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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