New Approaches in the Analysis of Amadori Compounds

7 TH Wartburg Symposium on Flavor Chemistry and Biology Eisenach, April 21 st - April 23 rd, 2004 New Approaches in the Analysis of Amadori Compounds T. Davidek, I. Blank, K. Kraehenhuehl, J. Hau and S. Devaud Nestlé Research Centre Lausanne, Switzerland 1

2 Structure of the presentation Introduction Analysis of Amadori compounds by HPLC Analysis of Amadori compounds using HPAEC-ECD Separation of glucose-based Amadori compounds Simultaneous analysis of Amadori compounds, precursors and degr. products Separation of Amadori compounds on CS-17 column coupling to ECD coupling to MSD Analysis of Amadori compounds by CE-MS

3 Analysis of Amadori compounds by HPLC HPLC (refractometer, UV-VIS) Rapid (no derivatisation prior to analysis) Generally low separation efficiency and sensitivity/selectivity An efficient HPLC method was developed by Eichner and co-workers (1989, 1990) DEAE-Si Post-column derivatisation with TTC

4 Analysis of Amadori compounds using HPAEC HPAEC PAD Separation of 8 glucose derived Amadori compounds 0.500 PAD Response (μc) 0.400 0.300 0.200 0.100 Glucose Fru-Pro Fru-Ala Fru-Val Fru-Gly Fru-Leu & Ile Fru-Met Fru-Phe Column: Carbopac PA-1 (strong anion exchange column) Detection: Electrochemical detector (PAD) Eluents: Gradient of Na and CH 3 CNa in water Equipment: Dionex DX 500 system Sample: Glucose + 8 amino acids 4h 70 C in Me 0.100 0.0 20.0 40.0 60.0 Time (min)

Simultaneous analysis of Amadori compounds, precursors and degradation products HPAEC DAD ECD ECD Response (µc) 0.160 0.100 0.050 Chromatogram of Fru-Gly heated 1h at 120 C (ph 8) A Electrochemical detection Glucose Mannose Glycine Fru-Gly R N C H R N C H R + H 2 N C H 2 A R H 2 N C Hexose sugar Amino acid C 2 H 2 4 5 DAD Response (mau) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min 40.0 9.00 12.0 5.00 2.50 B UV (λ=285nm) 0.0 5.0 10.0 15.0 20.0 20.0 25.0 30.0 35.0 40.0 min 5.0 C (Davidek et al. 2003) UV (λ=350nm) R N C H Fru-Gly R N C H B R H 2 N C Melanoidins Acids, Flavour 3 H 2 5

Analysis of Amadori compounds using HPAEC Advantages Rapid - no derivatisation and no or limited clean-up High separation efficiency High selectivity and sensitivity Simultaneous detection of parent compounds and degradation products Particularly suitable for analyses of model systems (monitoring of known compounds) Limitations No unequivocal identification Eluents not compatible with MS detection Not suitable for analysis of pentose based Amadori compounds 6

Analysis of xylose-based Amadori compounds using column CS-17 water Column CS-17 ECD Na ECD Response (µc) 0.006 0.004 0.002 Chromatograms of reaction mixtures containing xylose and amino acid Xyl / Ala+Ile 70 C; 4h Me Xyl-Ala Xyl-Ile 2.0 1.0 Xyl / Gly 80 C; 30min Phosphate ph 6 Xyl-Gly mol of Xyl-Gly / mol of xylose 0,15 0,1 0,05 Formation of Xyl-Gly 0 0 0 4 8 min12 0 4 8 min12 0 0 20 40 60 80 100 120 Time (min) 7

Analysis of Amadori compounds using column CS-17 (HPCEC) Chromatograms of Fru-Gly and Xyl-Gly 0.045 ECD Response (µc) 0.025 0.012 0 0 Xyl-Gly Fru-Gly 4.0 8.0 min 12.0 Co-elution of glucose-based and xylose based Amadori compounds Lower separation efficiency as compared with CarboPac PA-1 column Chromatograms of Reaction mixtures containing 8 amino acids and glucose or xylose ECD Response (µc) 0.00919 0.00500 Glucose Fru-Pro Fru-Ala Fru-Val Fru-Gly Fru-Met Fru-Leu Fru-Ileu Fru-Phe 0.0244 0.0100 Xylose Xyl-Ala Xyl-Val Xyl-Gly Xyl-Pro Xyl-Met Xyl-Leu Xyl-Ileu Xyl-Phe 0 0 4.0 8.0 min 12.0 0 0 4.0 8.0 min 12.0 8

9 Analysis of Amadori compounds using HPCEC-MS/MS HPCEC: 0.1% HC MS conditions: Column CS-17 Na MS/MS an integrated Agilent-1100 system (Agilent, Palo Alto, CA). a IonPac CS-17 ion exchange column Flow rate 0.25 ml/min a Q-Trap tandem mass spectrometer (AB/MDS Sciex, Concord, Canada). positive electrospray (EPI) mode enhanced scan, enhanced product ion or MRM (SRM) program Turboionspray temperature 450 C Gas speed 1: 35 L/h; gas speed 2: 60 L/h DP (declustering potential) : 40 CE = - 20 ev; LIT: 20ms

10 Fragmentation of Amadori compounds +ESI-MS/MS Spectrum of [M+H] + ion of Fru-Gly Rel. Int. (%) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 97.1 88.0 76.0 99.1 -H 2 C -C 154.1 156.1 -C 126.1 143.1 174.2 184.2 220.2 202.2 -H 2 -H 2 -H 2 [M+H] + 238.2 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z, amu Rel. Int. (%) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% H 2 C CH 2 +ESI-MS/MS Spectrum of [M+H] + of Xyl-Gly -C R NH CH -C 108.1 154.2 88.1 126.1 97.1 144.2 C 172.2 -H 2 190.2 -H 2 -H 2 [M+H] + 208.2 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z, amu

Analysis of Amadori compounds in sugar-aa mixture by HPCEC-MS/MS Glucose derived Amadori compounds Xylose derived Amadori compounds 2.5e4 Fru-Pro 2.5e4 Xyl-Pro 2.3e4 Fru-Ala 1.1e4 Xyl-Ala 1.4e4 Fru-Gly 1.8e3 Xyl-Gly Intensity, cps 6.7e4 Fru-Val Intensity, cps 1.6e4 Xyl-Val 6.0e4 Fru-Leu/Ile 2.2e4 Xyl-Leu/Ile 4.3e4 Fru-Met 1.0e4 Xyl-Met 4.5e4 Fru-Phe 7.8e3 Xyl-Phe 11 5 10 15 Time, min SRM mode ([M+H] + [M+H-H 2 ] + ) 5 10 15 Time, min

Analysis of Amadori compounds in dried tomatoes by HPCEC-MS/MS SRM mode ([M+H] + [M+H-H 2 ] + ) 9.0e4 Fru-Glu 1.7e4 1.6e3 Fru-Ala Fru-Gly 0.400 Electrochemical detection Intensity, cps 1.4e4 2.0e4 Fru-Val Fru-Leu/Ile ECD Response (µc) 0.200 2.4e3 Fru-Met 0 4.0 8.0 min 12.0 3.3e4 Fru-Phe 5 10 15 Time, min 12

13 Analysis of Amadori compounds in dried tomatoes by HPCEC-MS/MS SRM mode ([M+H] + [M+H-H 2 ] + ) 9.0e4 1.7e4 1.6e3 1.4e4 Fru-Glu Fru-Ala Fru-Gly Fru-Val Rel. Int. (%) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% MS/MS Spectrum of [M+H] + ion of Fru-Glu in dried tomatoes 226.2 274.2 292.2 148.1 246.3 310.2 130.1 160.2 256.3 84.0 97.0 208.1 238.2 60 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z, amu Intensity, cps 2.0e4 Fru-Leu/Ile 100% 90% 80% MS/MS Spectrum of reference compound (Fru-Glu) 292.2 2.4e3 3.3e4 Fru-Met Fru-Phe 5 10 15 Time, min Rel. Int. (%) 70% 60% 50% 40% 30% 20% 10% 226.2 274.2 148.1 246.3 160.2 310.2 208.1238.2 256.2 97.1 130.2 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z, amu

14 Separation of Amadori compounds using CE-MS CH 3 CNH 4 CE MS/MS CE: Equipment: Hewlett-Packard HP 3D CE System equipped with the manufacturer's CE-MS interface kit uncoated fused silica capillary (132 cm, with 50 μm i.d., 375 μm o.d.) Capillary: Run buffer: 20 mmol/l ammonium acetate in water (ph 6.9) Conditions: 30kV; 10 mbar pressurised capillary MS: Equipment: Finnigan TSQ 700 triple quadrupole mass spectrometer scan mode Sheath flow 2 µl/min (20% run buffer / 80% 2-propanol) No sheath or auxiliary gas used Collision gas - Argon at 4 hpa (3.0 mtorr)

15 Detection of Amadori compounds using CE-MS Extracted trace [M+H] + 32.91 Fru-Phe Sample: Glucose + 6 amino acids 4h 70 C in Me 32.56 Fru-Met 31.55 31.69 Fru-Ile MS/MS spectrum of [M+H] + ion of Fru-Pro 100 260 Fru-Val 116 (H) C 26.0 28.0 30.0 31.05 Fru-Pro 31.29 Fru-Gly 32.0 34.0 36.0 40.0 Time (min) Relative Abundance (%) 50 0 128 50 NH 232 116 128 100 150 200 Mass (m/z) -C -H 2 242 232 250 -H 2 [M+H] + 278 300

Conclusions HPAEC-ECD (CarboPac PA-1) for the analyses of hexose-based Amadori compounds in model systems Very useful for kinetics studies No unequivocal identification Not suitable for analysis of pentose-based Amadori compounds HPCEC-ECD (CS-17) for the analyses of pentose-based Amadori compounds in model systems High sensitivity Low separation efficiency No unequivocal identification HPCEC-MS (CS-17) Universal method for the analyses of pentose and hexose-based Amadori compounds Sensitive (0.2-0.4 ppm in SRM mode) and highly selective Very useful for analysis of food samples Expensive (parent sugars not retained) CE-MS 16 Alternative method to HPIC-MS

7 TH Wartburg Symposium on Flavor Chemistry and Biology Eisenach, April 21 st - April 23 rd, 2004 Back up slides 17

Methods for the analysis of Amadori compounds Gas Chromatography High separation efficiency and sensitivity Sugar and amino acid may be analysed in the same run Time consuming - need derivatisation and clean up Separation of 11 Amadori compounds on V-101 in the form of trimethylsilyl-oxim derivatives Eichner and co-workers (1990) 18

High Performance Anion Exchange Chromatography Huyghues-Despointes and Yaylayan (1994) Ge and Lee (1996) Detection of Amadori compounds (Fru-Pro, Fru-Trp) using PAD Separation of Phe, Fru-Phe and Glu on Carbopac PA-1 19