Preparation of external standards 

Both Si-HPLC and Cl8-HPLC rely on the availability of external standards for accurate identification and quantitation of individual compounds. Symmetrical thiosulphinates can be synthesised from the corresponding commercially available disulphides by oxidation with 3-chloroperbenzoic acid. The unsymmetrical thiosulphinates are prepared in a similar manner from their unsymmetrical disulphide precursors to yield pairs of configurational thiosulphinate isomers: allyl methyl/methyl allyl, 1-propenyl allyl/allyl l-propenyl, and methyl 1-propenyl/1-propenyl methyl (the first named residue is linked to the thio and the second to the sulphinate). The preparation of the unsymmetrical disulphides is detailed in a number of publications and involves methoxycarbonylsulphinyl chloride, prepared from chlorocarbonylsulphinyl chloride and methanol, being reacted with a thiol to give an intermediate methoxycarbonyl alkyl disulphane. This intermediate product is in turn reacted with a second thiol in the presence of triethylamine to yield the disulphide. Subsequent distillation followed by TLC yields pure products. In commercial applications it is the quantitation of the principal thiosulphinate, allicin and its precursor alliin, that is important and to that end a number of methods for the preparation of pure standards of these two compounds have been published. Mayeux et al describe a simple method whereby allicin can be synthesised by oxidising diallyl disulphide with acidic hydrogen peroxide and purified using a Si-TLC plate developed in hexane/ethyl acetate immersed in ice water. Alternatively pure allicin can be isolated by Si-TLC of the ethyl acetate extract of an aqueous solution of a quality commercial garlic powder or an homogenate of fresh garlic. (If fresh garlic is used then subsequent C18-TLC is necessary to separate the 1-propenyl allyl thiosulphinate which co-elutes with allicin on Si-TLC) Recent work has led to the development of a method for the simultaneous determination of alliin and allicin by ion-pair reversed-phase LC. The preparation involves the drying of fresh garlic slices followed by extraction with 80% methanol. The methanol is subsequently concentrated in a rotary evaporator and then partitioned with diethyl ether. The diethyl ether layer is discarded and the aqueous layer loaded onto a pre-equilibrated ion-exchange resin and allowed to drain. The resin is washed with water until washes are neutral with litmus paper and then the alliin retained on the resin is eluted with ammonium hydroxide. After freeze drying the alliin is crystallised from 70% ethanol and repeated recrystallisation gives white needles of pure alliin. Alliinase is extracted from fresh garlic homogenates and its action on alliin yields pure allicin. Both alliin and allicin prepared in this way are used as external standards for the simultaneous quantitation of the compounds in garlic samples. Aqueous extracts of the samples under investigation were injected into a TSK-gel column (150 x 4.7 mm), the mobile phase being (A) 0.01M phosphate buffer (pH 2.5) + 5 mM heptanesulphonic acid and (B) 0.01 M phosphate buffer (pH 2.5) + acetonitrile (50:50). Gradient elution and temperature control (40°C) were employed with this method which provides for a rapid and straightforward quantitative examination of garlic samples (bulbs, oils, powders, spice mixes, etc) (Figure 7).

LC-MS

During the past eight years great efforts have been made to combine the 'gentle' separation afforded by HPLC with the powerful identification capabilities of MS. Whilst early work gave promising results, it was some time before a successful method for use with alliums was published. Ferary et al used LC-MS to examine allium odours to determine whether or not they contained degradation compounds of thiosulphinates. Using a cryotrapping technique they made direct injections of aqueous solutions into the HPLC which was fitted with a UV detector and coupled to a mass spectrometer. A number of different commercial coupling and ionisation systems were tested and although best results were obtained using atmospheric pressure chemical ionisation (APCI), some degradation of thiosulphinates was observed,
Calvey et al have most recently reported on an improvement to the above method using reversed phase LC-MS and LC tandem MS (LC-MS-MS) with APCI coupling. All of the major thiosulphinates were readily characterised by this technique and for the first time trace amounts of propyl compounds were found in garlic.(The low UV absorbance, low concentration and thermal lability of these compounds had made previous detection by other means difficult). Using LC-APCI-MS the data based solely on the protonated molecule and adduct ions was often ambiguous but the MS-MS spectra show that the regioisomers can be uniquely distinguished (Table 2, Figure 8). 

Table 2. APCI-MS-MS data of thiosulphinates found in extracts of Allium spp.

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© Mike Watson 2005