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Mechanisms of formation of sulphide derivatives (continued)

Another interesting result is the formation (Figure 4) of compounds (20) and (23) by the thermal degradation of diallyl disulphide (11). Step (I) shows how diallyl disulphide undergoes thermal degradation to allyldithio (12) and allyl (13) radicals. Addition of the allyl and allyldithio radicals to the S-S bond of diallyl disulphide affords diallyl sulphide (15) and diallyl trisulphide (16) respectively. Allyldithio radicals (12) can also react with diallyl disulphide according to a Markovnikov addition, a process opposite to that encountered in intermolecular radical addition to simple olefins (Step II) . After rearrangement, the intermediate radical (17) gives thioacrolein (19) and a new radical (18). Addition of (18) to diallyl disulphide generates 6-methyl-4,5,8,9-tetrathiadodeca-1,11-diene (20) and this process is favoured over the formation of 4,5,9,10-tetrathiatrideca-1,12-diene (23) shown in step (III). Addition of the allyldithio radical to the terminal methylene of the allyl group of diallyl disulphide leads to an intermediate radical (21) and after rearrangement to radical (22). Addition of (22) to the S-S bond of diallyl disulphide gives (23) and an allylthio radical.
It is clear therefore that GC techniques which have commonly employed high injector port temperatures have not given a true picture of the primary flavour compounds present in garlic tissue and that many of the compounds detected by GC-MS over the last twenty-five years were 'artefacts of analysis'. By the late 1980's HPLC studies had shown the primary flavour compounds of garlic to consist almost exclusively of thiosulphinates and in 1992 a definitive study of the effect of GC conditions on thiosulphinates was undertaken. Initial experiments with GC-MS were conducted with synthetic samples and used a 12 m x 0.2 mm capillary column with on-column injection but it was soon noted that narrow bore columns rapidly lost resolution, presumably due to the deposition of non-volatiles, rendering them useless. Further studies showed that the use of wide bore (0.53 mm i.d.) columns did not suffer the same limitations and gave excellent resolution of most C2 - C6 thiosulphinates. As was expected, allied to the need for a wide bore column was the use of low GC temperatures, the GC injector and oven being initially cooled to 0°C and the GC-MS transfer line to 100°C which ensured minimum degradation of samples.

HPLC

During the late 1980's and early 1990's when GC-MS results were being subjected to increasing scrutiny, HPLC techniques capable of identifying the primary flavour compounds of garlic extracts without thermal degradation, were being developed. Although some weaknesses exist with the HPLC methods -incomplete separation of some peaks, variable retention time leading to possible misidentification of peaks and compounds having minor UV activity being overlooked - techniques are now highly developed and capable of presenting a true picture of the primary flavour compounds of allium species. HPLC analysis of allicin in garlic extracts was first reported in 1985 by Miething, who analysed diethyl ether extracts of garlic and garlic products by normal phase (Si) HPLC. This method suffers from the fact that allicin in very unstable in ether and other organic solvents that are necessary for Si-HPLC and in 1987, Jansen et al first reported on reverse-phase (C18) HPLC analysis of allicin in aqueous garlic extract.

Reversed-phase HPLC (C18-HPLC)

The separation, quantitation and variation in the amounts of all the detectable thiosulphinates present in garlic clove homogenates was first reported in 1990 together with a standard method for the quantitation of allicin using an external standard. In this report all possible thiosulphinate combinations were synthesised and Figure 5 shows that most of them were separable from each other. The separation of the allyl methyl/methyl allyl and methyl 1-propenyl/1-propenyl methyl pairs was however poor but this was subsequently improved upon by normal phase HPLC (Figure 6).
With this method sample preparation was relatively simple and filtered, aqueous homogenates were injected directly into the HPLC. The mobile phase for C18-HPLC consisted of a 50/50 mix of methanol and water and direct injections of methanol were frequently employed to extend guard column life. Hexane/isopropanol (95:5) was used for normal phase HPLC. Although the presence of the propyl group had been suggested this study was one of the first to confirm its absence from garlic clove homogenates. Although it was known that allyl propyl thiosulphinate co-elutes with 1-propenyl allyl thiosulphinate, its existence was ruled out because its isomer, propyl allyl thiosulphinate, could not be detected and no H-NMR signals characteristic of propyl groups could be found. The only thiosulphinate expected, but not found, was di-1-propenyl thiosulphinate. This compound was discovered to be highly unstable and has been shown to rapidly form two dimethyl dithiabicyclohexane oxides called zwiebelanes. This method was further extended by Lawson et al to identify and quantify a number of thiosulphinate breakdown products including sulphides, vinyl dithiins and ajoene.

Normal phase HPLC

As a result of comparative studies, Block et al concluded that the best peak resolutions were obtainable using normal phase HPLC with 2-propanol/hexane gradients. Aware of the instability of thiosulphinates in organic solvents Block's work concentrated on extraction and distillation procedures and comparative assessments. By careful planning of sample preparation, rapid extraction and analysis he was able to present a method comparable in stability to C18-HPLC methods but with the high resolution of Si-HPLC.
The preparation of fresh extracts for Si-HPLC can present a number of problems particularly with emulsion formation and the presence of plant pigments and waxy materials. This work experimented with distillation and extraction procedures and was able to provide methods with excellent quantitative and qualitative agreement. Distillation was performed with high vacuum at room temperatures and aqueous condensates were collected at -196°C. It was found that HPLC and NMR spectroscopic analysis of the CH2Cl2 extract of the salt-saturated condensate gave good qualitative thiosulphinate composition profiles. It is believed that this method of 'room temperature distillation' succeeds because of the stabilising effect of water, through hydrogen bonding, of the thiosulphinates. Extracts of aqueous homogenates were again undertaken with CH2Cl2 and performed quickly and at low temperatures: all analyses were undertaken within 30 minutes of extraction and reproducibility was excellent.

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© Mike Watson 2005


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