Ionpair HPLC method for simultaneous analysis of alliin

Ion-pair HPLC method for simultaneous analysis of alliin, allicin and dipeptide precursors in garlic products using MSn and UV. Christidès J. P. (1), Arnault I. (1), Doussineau T. (1), Mandon N. (1), Haffner T. (2), Kahane R. (3) & Auger J. (1). Université François Rabelais, IRBI CNRS UMR 6035, Parc de Grandmont, 37200 Tours. France. Head Product Development, Lichtwer Pharma AG, Wallenroder Straße 8 -10, D-13435 Berlin, Germany. (3) Coopd'Or R&D, INRA, laboratory of Physiology and in vitro culture, 21100 Bretenieres. France (1) (2) INTRODUCTION Table 1: Name and chemical structure of garlic compounds analysed Many beneficial health related biological properties are attributed to sulphur compounds of garlic (J Nutr 2001: 131 supl ). and particularly to the thiosulfinates(Ti). When the clove of garlic is cut or crushed, the enzyme alliinase is released and transforms alliin (Al. CSO) into allicin (Ti. Al 2) which is very unstable giving rapidly the other related organosulphur compounds like (poly)sulfides, thiosulfonates, vinyl-dithiines or ajoens. (Block, E, 1985: Sci Am 252: 94 -99) Furthermore γ-glutamyl-S-alk(en)ylcysteines appear to provide a reserve for alk(en)ylcysteine sulfoxides (Lawson et al 1991: J of Natural Products, 54, 436 -444 ) (Fig 1) (table 1). The quality of garlic and garlic products is usually associated only to their alliin and allicin contents, Until now no analytical method was able to quantify simultaneously all the sulphur compounds implicated in allicin potential until dipeptide precursors. Good RP C 18 HPLC methods were able to separate dipeptides and allicin but alliin was not sufficiently retained (Mutsch-Eckner et al 1992: J Chromatogr, 625, 183 -190). Compound Al. CSO 2 γ-glutamyl-S-allyl cysteine GLUAl. CS 3 γ-glutamyl-S-trans-1 propenyl cysteine GLUPe. CS 4 γ-glutamyl phenylalanine GLUPhe. Ala 5 diallyl thiosulfinate; allicin Ti. Al 2 NH 2 S COOH S O R S S R CO 2 H O O COO C H O γ-glutamyl-S-alk(en)yl-L-cysteine O H O Allinase H 2 N R N HOOC H 2 N γ-glutamyltranspeptidase H Chemical structure Abbreviation S-allyl-L-cysteine sulfoxide; alliin 1 We have focused here on the use of ion-pairing reagents to succeed in obtaining a good separation of all the compounds, due to they common ionisable nature. (Fialaire et al. 1993: J liq chromatogr, 16, 3003). [Synthesis of dipeptides was validated usinga Thermo Finnigan LCQ quadripole ion trap mass spectrometer equipped with a electrospray ionization (ESI) source. [M+H]+ ions were formed using ESI in positive mode (fig 2). Synthetic reference compounds were characterized by the same chromatographic method using a diode-array UV detector. ] Name S R Dialk(en)yl thiosulfinates S-alk(en)yl-L-cysteine sulfoxides R=Trans-1 -propenyl, allyl, methyl, propyl MATERIAL AND METHODS Material: Ion-pair HPLC: pump: 616 Waters. ; UV detector: diode-array DAD 966 Waters. MSn analysis: mass spectrometer: Thermo Finnigan LCQ quadripole ion trap. Methods: Ion-pair HPLC: Column: Hypurity Elite C 18; 150 x 3 mm, 3µm (Thermo. Quest, Hypersil) flow: 0, 4 ml/mn; temperature: 36°C; UV detection at 208 nm Eluent A: 20 m. M sodium dihydrogen phosphate + 10 m. M heptane sulfonic acid, adusted at p. H 2. 1 with orthophosphoric acid (85%). Eluent B: eluent A/acetonitrnile 50% (v/v). Gradient: Time (mn) %A %B 0 100 0 26 0 100 5 70 30 28 0 100 25 46 54 30 -40 100 0 MS parameters Ion Trap: LCQdeca Thermo finnigan Mode: positiv electrospray ionisation Flow rate: 10 µl/min Solution: amoniumformiat buffer p. H 3 Sheat gas flow rate: 30% Capillary Temp: 250°C Aux. gas flow rate: 50% Capillary Voltage: 23 V Ion Spray Voltage: 5 k. V Tube lens offset: 0 V Fig. 1 : Biosynthesis of dialk(en)yl thiosulfinates from γ-glutamyl-S-alk(en)yl-L-cysteine via S-alk(en)yl-L-cysteine sulfoxides 0, 5 1 5 0, 4 2 0, 3 3 4 0, 2 0, 1 0 0 -0, 1 0 5 10 15 20 0 25 5 10 15 20 25 Same garlic powder: active alliinase activity (b) Garlic powder- inhibited alliinase activity (a) Fig 3: Chromatograms of garlic powders RESULTS -We obtained good profiles of garlic powder crude (fig 3 a) or incubated (fig 3 b). 100 150 200 250 144, 9 162. 0 60 m/z 100 MSn spectrum of γ-glutamyl-S-allyl cysteine (2) MS 1 162. 0 -Application: alliin concentration of garlic powder increases significantly with sulfur fertilisation (fig 4). 18, 0 291. 0 0 100 140 180 220 260 m/z -Alliin and GLUACS linearity are respectively R 2=0. 9576 and R 2=0. 9444. MS 4 72. 9 MS 2 140 m/z 180 54. 9 0 60 16, 0 100 140 m/z MS 3 144. 9 290. 9 180 MS 4 54. 9 98. 9 170. 0 145. 0 122. 0 0 100 150 200 250 291. 0 273. 1 0 100 140 180 220 260 m/z 72. 9 116. 1 161. 8 58. 2 115. 4 0 60 80 100120140 160180 0 m/z MSn spectrum of γ-glutamyl-S-trans-1 -propenyl cysteine (3) MS 1 295. 1 200 MS 2 MS 3 120. 0 166. 0 148. 9 120. 278. 1 166. 1 0 100 166. 0 300 m/z 00 100 200 300 m/z 0 60 100 140 m/z 180 MSn spectrum of γ-glutamylphenyl alanine (4) Fig 2: MSn spectrums of dipeptides mg/g dry powder 144. 9 214. 1230. 9 0 MS 3 MS 2 161. 9 291. 0 concentration of alliin in MS 1 14, 0 12, 0 10, 0 8, 0 0 100 200 400 Sulfur fertilisation level 116. 9 145. 0 126. 9 60 80 100 120 140160 180 m/z Fig 4: Alliin variation with sulfur fertilisation CONCLUSION - This new gradient ion-pair HPLC method without sample derivatization allows simultaneous quantification of alliin, allicine and dipeptides. - It requires no sample preparation and only standard instrumentation. - This method is well adapted to routine analysis and quality control of garlic products. This work was financially supported by grant QLK 1 -CT-1999 -0498 from the European Union under the Quality of Life program.