die Autohydrolyse der Enzyme, insbesonders von Pepsin, wurde
MeSO in der Blindprobe der enzymatischen Hydrolyse detektiert. Deshalb wurde
dieser Blindwert bei der späteren Quantifizierung berücksichtigt. Die Verwendung
einer Festphasenextraktion (SPE) zur Entfernung der Enzyme und zur Aufreinigung
der Proben hatte einen Verlust der Analyten, besonders von LacLys zur Folge.
Deshalb wurde die Ultrafiltration einer SPE vorgezogen.
Vor der sauren Hydrolyse musste LacLys mittels Natriumborhydrid reduziert werden,
um die artifizielle Bildung des CML während der sauren Behandlung zu vermeiden.
Als Folge davon verhindert die Reduzierung den Nachweis und die Quantifizierung
von LacLys paralell zu CML und MeSO. Weiterhin konnte LacLys von
Natriumborhydrid nicht vollständig reduziert werden. Folglich konnte die Bildung von
CML aus LacLys nicht vollständig verhindert werden, was eine Überschätzung von
CML nach sich ziehen würde. Deshalb wurde auf die saure Hydrolyse verzichtet.
Weiterhin wurde eine deutliche Minderung des MeSO-Signals bemerkt, wenn
Natriumborhydrid vor der sauren Hydrolyse verwendet wurde. MeSO könnte in
Gegenwart von Natriumborhydrid weitere Reaktionen eingehen.
Die Analyten LacLys, CML und MeSO wurden durch das
Standardadditionverfahren quantifiziert, da für LacLys und MeSO keine
isotopenmarkierten Standards verfügbar sind, die als interne Standards benutzt
werden können. Für die Validierung wurde die Wiederholbarkeit von Messungen von
Proben mit hohen und niedrigen Modifikationsraten innerhalb eines Tages und
zwischen verschiedenen Tagen bestimmt. Außerdem wurden die Wiederfindung
sowie die Nachweis- und Bestimmungsgrenze für jeden Analyten bestimmt. Durch
die Identifizierung jedes Analyten über zwei charakteristische Massenübergänge
wurde eine hohe Selektivität der Methode erreicht. Die LC-ESI-MS-MS Methode
zeigte eine gute Präzision. Die Variationskoeffizienten von 4 bis 8 Proben, welche
die Analyten LacLys, CML and MeSO enthielten, lagen innerhalb eines Tages und
zwischen verschiedenen Tagen unter 10 %. Um die Wiederfindung zu untersuchen,
wurden drei unterschiedlich hohe Konzentrationen jedes Analyten unmittelbar vor
CHAPTER 5. ZUSAMMENFASSUNG 148
der enzymatischen Hydrolyse zu UHT-Milch zudotiert. Die Messungen erzielten
meist eine sehr gute Wiederfindung. Beispielsweise lagen die Wiederfindungen von
CML in einem optimalen Bereich zwischen 96,6±9,4 % und 101±5,4 %. Die
Wiederfindungen von LacLys lagen zwischen 110,5±9,6 % und 111,6±10,6 %,
während die Wiederfindungen von MeSO zwischen 107,8±10,6 % und 113,1±10,8
% lagen. Da die Wiederfindungsraten alle unter 115 % lagen, wurden sie als
akzeptabel angesehen.
Die Nachweisgrenze (LOD) und die Bestimmungsgrenze (LOQ) von LacLys wurden
ermittelt, indem die UHT-Milchproben bis zum Bereich, in welchem das Signal-
Rausch-Verhältnis bei ≤ 3 (qualifier) bzw. ≤ 10 (quantifier) lag, verdünnt wurden.
LOD und LOQ von LacLys lagen bei 4 bzw. 6,5 pg LacLys on column und von MeSO
bei 1 bzw. 2 pg on column, wobei diese Werte nur über den Quantifier von MeSO
(m/z 166,1→ 74) berechnet wurden. LOD and LOQ von CML wurden über ein
Zusatz bestimmter Mengen von CML zu 25 µg Rohmilchprotein bestimmt. Es
wurden LOD und LOQ von 4 und 8 ng pro mg Protein bestimmt, was 20 und 40 pg
CML on column entsprachen. Die auf diese Weise validierte Methode wurde
eingesetzt, um die wichtigsten Glykierungs- und Oxidationsprodukte LacLys, CML
und MeSO in Milchprodukten nachweisen zu können. Durch die hohe Selektivität der
Methode können auch geringe Mengen an Modifikationen in schonend verarbeiteter
Milch detekiert und quantifiziert werden.
Im letzten Teil der Arbeit wurde die Methode angewendet, um die Analyten
LacLys, CML und MeSO in Milchprodukten zu quantifizieren. Dazu wurden
verschiedene Proben wie z.B. Rohmilch, schonend erhitzte Milch wie pasteurisierte
Milch sowie stark erhitzte Milch wie Sterilmilch analysiert. Zusätzlich, wurden auch
verschiedene Proben von Babymilchnahrungen analysiert. Die
Analytenkonzentrationen in den Proben waren eindeutig von der angewandten
Prozessierung abhängig. Der niedrigeste Gehalt an Modifikationen wurde in
Rohmilch detektiert und die höchsten in Sterilmilch und Kondensmilch. Die einzige
Ausnahme stellt MeSO dar. Hier war der Gehalt in Rohmilch höher als der Gehalt in
schonend erhitzter Milch. Hohe Modifizierungsraten, vor allem durch Oxidation,
wurden in Babymilchnahrung gefunden. Dabei wurden tendenzielle höhere Gehalte
in hypoallergener als in normaler Babymilchnahrung gefunden.
Hohe Modifizierungsraten von Methionin und Lysin verhindern ihre physiologische
Verwertung als essentielle Aminosäure und reduzieren die Proteinverdaubarkeit.
CHAPTER 5. ZUSAMMENFASSUNG 149
Durch die Überwachung der LacLys-, CML- und MeSO-Gehalte mit Hilfe der neu
entwickelten LC-ESI-MS-MS Methode, kann die physiologische Wertigkeit der
Proben evaluiert und die Produktionsparameters überprüft werden.
150
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List of Tables
Table 1: Milk components and their concentrations in whole milk (Haug, Hostmark et al. 2007) ........................................................................................................... 11
Table 2: Composition of casein micelles (Belitz. H. D 2009) .................................... 12
Table 3: Composition, abundance and biological function of whey proteins. Adapted from (Wit 1998) ................................................................................................ 13
Table 4: Amount of methionine (0.25 M), HCl and H2O2 (30%) used to form MeSO 46
Table 5: Optimized MS-parameters dependent on the LC-flow ................................ 54
Table 6: Monitored transitions of LacLys, 5-OH-Trp, CML and MeSO and the optimized MS-parameters dependent on these compounds ............................ 55
Table 7: Mass spectrometric compound-dependent parameters of ornithine ........... 82
Table 8: The quantifier and qualifier fragments of LacLys, MeSO and CML ............ 97
Table 9: Within-day repeatability of the method for MeSO, LacLys and CML ........ 101
Table 10: Between-day repeatability of the method for MeSO, LacLys and CML .. 102
Table 11: Recovery of LacLys ................................................................................ 103
Table 12: Recovery of MeSO ................................................................................. 104
Table 13: Recovery of CML .................................................................................... 105
Table 14: MeSO, LacLys and CML contents in some dairy products determined by LC-ESI-MS-MS .............................................................................................. 110
Table 15: MeSO content in different milk products reported in literature ................ 113
Table 16: CML content in different milk products reported in literature .................. 113
Table 17: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the within-day repeatability for UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 50 µg/ml ................................................................................ 133
Table 18: Concentrations of LacLys, MeSO and CML which were used for standard addition to investigate the between-day repeatability in the samples of UHT milk and heated UHT milk. The concentration of the sample, in which the analytes should be quantified, was 75 µg/ml ................................................................ 134
Table 19 : The amounts of LacLys, CML and MeSO in 30 µl of the standards solutions which were spiked to each protein sample before enzymatic hydrolysis ....................................................................................................... 135
Table 20: The concentrations which were used for standard addition during the determination of the recovery of of LacLys, MeSO and CML. The concentration of the sample in which CML should be quantified was 150µg/ml and it was 25µg/ml to quantify LacLys and MeSO .......................................................... 135
160
Table 21: Amounts of CML which were spiked into 25 µg of raw milk protein hydrolyzate in order to determine LOD and LOQ ........................................... 136
Table 22: Concentrations of milk samples which were quantified. The concentration varied between 25µg/ml to quantifiy both MeSO and LacLys to 900µg/ml to quantify CML in raw milk proteins .................................................................. 137
161
List of Figures
Figure 1 : The early phase of the Maillard reaction .................................................... 2
Figure 2: Phases of the Maillard reaction ................................................................... 3
Figure 3: Some structures of AGEs ............................................................................ 5
Figure 4: Early stage of the Maillard reaction in milk ................................................ 18
Figure 5: Formation of CML through different pathways (Ferreira, Ponces Freire et al. 2003) ........................................................................................................... 19
Figure 6: Degradation of Amadori product under acidic, neutral or alkaline conditions ......................................................................................................................... 20
Figure 7: Schematic illustration of tandem mass spectrometry in MRM mode [adapted from (Zimmer 2003)] ......................................................................... 25
Figure 8: Degradation of LacLys via oxidative cleavage, acid hydrolysis and heating in the presence of oxalic acid ........................................................................... 28
Figure 9: Products of methionine oxidation .............................................................. 30
Figure 10: Lysine oxidation to form lysine aldehyde ................................................. 30
Figure 11: Some cysteine oxidation products .......................................................... 32
Figure 12: The chromatogram of the reaction mixture of FMOC-lys and lactose shows the formation of a new product with m/z of 693 indicating the formation of FMOC-LacLys .............................................................................................. 34
Figure 13 : MS2 spectrum of the synthesized LacLys .............................................. 34
Figure 14: Fragmentation pathway of lactulosyllysine .............................................. 35
Figure 15: Illustration of affinity chromatography. [Adapted according to the handbook (Affinity Chromatography) from Amersham Biosciences] ................ 37
Figure 16: The interaction of m-aminophenylboronic acid with lactulosyllysine ........ 38
Figure 17: Functional group packed in ZIC-HILIC columns ...................................... 40
Figure 18: Chromatogram of the reaction mixture of FMOC-Lys and lactose separated on a ZIC-HILIC column ................................................................... 40
Figure 19: Chromatogram and mass spectrum of FMOC-LacLys obtained after fractionation of the reaction mixture of FMOC-Lys and lactose on a ZIC-HILIC column. ............................................................................................................ 41
Figure 20: Synthesis of LacLys ................................................................................ 41
Figure 21: 1H-NMR spectrum of the synthesized LacLys shows the signal of α-C and ε-C of lysine as well as the expected signal of the bond between lactose and lysine ................................................................................................................ 43
Figure 22: Signal of the aldehyde proton of DMF which appeared at 7.926 ppm ..... 44
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Figure 23: Chromatogram of the reaction mixture of Cbz-lysine and iodoacetic acid shows the formation of Cbz-CML ..................................................................... 44
Figure 24: Synthesis of CML .................................................................................... 45
Figure 25: Chromatogram of the reaction mixture of methionine and H2O2 which was incubated at 21 °C. The chromatogram shows the form ation of MeSO and MeSOO. ........................................................................................................... 46
Figure 26: Chromatogram and mass spectrum of the reaction mixture of Cbz-lysine and urea extract of ESM shows unreacted Cbz-lysine as well as the formation of unknown compound. The unknown compound showed m/z 280.2 corresponding to the protonated Cbz-lysine aldehyde with a mass difference of -1Da compared to Cbz-lysine........................................................................... 48
Figure 27: Chromatogram of the reaction mixture of cysteine and H2O2 (recorded at 250 nm) shows the scavenger methionine and a new product with m/z 241.2 which was assigned to cystine ......................................................................... 49
Figure 28: Reaction of lysine with lactose during heating of whey protein ............... 51
Figure 29: Some protein oxidation products ............................................................. 52
Figure 30: Ion optic in the vacuum chamber of an MS-MS triple quadrupole system (Manual user API 4000 QTRAP, 2008). Q1: first quadrupole; Q2: collision cell; Q3: third quadrupole; ST: stubby lens between Q0 and Q1; ST2: stubby lens between Q1 and Q2; ST3: stubby lens between Q2 and Q3; RF: radio frequency; CEM: channel electron multiplier .................................................... 54
Figure 31: Product ion mass spectrum of CML and the corresponding fragments ... 56
Figure 32: Product ion mass spectrum of MeSO and the corresponding fragments 56
Figure 33: Product ion mass spectrum of LacLys and the corresponding fragments 57
Figure 34: Product ion mass spectrum of 5-OH-Trp and the corresponding fragments ......................................................................................................................... 57
Figure 35: LC-MS-MS chromatogram of the standards mixture, obtained in MRM mode, on a C18 column. The standard mixture contained MeSO, 5-OH-Trp, LacLys and CML .............................................................................................. 59
Figure 36: A centrifugal concentrator device which was used for the clean-up of the enzymatic hydrolyzate ..................................................................................... 60
Figure 37: Extracted ion chromatogram (XIC) of glycated α-LA shows the formation of CML (right). LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of LacLys (left). α-LA and lactose were heated in a milk matrix at 60 °C for 3 (A), 7 (B) and 14 (C) da ys and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. CML formation increased during the whole incubation period, while the highest signal of LacLys was obtained after 7 days of incubation. ........................................................................................... 62
Figure 38: LC-MS-MS chromatogram of glycated α-LA obtained in MRM mode shows the formation of MeSO. α-LA and lactose were heated in a milk matrix at 60 °C for 3 (1 a), 7 (2 a) and 14 (3 a) days and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. MeSO was detected in native α-LA (standard) and in α-LA heated without lactose for 3 (1 b), 7 (2 b) and 14 (3 b) days ................................................................................................................. 63
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Figure 39: LC-MS-MS chromatogram of oxidized α-LA obtained in MRM mode shows the formation of MeSO. 5-OH-Trp was not detected. α-LA and H2O2 were heated in a milk matrix for 10, 20 and 30 min at 120 °C (1a, 2a and 3a, respectively) and subjected to enzymatic hydrolysis prior to LC-MS-MS analysis. LC-MS-MS spectra obtained in MRM mode of native α-LA (standard) and α-LA heated for 10, 20 and 30 min at 120 °C (1b, 2b and 3 b, respectively) which were used as control .............................................................................. 65
Figure 40: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of MeSO and LacLys. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. LacLys and MeSO were identified by their specific mass transitions (LacLys: m/z 471.1→225.1, m/z 471.1→128.1, m/z 471.1→84.1; MeSO: m/z 166.1→74, m/z 166.1→56, m/z 166.1→102.1). ............................ 69
Figure 41: The increase of the MeSO signal and the stability of the unknown peak during heat treatment of milk............................................................................ 70
Figure 42: Relative signal of MeSO and LacLys in different milk samples ............... 71
Figure 43: Formation of MeSO during the different steps of enzymatic protein hydrolysis. A blank without sample protein was subsequently hydrolysed by pepsin, pronase and aminopeptidase. The enzymes remained in the samples throughout hydrolysis (A). Pepsin was removed before further hydrolysis (B). Pepsin and pronase were removed before further hydrolysis (C) .................... 72
Figure 44: The decrease of MeSO and LacLys signal intensities after the removal of pepsin from milk hydrolysate after 24 hours of enzymatic hydrolysis ............... 72
Figure 45: LC-MS-MS chromatogram of sterilized milk obtained in MRM mode shows a clear formation of MeSO and LacLys and a possible formation of CML. After the removal of lactose and fat, milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. MeSO and LacLys were identified by their specific mass transitions. Only one mass transition of CML was detected (m/z 205.1→ 84.1). ............................................................ 73
Figure 46: LC-MS-MS chromatogram obtained in MRM mode of 5-OH-Trp, MeSO, CML and LacLys (standard mixture) on a ZIC-HILIC column 150*2.1mm, 3.5 µm .................................................................................................................... 75
Figure 47: Comparison of the signal intensities of the standards MeSO, CML, 5-OH-Trp and LacLys on C18 and ZIC-HILIC columns ............................................. 76
Figure 48: LC-MS-MS chromatogram of UHT milk obtained in MRM mode shows the formation of CML. Milk proteins were subjected to enzymatic hydrolysis prior to the LC-MS-MS analysis using a ZIC-HILIC column. An unknown peak with the same specific mass transitions (m/z 205.1→ 84.1, m/z 205.1→ 130.1) appeared after 5 min. ....................................................................................... 77
Figure 49: The increase of CML signal and the stability of the signal of the unknown compound during heat treatment of milk .......................................................... 78
Figure 50: Ornithine formation from arginine ............................................................ 81
Figure 51: Some derivatives of imidazolone formed from the reaction of arginine and dicarbonyls ....................................................................................................... 82
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Figure 52: Product ion scan of ornithine shows the fragments and their proposed structures ......................................................................................................... 83
Figure 53: LC-MS-MS chromatogram of the standard ornithine (A) and UHT milk proteins (B) obtained in MRM mode. Milk proteins were subjected to enzymatic hydrolysis prior to LC-MS-MS analysis using a C18 column. Panel B shows a peak at the retention time of ornithine which showed only one of its characteristic fragments (m/z 133.1→ 70.1). The second fragment, which was characteristic for the standard (m/z 133.1→ 116), was not detected ............... 84
Figure 54: Peak areas obtained for the quantifier fragment (m/z 133.1→ 70.1) of the compound detected at the retention time of ornithine ...................................... 84
Figure 55: Reduction of lactulosyllysine with borohydride to form a product stable during acidic hydrolysis .................................................................................... 87
Figure 56: Protein samples hydrolyzed with a cocktail of enzymes for 72 hours or 27 hours were analyzed by ninhydrin assay. Enzymatic hydrolysis of sterilized milk and UHT milk heated for 10, 20, 30 and 60 min (120 °C) for 72 hours resulted in higher absorbance indicating a higher content of free amino acids .............. 88
Figure 57: LC-MS-MS signal intensity of MeSO, CML and LacLys obtained after the enzymatic hydrolysis of sterilized milk sample for 72 hours or 27 hours .......... 88
Figure 58: Decrease of the signal intensity of the standards MeSO, CML and LacLys after purification by SPE column compared to control. The samples were analyzed by LC-MS-MS. The highest loss was noted for LacLys .................... 89
Figure 59: Signal intensity of MeSO, CML and LacLys detected by LC-MS-MS in UHT milk which was heated for 60 min at 120 °C. The sample was purified either by ultrafiltration or by SPE, and then dried by Speed-Vac ..................... 90
Figure 60: Signal intensity of CML detected by LC-MS-MS in the samples of heated UHT milk which were dried either by lyophilization or Speed-Vac after purification ....................................................................................................... 90
Figure 61: Decrease of the signal intensity of CML in heated UHT milk and sterilized milk in which LacLys was reduced by NaBH4 prior to enzymatic hydrolysis. The samples were analyzed by LC-MS-MS ............................................................ 91
Figure 62: LC-MS-MS signal intensity of the analytes MeSO, CML, and LacLys which were detected in (A) HA-infant powder formula and (B) sterilized milk. The diagrams show an increase of CML in the acidic hydrolyzate compared to both enzymatic hydrolyzates. LacLys was not detected after the reduction by NaBH4 in the acidic nor the enzymatic hydrolyzates. Clear decrease of MeSO signal was found in the acidic hydrolyzate ....................................................... 92
Figure 63: Protein samples of UHT milk heated for 10, 20, 30 and 60 min (120 °C), sterilized milk, HA-infant powder formula and lactalbumin. The samples were analyzed by ninhydrin assay after acidic or enzymatic hydrolysis for 72 hours. The absorbance at 570 nm of the enzymatic hydrolyzate (3µg protein) was higher compared to the acidic hydrolyzate (10µg protein) ............................... 93
Figure 64: Schematic illustration shows the method of standard addition. (http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/3/anc/croma/kalibrierung.vlu/Page/vsc/de/ch/3/anc/croma/datenauswertung/quantitativ/standardaddition/standardadditionm80ht0801.vscml.html, date 9/2/2011) .................................. 100
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Figure 65: An unknown peak appeared at 8.08 min and showed the quantifier fragment of LacLys ........................................................................................ 106
Figure 66: The chamber for solid phase extraction with the cartridges .................. 130