Günther K. BonnInstitute of Analytical Chemistry and Radiochemistry, Leopold-Franzens

University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria

Austrian Drug Screening Institute – ADSIInnrain 66, A-6020 Innsbruck, AustriaOffice: Sensengasse, Vienna, Austria

Grundlagen und Anwendung moderner Trennverfahren

Teil 1 - Extraktion

Genome Transcription Proteome Metabolome

Transcription TranslationPost-translational Modification

DNA RNA Proteins Modified Proteins Biological Function

Environment

~30,000 Genes > 1,000,000 ProteinsPathways1500-2000Metabolites

Herausforderung Moderner TrennverfahrenGenomics – Proteomics - Metabolomics

2

Genomics

Proteomics

MetabolomicsStationary PhasesMaterial Science

Separation

InstrumentationMS, NMR, Spectroscopy

New Analytical ApproachesBionalysis

DiagnosticsBiomarker Discovery

Purification and Enrichment

Data InterpretationData Analysis

Therapy

Omics - Overview

Stationary PhasesMaterial Science

Separation

Omics - Overview

N Ca

Traditional techniques

(magnetic resonance,Ultrasound, CT…)

SAMPLEbiofluids

Genomic

Proteomic

MetabolomicA

naly

sis NMR, LC-MS,GC-MS, PCR,

Imaging, rt-PCR,ELISA, Chip-Array,…

Screening

DiagnosticsBiomarker Discovery

Therapy

Fällung• Spezifische Fällung von

Phosphoproteinen und Phosphopeptiden

Extraktion

• Festphasenextraktion• Anreicherung von

Proteinen und Peptiden• Entsalzung

Chromato-graphie

• Affinität• Chromatographie• HPLC/UHPLC

Elektro-phorese

• Gel Elektrophorese• Kapillarelektrophorese

Massen-spektro-metrie

• Quadrupol• Ionenfalle• TOF

Moderne Trennverfahren

Überblick

5

What is proteomics

Proteomics is a recent approach to studying proteins

The study of all proteins created or used by an organism

Goal: Identification, quantification and characterisation of all proteins in a given biological systems at a given time point.

6

Proteomics contributes to our understanding of health and disease

Up- and/or down-regulation of protein abundance Protein-protein interactions Post-translational modifications Leakage of proteins from diseased cells or tissues Biomarker discovery

7

Bioanalysis - Serum - Biomarker

8

Bioanalysis - Complexity of Human Serum

22 proteins are approx. 99% of the whole serum proteome

90% 10%

9

Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845-867

Protein abundance in human plasmaDynamic Range

Warum das Proteom untersuchen ?

Das Genom sagt, was potentiell in einer Zelle passieren könnte,

das Proteom sagt, was tatsächlich passiert.

11

12

Genomics/Proteomics

13

Transgenomic Wave-System - Mutation Analysis

Transgenomic Wave HPLC-System

Bonn, Gunther; Huber, Christian; Oefner, Peter. Separation of nucleic acid fragments with alkylated nonporous polymer beads. PCT Int. Appl. (1994), 30 pp.

Mutation Detection by Denaturing HPLC using PackedColumns and Monolithic Capillaries

Packed Column Monolithic Capillary

column: 50 x 4.6 mm I.D. 50 x 0.2 mm I.D.flow rate: 1 ml/min 3.0 µl/mininjection vol.: 5 µl 500 nl

0

157.8 °C

0 5 10time [min]

UV,

254

nmm

AU

UV,

254

nmm

AU

time [min]

0

156 °C

0 2 4 6

Festphasenextraktion - Prinzip

16

Festphasenextraktion - Prinzip

17

Nernst distribution coefficient

Konzentration des Analyten in der stationären Phase

Konzentration des Analyten in der mobilen Phase

Background of chromatographic methods

Important chromatography methods in proteomics

• Ion Exchange Chromatography(IEC)

• Size Exclusion Chromatography(SEC)

• Affinity Chromatography(AC)

• Reversed Phase Chromatography(RPC)

Ion Exchange Chromatography

• Separation is based on charge differences• Reversible interaction between oppositely charged solute and

chromatographic medium• Elution: increasing salt concentration or pH change• Solute molecules are eluted in a concentrated form• Ion exchange types:

– Anion Exchange Chromatography: negatively charged solute molecules compete with negatively charged mobile phase ions for the positively charged sites of the stationary phase

– Cation Exchange Chromatography: positively charged solute molecules compete with positively charged mobile phase ions for the negatively charged sites of the stationary phase.

Ion Exchange Chromatography

Carboxylic acidWeak cation

Sulfonic acidStrong cation

Tertiary amine

Secondary amine

Primary amineWeak anion

Quaternary AmineStrong anion

TypeFunctional groupType of Exchanger

Operating

N+ CH3

NH2

NH

N

SO3-

COO-

Size Exclusion Chromatography

• Solute molecules are separated by their size• Stationary phase has pores of well defined size• Retention is a function of solute penetration into the

pores that is proportional to the hydrodynamic volume of the solute

• No selective interaction with the stationary phase• Particularly useful for buffer exchange

Size Exclusion Chromatography

Affinity Chromatography

• Solute molecules are separated on the basis of specific reversible binding to an affinity ligand attached to the stationary phase.

• Utilizes very specific stationary phases such as antibodies, lectins, etc.

• Desorption is performed by adding a competitive ligand to the elution buffer system, or changing ionic strength, pH or polarity.

• The availability of the affinity ligand defines its applicability.

• Very specific for the solute molecule.

Affinity Chromatography

Most time a spacer is necessary to bind the affinity ligand to the stationary phase

Affinity ligands and applications

LIGAND • Avidin• Aprotinin• Biotin• Concanavalin A• Gelatin• Glutathione• Heparin• Iminoacetic acid• Lysine• Protein A• Phophorylethanolamine• Protein G• Protamine

APPLICATIONS• Biotin derivatives• Serine proteases• Avidin• Glycoproteins, Oligosaccharides• Fibornectine enzymes• Enzymes related to glutathione• Blood coagulation factors• Interferon, serum proteins• Plasminogen, polysaccharides• Human IgG• C-reactive protein• IgG immune complex• IgM

Curtesy of Dr. R. Bishoff

Reversed phase chromatography

• Solute separation is based on reversible hydrophobic interactions with a hydrophobic stationary phase

• Commonly used stationary phases are silica or polymer based with different chain length hydrocarbon ligands

• Due to strong binding, organic solvents are necessary for elution, sometimes with such additives as ion pairing agents.

• During RP-HPLC, proteins may get denatured or loose their biological activity

Je länger ein Stoff in der stationären Phase verbleibt, desto größer wird der

Kapazitätsfaktor und damit auch die Retentionszeit des Analyten. Der Kapazitätsfaktor

gibt an, um wieviel länger sich Moleküle an der stationären Phase im Vergleich zur

mobilen aufhalten. Mit Bruttoretentionszeit (tR) und Totzeit (t0) gilt:

Ein hoher Kapazitätsfaktor beschreibt ein hohes Retentionsverhalten!

Kapazitätsfaktor

Oasis Material (Waters)

Example

Oasis Material (Waters)Alternative to C18

IMACImmobilized metal-ion chromatography

Example

for specific binding of phosphopeptides!

Immobilized metal ion/Metal Chelate affinity chromatography is separation techniquethat is based on coordinate covalent binding between proteins and metal ions. Proteinshave a wide variety of amino acids composition which, in effect, generates a range ofdifferent affinities towards metal ions. However, not many naturally occurring proteinshave affinity for metal ions, so the technique is mainly used to purify recombinantproteins. For example proteins can be engineered to contain a poly-histidine tail(histidine can generally act as a ligand towards divalent metal cations). If the stationaryphase is immobilized with divalent metal cations, a mixture of proteins can beseparated based on their ability to interact with the metal ions. Those proteinscontaining a higher number of histidine residues would be able to bind to the columnmore tightly than those with fewer histidine residues.

Several different types of immobilized metal ion column have been developed toseparate various proteins (e.g. Fe, Co, Cd, Ni, or Zn). Protein separation in IMACgenerally depends on the strength of the metal ion-protein bond. Thus, choosing thetype of immobilized ion is crucial to the success protein separation. By far the mostwidely-used technique is to use an immobilized nickel column, and to engineer poly-histidine tags of six or more residues onto the recombinant proteins of interest. Onething to keep in mind is that the binding between metal ion and protein must bereversible, allowing elution of bounded protein at later steps. Three different elutionstrategies can be applied to IMAC competitive elution, stripping elution and pHAdjustment.

MACHEREY-NAGEL´s concept

Protino Ni-IDA/TEDProtino Ni-IDA/TED – purification of His-tag proteins

Protino Ni-TED

Protino Ni-IDA

TED (tris(carboxymethyl)ethylene diamine)

IDA (iminodiacetic acid)

MOACMetal Oxide Affinity Chromatography

O OP

O OR

TiO2 TiO2

Mechanism: Bridging Bidentate

for specific binding of phosphopeptides!

Example

DHB as “excluder“ or “displacer“

Karl Mechtler et. al

poly(divinylbenzene)

TiO2 < 100 nm

ZrO2 < 100 nm

Preparation of - Hollow MonolithTM

O OP

O OR

TiO2 TiO2Mechanism: Bridging Bidentate

Hollow MonolithTM

Example

Enrichment of Phosphopeptides

... embeddedTiO2/ZrO2

... Phosphorylated Peptides

Enrichment of in vitro phosphorylated ERK1 digest

MALDI MS spectra:

1.) before enrichment (A)

2.) after enrichment with poly(DVB)-TiO2/ZrO2 tips (B)

Signals at m/z 2252.25 and m/z 2332.23 correspond to phosphorylated peptides

Collaboration with Prof. Lukas Huber, Biocenter - Innsbruck

Mono-phosphorylated peptide (m/z 2252.25) identified as RIADPEHDHTGFLTEpYVATRW (SwissProt as database)

MS/MS analysis of enriched phosphopeptides from ERK1 digest

Identification of in vitro phosphorylated ERK1MS/MS Analysis

Activating: ACN/0.1% TFA twice

Equilibrating: 1) H2O/0.1% TFA two times. 2)50% ACN/0.1% TFA containing DHB (20 mg/mL)

Loading: 50% ACN/0.1% TFA containing DHB (20 mg/mL)

Washing: 50% ACN/0.1% TFA containing DHB (20 mg/mL) ,x5Additionally, two washing steps with 80% ACN/0.1% TFA and one washing step with deionized water were performed.

Eluting: 20% ACN/ 50 mM H3PO4, ca 1% NH4OH (pH 10.5)

matrix composition:10 mg/ml DHB80% ACN0.2% H3PO41 % TFA

0

1

2

3

4

5

4x10

Inte

ns. [

a.u.

]

2567.319

1709.807

1502.872

2963.477

2647.3481993.086

2077.040

0

1

2

3

4

5

4x10

Inte

ns. [

a.u.

]

Sample before enrichment

Sample after Ti/Zr-Tipenrichment.8 P-peptides detected

2586.314

ZipTip-MC tips,4 P-peptides detected

2647.1151

2

3

4

5

Inte

ns. [

a.u.

]

1400 1600 1800 2000 2200 2400 2600 2800

m/z

1501.669

1992.682

2587.218

x10 4

0

8 phosphopeptides in p57 protein could be detected after Ti/Zr-Tip enrichment, only 4 with ZipTip MC IMAC

1- 21 2567 GSYPYDVPDYASLEFTVLRPR phosphorylated1- 21 2646 GSYPYDVPDYASLEFTVLRPR 2xphosphorylated

Collaboration with Biocenter Innsbruck

P57 peptides detected after phosphopeptides enrichment#1_2183_ xxxxxx_HAp57wt +src _TiZr

Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence32 - 44 1485.7451 1484.7378 1484.7260 0.0119 0 R.SLFGPVDHEELSR.E 50 - 60 1272.6811 1271.6738 1271.6106 0.0633 0 R.LAELNAEDQNR.W 50 - 72 2883.3306 2882.3234 2882.2929 0.0304 1 R.LAELNAEDQNRWDYDFQQDMPLR.G Oxidation (M) 50 - 72 2963.3517 2962.3444 2962.2593 0.0852 1 R.LAELNAEDQNRWDYDFQQDMPLR.G Oxidation (M); Phospho (Y) 61 - 72 1709.6695 1708.6622 1708.6593 0.0029 0 R.WDYDFQQDMPLR.G Oxidation (M); Phospho (Y) 61 - 76 2076.8559 2075.8486 2075.8561 -0.0075 1 R.WDYDFQQDMPLRGPGR.L Oxidation (M); Phospho (Y) 77 - 92 1912.9939 1911.9867 1911.9003 0.0864 0 R.LQWTEVDSDSVPAFYR.E 77 - 92 1992.9086 1991.9013 1991.8666 0.0347 0 R.LQWTEVDSDSVPAFYR.E Phospho (Y) 266 - 278 1502.7793 1501.7721 1501.7582 0.0139 1 K.KLSGPLISDFFAK.R Phospho (ST) 287 - 312 2587.1004 2586.0931 2586.1422 -0.0490 0 K.SSGDVPAPCPSPSAAPGVGSVEQTPR.K Phospho (ST)

Automation of Sample Preparation

• Specific enrichment• Purification• Desalting

1. sample loading

laser

2. sample spotting

3. sample analysis 4. data processing

A collaboration with PhyNexus Inc., San Jose, CA, USA

α-caseinPhospho TiZrTiO2/ZrO2PhyNexus

ZipTipMC-Fe3+

Millipore

MonoTipTiO2

GL Sciences

TopTipTiO2

Glygen

TopTipZrO2

Glygen

total number ofphosphopeptides 20 7 11 11 9

ß-caseinPhospho TiZrTiO2/ZrO2PhyNexus

ZipTipMC-Fe3+

Millipore

MonoTipTiO2

GL Sciences

TopTipTiO2

Glygen

TopTipZrO2

Glygen

total number ofphosphopeptides 5 4 5 2 1

Comparative study with commercial products

+

+

[60]fullerenoacetylchloride[811,96]

[60]epoxy fullerene[736,64]

aminopropylSilica fullerene bonded Silica

aminopropylSilica

SiHO

SiOH2N Si

OH

OH

SiHO

SiHO

SiOH2N Si

OH

OH

SiHO

Synthesis of C60-Silica

Fullerene C60-amino silica

fullerene bonded Silica

Example

peptides with multiple phosphorylations – MS

monophosphorylated peptides

Single negatively charged group higher charge states in positive

ionization mode Single loss of phosphoric acid

peptides with multiplephosphorylated amino acids

multiple negatively charged moieties

low charge states preferred multiple losses of phosphoric acid

occur

PO

OHO

O

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

PO

OHO

OP

OOH

O

O

PO

OHO

O

PO

OHO

O

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica

peptides bind to C60-fullerene silica

1

2

Elution of multi-phosphorylated peptides with 0.1 M NH4OH

3Elution of mono-phosphorylated peptides with 20% ACN and 1% TFA

4

Elution of non-phosphorylated

peptides with 80% ACN and 1 % TFA

Figure 1: Batch experiment, (A) fractionation of multi-phosphorylated peptide by elution with 0.1 M NH4OH and (B) fractionation of mono-phosphorylated peptide by elution 1% TFA in 20% ACN

Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica

Elution with 0.1 M NH4OH

Elution with 1% TFA in 20% ACN

Elution with 1% TFA in 80% ACN

Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica

α-casein tryptic digest

Protein Verdau

DTT Dithiothreitol

68

Protein Verdau

(DTT Dithiothreitol)

(CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate)

69

Trypsin Mostly, proteomics scientists use trypsin to

generate peptides− Trypsin cleaves after the positively charged amino

acids arginine [R] and lysine [K], generating multiply positively charged peptides

− Most tryptic peptides have an ideal size for mass spectrometry (500-4000 Da)

− Tryptic peptides fragment very well in the mass spectrometer, generating good quality sequence information

Protein Verdau

70

Peptide Mass Fingerprint

++

++

+++

trypsin

Inte

nsity

m/z

++

++

+++

71

Monolithic Extraction Tips for Enzymatic Digestion

Trypsin in TrisHCl-buffer =

2h , 5°C

GMADVBAIBNToluolDecanol

=+

2h , 95°C

+

BSAα-caseine

+ DTT

Enzyme-Tip

=> =>

+ iodoacetamide

15mins, RT, in dark

15 mins, 60°C

15 mins, 20°C, in

dark

=>

30 mins (in-tip) 60 mins (literature)

DenaturationProtein

Monolithic Extraction Tips for Enzymatic Digestion

Microwave-Assisted In-Tip Digestion

Total Time 45 min!conventional digestion time: 6-10 h

Results - Comparison Study

α-casein digested, eluted from enzyme-Tips with Tip-Technology

Glygen Corp.

Tip

GL SciencesGlygen Corp.

Glygen Corp.

Company

0

10

20

30

40

50

60

70

80

90

a-casein myoglobin BSA

NT1CARTRY

NT1TRY

NT1C18TRY

MonoTip™

GMA/DVB-Tips

sequ

ence

cove

rage

[%]

Desalting of Proteins – Why?

Spin Columns

C18-reversed phase

dialysis

Extraction Tips

Spin Columns

ZipTips (Millipore)

Fullerene C60-Silica for Desalting Biological Samples

Proteines

MALDI-MS spectra of the peptides and proteins which were eluted from Kovasil 300 Å C60-Silica

breakthrough breakthrough

sample sample

elution elution

Peptides

5734.63

8565.632867.10

16952.4014306.6130

1.0

2.0

3.0

4.0

0

1.0

2.0

3.0

4.0

8566.35

14291.4292866.33 16943.00311466.50

0

1.0

2.0

3.0

4.0

1 0

A

B

C5734.73

4282.79

1296

.44

1347

.53

1619

.48

2094

.02

1757

.75

2466

.10

0.0

1.0

2.0

0.0

1.0

2.0

1347

.32

1619

.34

2465

.92

2093

.66

1758

.47

0.0

1.0

2.0

1046.54

1046.72

A

B

C

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

0.0800 1000 1200 1400 1600 1800 2000 2200 2400

m/z

immobilization washing elution

before

supernatant

elution

before

supernatant

elution

C60-amino-silica

Vallant Rainer M; Szabo Zoltan; Bachmann Stefan; Bakry Rania; Najam-ul-Haq Muhammad; Rainer Matthias; Heigl Nico; Petter Christine; Huck Christian W; Bonn Gunther K. Analytical chemistry (2007), 79(21), 8144-53.

Fullerene C60-amino silica for Desalting

C60-Amino Silica vs. ZipTip® C18 (Millipore)

ZipTips C18supernatant

ZipTips C18elution

1140

.52

1.0

2.0

3.0

4.0

0.0

0.2

0.4

0.6

0.8

5.0

0.0

Inte

nsity

. [a.

u.]

m/z

Phosphopeptide in supernatantNo immobilization on commercial ZipTip ® (Millipore)

Phosphopeptide removed after washingNo immobilization of phosphopeptide

C60-Amino Silicasupernatant

C60-Amino Silicaelution

1.0

2.0

3.0

4.0

5.0

1140

.49

0.0

0.05

0.10

0.15

0.20

0.25

1110 1120 1130 1140 1150 1160 1170 1180

0.0

No Phosphopeptide in supernatantQuantitative binding of phosphopeptideon C60-Amino Silica

Phosphopeptide eluted from C60-Amino Silica

Desalting of hydrophilic Phosphopeptides

Enrichment of Phenolic Compounds using Hexagonal Boron Nitride

Martin Fischnaller, Rania Bakry, Günther Bonn

Institute of Analytical Chemistry and RadiochemistryAustrian Drug Screening Institute – ADSI

University of Innsbruck, Austria

Crystalline forms of Boron Nitride

β or cubic BN

• diamond analog

• stable

• very hard

• black

γ or wurzite BN

• lonsdaleit analog

• only stable under high

pressure

α or hexagonal BN

• graphite analog

• chemically inert

• nontoxic

Boron Nitride, a Novel Material for the Enrichment and Desalting of Protein Digests and the Protein Depletion

N

BN

B

NB

NB

NB

NB

NB

N

B

BN

BN

BN

B

BN

BN

BN

N

BN

0.1446 nm

Covalent bonds

Van der Waalsbonds

0.33

31 n

m

Boron

Nitrogen

Structure of α-BN

Arnold F. Holleman, N. W. Lehrbuch der Anorganischen Chemie; de Gruyter: Berlin, 2007; Vol. 102.

δ 0.93e δ -0.93e

Wettability of α-BN

SEM picture of BN with 60 m² surface area.

Wettability of α-BN

Published in: Hui Li; Xiao Cheng Zeng; ACS Nano 2012, 6, 2401-2409.DOI: 10.1021/nn204661dCopyright © 2012 American Chemical Society

50-67° high wettability

86° low wettability

Snapshots of a water droplet on (A) an atomically BN sheet in the end of classical molecular dynamics

Boron Nitride, a Novel Material for the Enrichment and Desalting of Protein Digests and the Protein Depletion

LC-ESI-MS

Incubation with trypticdigests

MALDI-MS

Washingandenrichment

Elution of peptides

Analysis of desalted and protein free peptide solutions

N

BN

B

NB

NB

NB

NB

NB

N

B

BN

BN

BN

B

BN

BN

BN

N

BN

BN

Boron Nitride, a Novel Material for the Enrichment and Desalting

Bradykinin Substance P Bombesin LHRH Oxytocin

Recovery rates [%]

A B A B A B A B A B

BN 60 71.65 81.67 70.13 73.59 56.12 90.88 92.13 87.46 85.92 95.24

BN 20 98.83 93.74 96.90 90.13 98.71 96.23 95.18 91.01 84.50 92.66

BN NANO 96.75 94.46 108.52 91.30 89.36 95.70 97.07 93.44 91.39 92.85

Graphiteparticle 85.54 83.60 31.70 64.17 47.98 52.78 32.15 76.28 76.33 94.39

Graphite 15 17.62 36.90 14.08 16.78 12.65 17.39 25.70 25.21 49.27 77.04

Recovery Study using different Peptides

A = 60% ACN in 1% TFA and 1% H3PO4; B = 60% ACN in 1% formic acid

0

5

10

15

20

25

30

0 10 20 30 40 50 60 70

BN 60 m²/g

BN 20 m²/g

Graphite 15 m²/g

Cequ [µg/ml]

Mad

s[µ

g/m

g]

Langmuir adsorption isotherms of

Bradykinin for BN 60, BN 20 and

graphite 15

max. loading capacity

mads [µg/mg]

mads/surface area

[µg/m2]

BN 20 m²/g 14.61 0.731

BN 60 m²/g 24.13 0.402

Graphite 15 m²/g 8.19 0.546

Boron Nitride, a Novel Material for the Enrichment and Desalting

MALDI Spectra:

The identified peptides are labeled with

asterisk. Sequence Coverage of desalted

BSA > 68 %

Boron Nitride, a Novel Material for the Enrichment and Desalting

0

20

40

60

80

100

Inte

ns. [

a.u.

]

0.0

0.2

0.4

0.6

0.8

1.0

5x10

Inte

ns. [

a.u.

]

1000 1500 2000 2500 3000 3500m/z

*

*

*

*

*

**

*

*

**

*

*

*

*

*

*

**

*

*

**

* **

*

*

*

*

*

**

*

**

* *

*

*

**

*

*

*

*A

B

x 1000BSA digest in urine

desalted urine sample1000 fold increase in signal intensity

N

BN

B

NB

NB

NB

NB

NB

N

B

BN

BN

BN

B

BN

BN

BN

N

BN

- Pyrrolizidine alkaloids (PA) are secondary plant metabolites (for plant protection)- 400 different PAs in approximately 6000 plant species are known

Contamination of plant products during harvest or through animals (for example bees)Examples for contaminated food: herbal teas, honey, salads etc.

- Problem: Hepatotoxic for animals as well as for humansSafety values for the maximum dose for drugs in Germany:- Application up to 6 weeks: 1 µg/day oral, 100 µg/day cortically - Application for more than 6 weeks: 0.1 µg/day oral, 10 µg/day cortically

Pyrrolizidine alkaloidsBackground

90

- General structure:

- Requirement(s) for toxicity:- 1,2-unsaturated necine- Esterification of at least one OH-group with a branched acid

Nearly all types are in coexistence with their N-oxides

Pyrrolizidine alkaloidsToxicity

91

- HPLC-MS (LOD ~ 1 ppb)- GC-MS (LOD = 3 ppb)

Otonecines and N-oxides (without derivatisation) not detectable- Double antibody ELISA (LOD = 0.1 – 1.5 ppb)

Antibodys only against some specific PAs- HPLC Evaporative Light Scattering Detection (ELSD) (LOD = 40 ppm)- Nonaqueous capillary electrophoresis (NACE) MS (LOD < 7.5 ppm)- Photometric detection with Ehrlich reagent (LOD = 10 ppm)

- General problem for PA analysis: Only about 25 standards are availableValidation is limited to a small spectrum of PAs

Isolation of PAs out of plant material with countercurrent chromatography (CCC) to get morestandards (Cooperation with Medical University of Lublin)

Pyrrolizidine alkaloidsMethods for detection

92

- Detection only possible in the low ppb-range (for GC- and HPLC-MS) for lower concentrations and for separation from other plant substancesenrichment is necessary

- Due to the chemical structure ion exchange is the enrichment method of choice- Automation with PhyNexus MEA 2 possible

PhyTip with Toyopearl SP-650 resin

Pyrrolizidine alkaloidsCation exchange

93

Extraction of the plant material

Enrichment of PAs with ion exchange

Detection with UPLC-MS

Pyrrolizidine alkaloidsProcedure

94

Pyrrolizidine alkaloidsCation exchange – Comparison of commercial products

• In terms of enrichment of pyrrolizidine alkaloids different commercial materials were tested

• Best recoveries (94-99%) were achieved with a polystyrene-divinylbenzene resin functionalized with sulfonate groups

• Further approach- Polystyrene DVB resin in PhyNexus tips and automation of the cation

exchange with PhyNexus MEA 2- Continuing comparison with other resins (in cartridges and tips)- Test of anion exchange resins and Immobilized Metal Ion Affinity

Chromatography (IMAC)

95

Pyrrolizidine alkaloids

• Development of a UPLC-MS/MS method for detection and quantification of standard PA´s

• Improvement of enrichment methodologies with ion-exchange

• Automation of enrichment method

Output

96

SPE of Phenolic Acids using Bismuth Citrate andZirconium Silicate Inside Micro Spin Columns

SPE of Phenolic Acids using Bismuth Citrate andZirconium Silicate Inside Micro Spin Columns

1,3,4,5-tetra-o-galloylquinic acid

CynarinChlorogenic acid

mono-caffeoylquinic-aciddi-caffeoylquinic-acid

Galloyl- and caffeoylquinic-acids are characterized by one free carboxylic group in the quinic acid moiety

Caffeoylquinic acids are the esters of quinic acids with caffeic, ferulic or p-coumaric acids

Galloylquinic acids are the esters of quinic acid with gallic acids

Their biological activity includes anti-HIV, antiasthamatic, bronchial hyper reactivity, allergicreactions and anti-inflamatory properties

Caffeoylquinic acids are antidiabetic, hypoglycemic, antihepatitis, hepatoprotective and anti canceragents

Proposed binding mechanism of bismuth citrate and zirconium silicate with carboxylic group of phenolic acids

Zirconium Silicate45 µm

Bismuth Citrate45 µm

Hussain, Shah, et al. Journal of Pharmaceutical and Biomedical Analysis (2013) in press

Tetragalloyl-quinic acids

Crystalline forms of Boron Nitride

β or cubic BN

• diamond analog

• stable

• very hard

• black

γ or wurzite BN

• lonsdaleit analog

• only stable under high

pressure

α or hexagonal BN

• graphite analog

• chemically inert

• nontoxic

BADGE·2H2O BADGE

BPF BPA

BPZ

Bisphenol derivatives

MW pKa log Kow R2 LOD[ng/ml]

LOQ[ng/ml]

BADGE·2H2O 376.44 14.7b 2.05b 0.999 27.0 82.0

BPF 200.23 7.5a 2.91a 0.999 27.0 82.0BPA 228.29 9.6a 3.32a 0.999 33.0 99.0BPZ 268.35 9.7b 4.53b 0.999 28.0 85.0BADGE 340.41 - 4.02a 0.999 30.0 90.0

Recovery for the enrichment of 5 Phenols

Phenol 4-Nitrophenol 2-Chlorphenol 2-Nitrophenol Dimethylphenol

[12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml]

87.51 94.74 108.08 91.29 100.71

93.97 102.95 114.70 95.20 111.82

91.36 101.91 113.24 96.88 105.84

Average 90.95 99.87 112.01 94.46 106.13

RSD 2.66 3.65 2.84 2.34 4.54

5 mg of BN was incubated with a phenol mixture and afterward eluted with80% acetonitrile in water.

• The French Agency for Food, Environmental and Occupational Health & Safety showed that

there are ‘recognized’ effects in animals (effects on reproduction, on the mammary gland, on

metabolism, the brain and behaviour) and other ‘suspected’ effects in humans (on

reproduction, metabolism and cardiovascular diseases). These effects could be observed, even

at low levels of exposure, during sensitive phases of an individual’s development.

• The French Agency for Food, Environmental and Occupational Health & Safety endorses the

conclusions of the Expert Committee on Assessment of the risks related to chemical

substances relating to the risks associated with BPA for human health, and on toxicological

data and data on the use of bisphenols M, S, B, AP, AF, F and BADGE.

Risk associated with bisphenols

Analysis of 5 bisphenol derivatives after enrichment with BN by LC and fluorescence detector

HPLC-FLD chromatogramms of (A) standard solution containing 5 bisphenolderivatives [100 ng/mL] and (B) bisphenol leached from baby bottle afterenrichment with h-BN-60.Chromatographic conditions: Shimadzu LC-10 Acvp, Phenomenex Luna 5u C18 250*4,6 mm, isocratic 3 min 30% B,gradient 30-70% B in 15 min, flow rate 1mL/min, 50°C, inj.: 30 µL,FLD: 230/303 nm

Recovery rates for the enrichment of 5 bisphenolderivatives

Recovery rates ± SD [%]

Concentration[ng/ml] BADGE·2H2O BPF BPA BPZ BADGE

0.5 90.01 ± 5.65 96.43 ± 8.17 111.93 ± 3.8 95.40 ± 5.87 85.38 ± 11.53

1 93.69 ± 2.57 98.88 ± 2.57 98.13 ± 4.05 100.90 ± 1.84 91.43 ± 4.47

2 99.47 ± 1.91 100.92 ± 1.13 95.88 ± 4.21 102.11 ± 3.23 97.48 ± 1.97

10 97.39 ± 2.41 102.47 ± 2.42 103.03 ± 4.21 101.67 ± 2.56 99.17 ± 4.15

100 98.67 ± 5.78 101.55 ± 4.45 101.54 ± 4.47 100.23 ± 2.64 87.51 ± 7.78

Found ± SD [ng/ml]Recovery rates ± SD [%]

BADGE·2H2O BPF BPA BPZ BADGE

river watera nd(92.13 ± 2.69)

nd(102.62 ± 3.73)

nd (107.14 ± 6.50)

nd(104.95 ± 1.92)

nd (98.74 ± 2.57)

drinking watera nd(90.16 ± 2.40)

nd(98.56 ± 3.51)

nd (99.16 ± 1.60)

nd (101.14 ± 2.58)

nd(96.68 ± 3.35)

colab 4.63 ± 0.14 (103.41 ± 4.39)

2.23 ± 0.21 (96.98 ± 2.39)

10.34 ± 0.41(96.93 ± 5.72)

nd (92.46 ± 6.58)

1.49 ± 0.04 (96.57 ± 3.84)

apple juiceb 1.93 ± 0.15(96.43 ± 3.63)

3.11 ± 0.30(90.77 ± 3.63)

nd (84.22 ± 2.20)

nd (98.74 ± 1.23)

1.36 ± 0.22(98.22 ± 2.71)

lemon sodab 8.14 ± 0.09(82.46 ± 3.01)

1.40 ±0.07(84.45 ± 5.44)

nd (97.60 ± 1.81)

nd (87.61 ± 2.94)

5.70 ± 0.04(94.00 ± 2.61)

citrus soft drinkb 5.32 ± 0.71 (103.90 ± 2.58)

nd(97.82 ±5.82)

1.04 ± 0.31(98.31 ± 5.67)

nd (98.05 ± 4.04)

1.56 ± 0.042 (99.63 ± 4.42)

herbal sodab nd (100.45 ± 5.15)

nd(99.07 ± 5.09)

nd (104.43 ± 6.20)

nd (105.07 ± 7.21)

nd (108.42 ± 4.83)

energy drinkb 4.46 ± 0.07 (98.54 ± 9.21)

1.87(91.55 ± 4.21)

5.57 ± 0.81(93.55 ± 5.84)

nd (100.76 ± 2.04)

nd (103.60 ± 1.91)

canned mushroom liquidb 20.42 ± 1.19(99.06 ± 6.88)

nd(91.35 ±0.79)

8.60 ± 0.37 (98.47 ± 8.37)

nd (103.71 ± 6.17)

1.81 ± 0.12 (99.63 ± 0.62)

pickled cucumber liquidb 1.81 ± 0.15 (97.53 ± 1.12)

2.23 ± 0.21(96.02 ± 2.45)

125.00 ± 2.18(109.12 ± 0.77)

nd (103.36 ±3.80)

1.44 ± 0.06 (92.67 ± 0.71)

pickled onion liquidb 2.98 ± 0.08 (105.80 ± 3.90)

nd(96.53 ± 8.20)

13.45 ± 0.63 (92.03 ± 4.70)

nd(97.91 ± 3.12)

1.74 ± 0.00 (95.20 ± 0.63)

urineb nd (100.45 ± 3.13)

nd(105.67 ± 0.68)

nd(92.80 ± 2.52)

nd(105.26 ± 4.00)

nd(103.70 ±3.45)

Determination of bisphenol derivatives

Leaching of BPA from polycarbonate products

leached BPA [ng/ml] ± SD (%)

Babybottle 1. treatment 20.79 1.05

Babybottle 2. treatment 19.82 0.72

muffin form 2.26 0.07

Siringe 0.85 0.03

Treatment conditions: boiling water for 1 h

Langmuir adsorption isotherms of Bisphenol A on h-BN-60

M ad

s[µ

g/m

g]

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10

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70

0 5 10 15 20 25 30

Cequ[µg/ml]

max. loading capacity of Bisphenol A = 60 µg/mg

Depletion of proteins with BN

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A

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x 10

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(A) MALDI-MS analysis of a human albumin proteinand those digest in a ratio of 50000-1 (1mg/ml-0,02 µg/ml) and a image of thecrystallization of the sample and matrixSequence coverage of HSA 36%

(B) MALDI spectra of the sample after SPE with BN(sequence coverage of 72%) and an image ofthe matrix crystallization

asterisk: Mascot identified peptides

Aufgabe 1

Analyt: Disaccharide

Welche Festphase? Warum?

Aufgabe 2

Analyt: Proteine

Welche Festphase? Warum?

Aufgabe 3

Analyt: Pestizide

Welche Festphase? Warum?