Grundlagen und Anwendung moderner Trennverfahren · 10 min, 70W microwave‐assisted digest...

25.01.2017

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Grundlagen und Anwendung moderner Trennverfahren

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

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

1

Fällung + ADSI

Komplexität von biologischen Proben

1. enorme Komplexität der Proben

2. geringe Konzentration der Zielanalyten

3. beschränkter dynamischer Bereich der analytischen Messgeräte

4. Störkomponenten (Detergenzien, Puffersysteme etc.)

MS

biologische Probe

Herausforderungen:

2

EinleitungAnalytik der Biomoleküle

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3

22 Proteine machen 99% des gesamten Serumproteoms aus!

90% 10%


1. Enorme Komplexität von biologischen Proben

z.B. Blutserum

4Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845‐867

Dynamischer Bereich von Blutplasma


2. Geringe Konzentration vieler Zielanalyten

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Selektivität

Empfind‐lichkeit

Geschwin‐digkeit

Hoher Probendurchsatz

Verbesserte Detektion

Gezielte Analyse

Effizienz

Effiziente Probenvorbereitung


Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:

• Fällung durch Aussalzen

• isoelektrische Fällung (Fällung am IEP)

• Fällung mit organischen Lösungsmitteln

• Co‐Präzipitation

Fällung von Proteinen

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Precipitation of Phosphoproteins by

Trivalent Lanthanide Ions

A Top‐Down Approach

7

Protein phosphorylation

Protein dephosphorylation

Berg JM et al. 2010

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Protein separation and isolation

2. Chromatographic techniques

IMACImmobilized Metal ion Affinity Chromatography

MOACMetal Oxide Affinity Chromatography

Berg JM et al. 2010 According to: Leitner A. 2010

Proposed Precipitation Mechanism

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Lanthanide Phoshates(Low Solubility Products)

solubility products [logKsp(M)]

ErPO4 -25.13 ± 0.11

EuPO4 -25.96 ± 0.03

TbPO4 -25.39 ± 0.04

Li, X. et al. Geochimica et cosmochimica acta,1997.61(8):p.1625-1633

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Dissolvation of pellet

MALDI-MS

on-pellet digest

denaturation, tryptic digestion30% formic acid

washing

precipitation

10 min, 70W microwave‐assisted digest

MALDI-MS

PeptidesProteins

Scheme for Precipitation of Phosphoproteins by Trivalent Europium-, Terbium- and Erbium- Ions

PrecipitantKH2PO4

Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133112

TOP‐DOWNBOTTOM‐UP

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Methods and instruments

MALDI‐TOF MS: linear mode

For TOP‐DOWN


MALDI‐TOF MS: reflector mode

Improved Resolution!

for BOTTOM‐UP

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Bovine milk composition

87,1%

4,9%3,9%

3,4%0,7%

water

carbohydrates

fats

proteins

minerals

38%

9% 29%

9%

4%9%2%

α‐S1‐casein

α‐S2‐casein

β‐casein

κ‐casein

α‐lactalbumin

β‐lactoglobulin

others

according to Pavia DL. 1990 / Eigel WN et al. 1984

Phosphoproteine

Milk proteins

protein (variant)mol. weight /

Daamino acids

c / g∙L‐1P

per molSH | S‐S per mol

pI

αS1‐casein (B 8P) 23,614 199 12 – 15 8 0 | 0 4.4 – 4.8

αS2‐casein (A 11P) 25,230 207 3 – 4 11 2 | 0 −

β‐casein (A2 5P) 23,983 209 9 – 11 5 0 | 0 4.8 – 5.1

κ‐casein (B 1P) 19,023 169 2 – 4 1 2 | 0 5.3 – 5.8

α‐lactalbumin (B) 14,176 123 0.6 – 1.7 0 0 | 4 4.2 – 4.5

β‐lactoglobulin (B) 18,363 162 2 – 4 0 1 | 2 5.1

serum albumin 66,267 582 0.4 0 1 | 17 4.7 – 4.9

according to: Eigel WN et al. 1984 / Fox PF et al. 1998

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Precipitation of Phosphorylated Proteins – Top‐Down Workflow

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Güzel, Y.; Rainer, M. (); Mirza, M.R.; Bonn, G.K. Highly efficient precipitation of phosphoproteins using trivalent Europium‐,Terbium‐ and Erbium Ions. Analytical and Bioanalytical Chemistry (2012) 403(5), 1323–1331.

Terbium

Wash 1

Wash 2

Pellet

Supernatent

milk

Erbium

Wash 1

Wash 2

Pellet

Supernatent

milk

Europium

PPC‐5 (m/z ~12–13 kDa)

α‐lactalbumin (m/z ~14.1 kDa)

β‐lactoglobulin (m/z ~18.3 kDa)

non‐phosphorylated milk‐proteins

αS1‐casein (m/z ~24.5 kDa)

β‐casein (m/z ~25.1 kDa)

phosphoproteins

αS2‐casein (m/z ~24.5 kDa)

κ‐casein (m/z ~20.0 kDa)

Wash 1

Wash 2

Pellet

Supernatent

milk

Precipitation of Phosphoproteins from Bovine Milk by Trivalent Europium-, Terbium- and Erbium- Ions


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Erbium

Wash 1

Wash 2

Pellet

Supernatent

eggwhite

Terbiumegg

white

Pellet

Wash 2

Supernatent

Wash 1

lysozyme (m/z ~14 kDa)

ovomucoid (m/z ~ 28 kDa)

ovoglobulins G2+G3 (m/z ~30‐45 kDa)

ovotransferrin (m/z ~80 kDa)

ovalbumin (m/z ~45 kDa)

non phosphorylated egg‐white proteins phosphoprotein

Europium

Wash 1

Wash 2

Pellet

Supernatent

eggwhite

Precipitation of Phosphoproteins from Egg-White by Trivalent Europium-, Terbium- and Erbium- Ions


2M Ln3+‐Chloride

UV/VIS

take supernatant

bicinchoninic acid (BCA)

chelation of two bicinchoninicacid molecules with Cu+1

reduction of Cu+2 to Cu+1 in the presence ofproteins (alkaline conditions)

colorimetric detection 562 nm

Recovery Study of Phosphoprotein

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Colorimetric assays: Bradford and BCA

Bradford reagent:Coomassie Brilliant Blue G‐250

BCA assay reaction

Lottspeich F. 2012

Bicinchoninsäure

Absorptionsmaximum von 562 nm

0

20

40

60

80

100

0,5 1 1,5 2 2,5 3

Rec

overy

[%

]

Volume Precipitant [µl]

Lanthanum

Europium

Terbium

Erbium

100%

Precipitation of Phosphoproteins by Trivalent Europium-, Terbium- and Erbium- Ions

Recovery Study

ccasein = 300µg/ml

cprecip. = 2M

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Precipitation of Phosphopeptides by

Trivalent Lanthanide Ions

A Bottum‐Up Approach

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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation

Workflow for the precipitation of phosphorylated peptides24

Bottom‐up

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MALDI mass spectra taken from digested milk peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. α‐S1 and β‐S2 refers to first and second subunits of α‐caseinrespectively. β‐C refers to peptides form β‐casein

Erbium

Holmium

Cer

25


MALDI mass spectra taken from egg white peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. Only phosphorylated peptides are labeled

Erbium

Holmium

Cer

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MALDI mass spectra of a sensitivity study using two synthetic phosphopeptides. A, representing 500 fmol/µL; B, 10 fold dilution (50 fmol/µL) and C, 100 fold dilution (5 fmol/µL)

500 fmol/µL

50 fmol/µL

5 fmol/µL

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High Sensitivity!

[M+H]+ Da Phosphopeptide Sequencesa Phosho‐

groups

ErCl3 HoCl3 CeCl3 LaCl3 EuCl3 TmCl3 TbCl3 TiO2

1254.52

1331.53

1411.50

1466.61

1594.70

1660.79

1832.83

1847.69

1927.69

1951.95

2061.83

2088.89

2432.05

2511.13

2556.10

2619.04

2678.01

2703.50

2720.91

2747.10

2856.50

2901.32

2935.15

2966.16

3008.01

3042.27

3087.99

3122.27

3132.20

EVVGSpAEAGVDAA (Ov‐(340–352))

EQLSpTSpEENSK (α‐S2‐(141–151))

EQLSpTSpEENSK (α‐S2‐(141–151))

TVDMESpTEVFTK (α‐S2‐(153–164))

TVDMESpTEVFTKK (α‐S2‐(153–165))

VPQLEIVPNSpAEER α(‐S1‐(121–134))

YLGEYLIVPNSpAEER (α‐S1)

DIGSESpTEDQAMEDIK (α‐S1‐(58–73))

DIGSESpTEDQAMEDIK (α‐S1‐(58–73))

YKVPQLEIVPNSpAEER (α‐S1‐(119–134))

FQSpEEQQQTEDELQDK (β‐C‐(33–48))

EVVGSpAEAGVDAASVSEEFR (Ov‐(340–359))

IEKFQSpEEQQQTEDELQDK (β‐C‐(33–48))

LPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(62–84))

FQSpEEQQQTEDELQDKIHPF (β‐C‐(48‐67))

NTMEHVSpSpSpEESpIISQETYK (α‐S2‐(17–36))

VNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(52–73))

LRLKKYKVPQLEIVPNSpAEERL(α‐S1‐(114–135))

QMEAESpISpSpSpEEIVPNSVEAQK (α‐S1‐(74–94))

NTMEHVSpSpSpEESpIISQETYKQ (α‐S2‐(17–37))

EKVNELSpKDIGSpESTEDQAMEDIK (α‐S1‐(50–73))

FDKLPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(59–84))

EKVNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(50–73))

ELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(17–40))

NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))

RELEELNVPGEIVESLSpSpSpEESITR (β‐C‐(16–40))

NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))

RELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(16–40))

KNTMEHVSpSpSpEESpIISQETYKQEK (α‐S2‐(16–39))

Mono

Mono

Di

Mono

Mono

Mono

Mono

Mono

Di

Mono

Mono

Mono

Mono

Mono

Mono

Tetra

Tri

Mono

Penta

Tetra

Di

Mono

Tri

Tetra

Tetra

Tetra

Penta

Tetra

Tetra

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Recovery of Phosphopeptides

23 20 19 20 21 12 14 18

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PP

P

PP

3.6% HCl

P P

P P

P

trypsin

Tryptic On‐Pellet Digest of Precipitated Phosphoproteins

Inte

nsity

m/z29

Workflow

Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins

Güzel, Y.; Rainer, M. (); Mirza, M.R.; Messner, C.B.; Bonn, G.K. Highly Selective Recovery of Phosphopeptides using Trypsin‐Assisted Digestion of Precipitated Lanthanide‐Phosphoprotein Complexes. Analyst (2013) 138(10), 2897‐2905.

LaPO4

PP

P

PP

washing

LaPO4

Overview of recovered phosphopeptides from a proteinmixture (lysozyme, cytochrome c, myoglobin, bovine serum albumin, a‐ and b‐casein) and bovine milk

proteinmixture milk 1:100 dilution

[M+H]+ Position Protein Phosphopeptide sequences Phospho groups LaCl3 CeCl3 TiO2 LaCl3 CeCl3 TiO2 LaCl3 CeCl31466.6 153‐164 α‐S2 TVDMESpTEVFTK mono + + ‐ + + + + +

1482.6 153‐164 α‐S2 TVDM*ESpTEVFTK mono + + ‐ ‐ + ‐ + +

1594.7 153‐165 α‐S2 TVDMESpTEVFTKK mono + + + + + + + +

1610.7 153‐165 α‐S2 TVDM*ESpTEVFTKK mono + + ‐ ‐ + ‐ + +

1660.7 121‐134 α‐S1 VPQLEIVPNSAEER mono + + + + + + + +

1832.8 104‐119 α‐S1 YLGEYLIVPNSpAEER mono + + + + + + + +

1847.6 58‐73 α‐S1 DIGSESpTEDQAMEDIK mono + + ‐ ‐ ‐ ‐ ‐ ‐

1927.6 58‐73 α‐S1 DIGSpESpTEDQAMEDIK di + + ‐ + + + + +

1943.6 58‐73 α‐S1 DIGSpESpTEDQAM*EDIK di + + + + + + + +

1951.9 119‐134 α‐S1 YKVPQLEIVPNSpAEER mono + + + + + + + +

1981.8 48‐63 β FQSpEEQQQTEDELQDK mono + + + + ‐ + + ‐

2000.0 118‐134 α‐S1 KYKVPQLEIVPNSpAEER mono + + ‐ + + ‐ ‐ +

2061.8 48‐63 β FQSpEEQQQTEDELQDK mono + + + + + + ‐ ‐

2080.0 118‐134 α‐S1 KYKVPQLEIVPNSpAEER mono + + + + + + + +

2432.0 45‐63 β IEKFQSpEEQQQTEDELQDK mono + + ‐ + + ‐ ‐ ‐

2548.2 119‐139 α‐S1 YKVPQLEIVPNSpAEERLHSMK mono + + ‐ + + ‐ + +

2560.1 44‐63 β KIEKFQSpEEQQQTEDELQDK mono + + ‐ + + ‐ ‐ ‐

2564.6 119‐139 α‐S1 YKVPQLEIVPNSpAEERLHSM*K mono + + ‐ + + ‐ + +

2598.0 52‐73 α‐S1 VNELSpKDIGSpESpTEDQAMEDIK di + + ‐ + + ‐ + +

2678.0 52‐73 α‐S1 VNELSpKDIGSpESpTEDQAMEDIK tri + + ‐ + ‐ + + +

2703.5 114‐135 α‐S1 LRLKKYKVPQLEIVPNSpAEERL mono + + ‐ + + + ‐ ‐

2716.2 130‐152 α‐S2 NAVPITPTLNREQLSpTSpEENSKK di + + ‐ + + ‐ + +

2720.9 74‐94 α‐S1 QMEAESpISpSpSpEEIVPNSpVEQK penta + + ‐ + + ‐ ‐ ‐

2747.1 17‐37 α‐S2 NTMEHVSpSpSpEESpIISQETYKQ tetra + + + + + + ‐ ‐

2856.5 50‐73 α‐S1 EKVNELSpKDIGSESTEDQAMEDIK di + + ‐ + + ‐ + +

2935.1 50‐73 α‐S1 EKVNELSpKDIGSpESTEDQAMEDIK tri + + ‐ + ‐ ‐ + +

2966.1 17‐40 β ELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + ‐ ‐ ‐

3008.0 61‐85 α‐S2 NANEEEYSIGSpSpSpEESpAEVATEEVK tetra + + + + + + + +

3024.2 16‐40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + ‐ ‐ ‐

3042.2 16‐40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + ‐ ‐ ‐

3087.9 61‐87 α‐S2 NANEEEYSpIGSpSpSpEESpAEVATEEVK penta + + ‐ + + ‐ ‐ ‐

3122.2 16‐40 β RELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + + + +

3132.2 16‐39 α‐S2 KNTMEHVSpSpSpEESpIISQETYKQEK tetra + + ‐ + + + + +

34 15 31 16 22

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Dephosphorylated HeLa cell lysate (1 mg/mL) with spiked α‐ and ß‐casein (5 μg/mL) after enzymatic on‐pellet digestion using trivalent cerium cations. α1, α2 and β correspond to the tryptic phosphopeptides deriving from αS1‐, αS2, and β‐casein, respectively

HeLa cell lysate ‐ digest

spiked cell lysate after precipitation


On‐pellet digest of precipitated α‐/ß‐casein from spiked cell lysates

crude saliva digest

precipitated fractionCeCl3

Rainer, M. (), Güzel, Y., Messner, C., & Günther Bonn (2014). Co‐Precipitation of Phosphorylated Proteins Using Trivalent Cerium‐, Holmium‐, and Thulium Cations. Current Pharmaceutical Analysis, 10(3), 175‐184.


On‐pellet digest of precipitated proteins from Human Saliva

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Conclusion

simple and fast method

highly selective for phosphorylated peptides and proteins

enables top‐down and bottum‐up phosphoproteomics

no stationary phase or resin required (reduced unspecific binding)

trypsin was observed to be not affected by the lanthanide ions

the amount of precipitant can be adjusted to each application

MS and LC‐MS compatible

allows automation using liquid handling robotics

Highly efficient precipitation of phosphorylated peptides and proteins using trivalent lanthanide ions

ADSIAnalytische AbteilungVO Moderne Trennmethoden

Innsbruck, 25.01.2017

25.01.2017

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• Medikamentenentwicklung

• Kosmetik

• Nutrition

Themenschwerpunkte

Phytokosmetik

(Phyto)-NutritionPflanzl.Lebensmittel-

zusatzstoffe

PhytoanalytikPhytopharmazie

Institut für Analytische Chemie

und Radiochemie

allows to see the Nature

Olive‐Net

Themenschwerpunkte „Phytovalley“ Tirol

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Medikamentenentwicklung

Ziel: Therapeutisch wirksames Pflanzenextrakt

1. Herstellung und Qualitätskontrolle von Pflanzenextrakten

2. In vitro Tests: ‐ Löslichkeitsexperimente

‐ Bioverfügbarkeit

‐ Toxizität

‐ Immunreaktionen

‐ Hepatische Metabolisierung

3. Charakterisierung der Inhaltsstoffe

4. Tiermodellversuche ausgewählter Kandidaten

Solubility Tests (WP 1)

• solubility in DMSO

• comparison of 100 mg/ml, 50 mg/ml, 20 mg/ml, 10 mg/ml 2 mg/ml

• determination by means of UHPLC-UV and visually

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MedikamentenentwicklungEndotoxinquantifizierung

• an integral part of gram-negative bacteria cell wall

• thermally stable (autoclaving is insufficient)*

• activate many transcription factors and septic shock mediators=> may lead to wrong results in cell culture experiments

• factors B and C in the LAL enzyme mix react with endotoxins, triggering a protease activity

• matching peptide releases p-nitroaniline upon cleavage

• direct quantification via UV/VIS absorbance measurement

Quantification via a LAL chromogenic assay

*Bang FB Biol Bull 1953 105 361**Amanda44 Wikipedia CC v3.0 license 2011 July 31st

Limulus Amoebocyte Lysate

**

MedikamentenentwicklungEndotoxinquantifizierung

• two-stage analysis (pre-testing and exact quantification)

• quantification limit of 0.5 EU/mg extract (10 EU/mg uncritical)

• false-positives due to β-1,3-glucans are eliminated by means of glucan blocker

• absorbance vs. concentration to eliminate quenching

Key facts of ADSI‘s workflow

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MedikamentenentwicklungAssoziierte Partner

MedikamentenentwicklungBioverfügbarkeit

In vitro Gastrointestinal‐Permeabilität

• PAMPAPermeabilitätsassay basierend auf einer Lipid Doppelschicht

Durchführung und LCMS Analytik am ADSI

• CaCo2Permeabilitätsassay auf Basis von colorektalen Darmepithelzellen

Durchführung extern, LCMS Analytik am ADSI

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MedikamentenentwicklungBioverfügbarkeit

• sophisticated tri-layer of phospholipids and hexadecane*

• isothermal experiment (25 °C)

• two scenarios: - proton gradient (pH4.5 → pH7.4)- pH-eqilibrated (pH7.4 → pH7.4)

• three replicates per time step, three observations (2h, 4h, 6h)

• two internal standards*: - phenazone (MW 189 g/mol)- sulfasalazine (MW 398 g/mol)

Optimization for plant extracts

*Chen X et al. Pharm Res 2009 25 1511

MedikamentenentwicklungParallel Artificial Membrane Permebility Assay

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Vasicine Pe ≈ 2∙10‐6 cm/s (HI) (pH7.4)

Pe ≈ 2∙10‐7 cm/s (LO) (pH4.5)


sum formula, ID, m/z, r.t., Pe, SD, RSD, graphical rating

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Datenintegration und Bioinformatik

• Toxizität und Metabolismus der Wirkstoffe

• Identifizierung der bioaktiven Substanzen

• Information über Wirkungsmechanismus

Biologische DatenAnalytische Daten

+

Datenintegration‐ und Bioinformatik‐Plattform

• Quantifizierung der bioaktiven Substanzen

NutritionBeispiel Olivenöl

Ziel: Aussage über Qualität; EFSA Health‐Claim

1. Extraktion phenolischer Komponenten

2. Saure Hydrolyse

3. Quantifizierung von Tyrosol und Hydroxytyrosol

4. Bestimmung der Routine‐Qualitätsparameter

‐Säurezahl

‐Peroxidzahl

‐Iodzahl

ADSI, InnsbruckUniversity of Athens

Greece

Institute of ChemicalMethodologies

Rome

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NutritionBeispiel Olivenöl

Health claim: Commission Regulation No. 432/2012

Commission Regulation No. 432/2012 of 16th of May, 2012

Correlation between Olive‐Polyphenols and Protection of the LDL‐Cholesterols forOxidation (Oxidized LDL is strong atherogenic)

NutritionWorkflow – determination of phenolic compounds in EVOO

Native Olive Oil Extra

Direct Quantification ofHydroxytyrosol und Tyrosol

Acid Hydrolysis (1M H2SO4)

Hydroxytyrosol Tyrosol

Quantification of Hydroxytyrosol andTyrosol as Parameter of all Derivates

(EFSA: ≥5mg/20g)

Extraction with 60% MeOH/Water

EFSA Claim: Polyphenoles out from Olive Oil protect LDL‐Cholesterolfrom oxidative Stress (min. 5mg Polyphenoles per 20 g

Olive Oil)

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NutritionAcid hydrolysis

Acid hydrolysis of phenolic compounds in EVOO

Lecture Prof. Skaltsounis, Innsbruck 2014

Acid hydrolysis with 1 M H2SO4

HPLC‐UV analysis of a oleuropein standard (0.3 mg/ml) before and after acid hydrolysis

HPLC‐UV chromatograms of a oleuropein standard before and after acid hydrolysis at 280nm

0

5

10

15

20

0

5

10

15

20

0 2 4 6 8 10 12 Time [min]

Absorbance [mAu]

Absorbance [mAu]

Oleuropein

Hydroxytyrosol

Before hydrolysis

After hydrolysis

NutritionAcid hydrolysis of phenolic compounds in EVOO

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Horizon 2020Bioactive Compounds from Olea Europaea: Investigation and

Application in Food, Cosmetic and Pharmaceutical Industry

Olive‐Net

Call: H2020‐MSCA‐RISE‐2016(Marie Sklodowska‐Curie Research and Innovation Staff Exchange)

Topic: MSCA‐RISE‐2016

Proposal number: 734899

Proposal acronym: Olive‐Net

Key words: olea europaea, olive mill waste, olive oil, extraction, analysis, profiling, cosmetics, inflammation, in vivo, ageing, supplements, nutraceuticals, oleacein, oleocanthal, obesity,

EU‐Evaluation Summary Report ‐ Results (0‐5)Excellence: 4.5, Impact: 4.6, Quality and Efficency of the Implementation: 4.7

Optimal capacity: yesFounded by the European Commision: September 2016

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List of Participants

UNIVERSITE D'AVIGNON ET DES PAYS DE VAUCLUSE France

UNIVERSIDAD COMPLUTENSE DE MADRID Spain

CONSIGLIO NAZIONALE DELLE RICERCHE Italy

ADSI‐Austrian Drug Screening Institute GmbH Austria

PHARMAGNOSE VIOTECHNOLOGIKI ANONYMI ETAIREIA Greece

UNI‐PHARMA KLEON TSETIS PHARMACEUTICAL LABORATORIES S.A. Greece

SERVIZO GALEGO DE SAUDE Spain

NATAC BIOTECH SL Spain

ALNuMed GmbH Germany

NEURO‐SYS SAS FranceECOLE NATIONALE D'AGRICULTURE DE MEKNES Morocco

INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE D ALGERIE Algeria

Rangsit University Thailand

ETHNIKO KAI KAPODISTRIAKO PANEPISTIMIO ATHINON Greece

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France

Natural ingredients and extracts discovery, technical extraction and purification technologies, analytical standards

Biomedical Research

Development of Drug Delivery Systems, API Synthesis, Clinical Studies, Drug Repositioning, Translational Research Novel routes of administration

Drug discovery, neurodegeneration

Development of functional foods and active ingredients from natural substances

Development of novel analytical methods, NMR and evaluation tools for rapid authenticity and quality testing

ICOA; Analytical strategies, molecular interactions and bioactivity tests

Laboratory for green extraction techniques of natural products

Physiology

Analysis, HPLC, CE‐technologysis

Extractions, Analytical methods, HPLC‐MS, MALDI, Screening technologies, Cell systems

Pharmacognosy and Natural Products Chemistry, Plant data base

Agrifood technology

ECOLE NATIONALE D'AGRICULTURE DE MEKNES

Oriental Medicine applications

Medicine and ApplicationsAgriculture

Extraction, Purification, Analysis

Expertise der Projektpartner im Olive‐Net EU‐Projekt

25.01.2017

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Olive‐Net Roadmap

Olivenöl Abfälle

Olivenkultivierung Olivenölgewinnung

25.01.2017

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Abfälle

Erzeugung von Wertstoffen aus Olivenöl‐Produktionsabfällen

Tocopherole Aldehyde

Polyphenole Flavonoide

Cyclische Mono‐ und Sesquiterpen‐kohlenwasserstoffe

Wertstoffe für Phytokosmetik‐, Pharma‐, Nahrungsmittel‐, Nahrungsergänzungsmittel

Abfälle

Fester Presskuchen, Waschwasser und einsehr dünnflüssiger Schlamm sind umwelt‐belastende Abfälle bei der Olivenölproduktion

Wertstoffe aus dem Abfall

Mehr als 2 Mio t/a Olivenöl werden in Europa produziert

Im Mittelmeerraum wird die Umwelt dadurchjedes Jahr mit über 30 Millionen TonnenPressrückstände, Waschwasser und Oliven‐blätter belastet.

Dort werden die flüssigen Reststoffe häufig inLagunen und Seen geleitet wodurch dasGrundwasser verunreinigt wird. Die Gewässerverfärben sich schwarz, sterben ab undstinken, da Olivenabfälle auch toxischeVerbindungen enthalten.

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