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).
(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
(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 (AlCSO) into allicin (TiAl2) 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).
Compound
Name
S-allyl-L-cysteine
sulfoxide; alliin
1
Chemical structure
Abbreviation
H2N
AlCSO
S
COOH
O
NH2
γ-glutamyl-S-allyl cysteine
2
N
GLUAlCS
S
HOOC
COOH
O
NH2
γ-glutamyl-S-trans-1propenyl cysteine
3
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 C18 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).
GLUPeCS
N
S
HOOC
COOH
O
NH2
γ-glutamyl phenylalanine
4
GLUPheAla
N
HOOC
O
diallyl thiosulfinate; allicin
5
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).
NH2
[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.]
S
S
Allinase
H2N
R
N
HOOC
TiAl2
γ-glutamyltranspeptidase
H
S
O
R
S
S
R
CO2 H O
O COOH
C
O
γ-glutamyl-S-alk(en)yl-L-cysteine
O
H
COOH
O
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 C18; 150x3mm, 3µm (ThermoQuest, Hypersil)
flow: 0,4 ml/mn; temperature:36°C; UV detection at 208nm
Eluent A: 20mM sodium dihydrogen phosphate + 10mM heptane sulfonic
acid, adusted at pH 2.1 with orthophosphoric acid (85%).
Eluent B: eluent A/acetonitrnile 50% (v/v).
Gradient: Time (mn) %A %B
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 pH 3
Sheat gas flow rate:
30%
Capillary Temp:
250°C
Aux.gas flow rate:
50%
Capillary Voltage:
23 V
Ion Spray Voltage:
5 kV
Tube lens offset:
0V
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
0,5
1
5
0,4
0,4
2
0,3
2
0,3
3 4
3 4
0,2
0,2
0,1
0,1
0
0
-0,1
-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 3a) or incubated (fig 3b).
100
180
54.9
0 60
16,0
100
m/z
m/z
162.0
140
MS2
140
m/z
MS3
144.9
290.9
180
MS4
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
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)
MS1
295.1
200
MS2
MS3
120.0
300
m/z
00
100
200
300
m/z
0 60
100
140
180
m/z
MSn spectrum of γ-glutamylphenyl alanine (4)
Fig 2: MSn spectrums of dipeptides
12,0
10,0
8,0
0
100
200
400
116.9 145.0
126.9
60 80 100 120 140160 180
Fig 4: Alliin variation with sulfur fertilisation
m/z
CONCLUSION
- This new gradient ion-pair HPLC method without sample derivatization allows
simultaneous quantification of alliin, allicine and dipeptides.
166.0
148.9
120. 278.1
166.1
0 100
166.0
14,0
Sulfur fertilisation level
72.9
m/z
mg/g dry powder
162.0
60
MSn spectrum of γ-glutamyl-S-allyl cysteine (2)
MS1
-Application: alliin concentration of garlic powder increases significantly with sulfur fertilisation (fig 4).
concentration of alliin in
291.0
0 100 140 180 220 260
m/z
-Alliin and GLUACS linearity are respectively R2=0.9576 and R2=0.9444.
MS4
72.9
18,0
144.9
214.1230.9
100 150 200 250
144,9
161.9
291.0
0
MS3
MS2
MS1
- 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 QLK1-CT-1999-0498
from the European Union under the Quality of Life program.
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