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Analytical
Methods
Advancing Methods and Applications
www.rsc.org/methods
ISSN 1759-9660
CRITICAL REVIEW
Clarke
Glucosinolates, structures and analysis
in food
PAPER
Schazmann et al.
A wearable electrochemical sensor for
the real-time measurement of sweat
sodium concentration
1759-9660(2010)2:4;1-7
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CRITICAL REVIEW
1. Introduction
Glucosinolates (GLS), b-thioglucoside-N-hydroxysulfates (cisN-hydroximinosulfate esters) are sulfur rich, anionic secondary
metabolites found almost exclusively within the plant order
Brassicales (Fig. 1). Various aspects of glucosinolates research
have been reviewed; nutraceutical compounds in broccoli,1 the
biochemical genetics of secondary metabolites in Arabidopsis
thaliana,2 the dietary role of glucosinolates,3 the role and effects
of glucosinolates of Brassica species,4 the enzymatic and chemically induced decomposition of glucosinolates,5 the biology and
biochemistry,6 their role in insect-plant relationships,7 their
2. Glucosinolate structures
The Food and Environment Research Agency (Fera), Sand Hutton, York,
YO41 1LZ, UK. E-mail: don.clarke@fera.gsi.gov.uk; Fax: +044-1904462133; Tel: +044-1904-462000
Electronic supplementary information (ESI) available: A database of
structures, formulae and accurate masses of both the 200 known, and
a further 180 predicted GLS, for use in mass spectrometry; Fig. S1.
Further glucosinolates; Fig. S2. Screening for cinnamoyl and benzoyl
esters; and Table S1. Some sources of Brassicale seeds in the UK. See
DOI: 10.1039/b9ay00280d
Glucosinolates are characterized by a core sulfated isothiocyanate group, which is conjugated to thioglucose, and
a further R-group. Both the glucose and the central carbon of the
isothiocyanate are often further modified. This results in
a diverse range of glucosinolate structures (Fig. 1). These are
broadly classified as alkyl, aromatic, benzoate, indole, multiple
glycosylated and sulfur containing side chains. The R chains may
then contain double bonds, oxo, hydroxyl, methoxy, carbonyl or
di-sulfide linkages. Since 2001 it has been generally agreed that
there are 120 distinct individual glucosinolates14 and this is still
almost invariably the quoted number.7 In a 2004 survey of seeds
screened for 66 intact glucosinolates, four were not included in
the accepted list of 120.15 Bellostas (2007) increased the number
to 133, but this list has not been recognized by most subsequent
researchers.16 As these three lists overlap incompletely, the Bellostas review adds a further 25 new structures raising the total to
149. Since this total has not been systematically reviewed since
2001, the number of reported glucosinolates is now approaching
200 (Fig. 1). A number of plants contain only a single glucosinolate, the majority contain 25, while 34 individual glucosinolates are reported in the seeds and leaves of a collection of
ecotypes of Arabidopsis thaliana.17 In some respects, the number
of possible structures is limited by the R-group being restricted to
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Fig. 1 (continued) Reported glucosinolates structures by chemical class. A thorough literature review has lead to a much greater number of individual
glucosinolates being characterized than previously thought. This reflects the absence of any major reviews or advances in this area since 2001. We
currently have listed 200 structures, for a LC-TOF-MS screening library, with e.g. 32 of these being of relevance to the UK diet.
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3. Taxonomic classification
Knowledge of the genetic-based regulation of the accumulation
of glucosinolates in plants is essential to explain the limited
occurrence of individual structures in the various plant species.
The mustards or cabbages are a family of flowering plants
(Angiospermae) known either as the Brassicaceae or Cruciferae.
Cruciferae is the older, but equally valid name, meaning crossbearing, because the four petals of the flowers are reminiscent of
a cross. Historically there has been confusion over the
This journal is The Royal Society of Chemistry 2010
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lineage with many small but distinct clades and a large Brassicaceae family containing 93% of species within the order. The
Brassicales order and Brassica genus are remarkable in that they
each contain more commercially important agricultural and food
crops than any other. With the minor exceptions of capers,
papaya, nasturtium and the horseradish tree (Moringa oleifera),
which are spread through the other orders, all of the species of
dietary importance are contained in the core Brassicaceae order.
This order is then divided into four clades and 25 tribes. With
horseradish and cresses in the Cardamineae and Lepideae tribes
of Clade 4. All other food genera (Alliaria, Bunias, Crambe,
Diplotaxis, Euruca, Raphanaus, Sinapis, Wasabi) are now
Fig. 2 Summarized taxonomy of the order Brassicales indicating the current relationship of all the glucosinolate producing families.3639,42 Numbers are
the approximate numbers of genus and or species in each branch. These numbers are in flux and question marks denote a lack of clear data. Each branch
is exemplified by a species used either in previous phylogenetics reclassifications, or as a food crop.
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Common
name
Ethiopian mustard
Indian mustard
Chinese mustard
Red giant mustard
Wrapped heart mustard
cabbage
Yellow mustard
Canola/rape seeds
Siberian kale
Rutabaga (swede)
Black mustard
Kale
Kai-lan (Chinese broccoli)
Cauliflower
Romanesco broccoli
White cabbage (drum)
Pointed cabbage
Red cabbage
Savoy cabbage
Brussels sprouts
Kohl rabi
Broccoli
Broccoflower
Komatsuna
Collard greens
Mustard spinach
Pak Choi (Cantonese)
Broad beak mustard
Mibuna
Choy Sum (false Pak Choi)
Chinese cabbage
Komatsuna
Senposai
Purple stem mustard
Turnip
Tatsoi (rosette Pak Choi)
Rapini (broccoli raab)
Common name
Number of consumers
Population mean
Consumer mean
Broccoli
Head cabbage
Cauliflower
Brussels sprouts
Kohl rabi
Chinese cabbage
Totals
743
737
767
212
0
26
1341
6.4
5.9
5.6
1.9
0.2
19.9
14.7
13.7
12.6
15.9
10.1
Consumer
max
80.7
112.1
158.7
72.7
66.4
158.7
Brassica species
Brassica oleracea var. italica
Brassica oleracea var. capitata f. alba
Brassica oleracea var. botrytis
Brassica oleracea var. gemmifera
Brassica oleracea var. gongylodes
Brassica rapa var. pekinensis
a
Reproduced and adapted from: Henderson L., Gregory J. Swan G. National Diet and Nutrition Survey: adults aged 1964 years. Volume 1: types and
quantities of foods consumed, The Stationery Office 2002.43
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5. Analysis
5.1 Stability
There appears to be little quantitative data documenting the
stability of glucosinolates during processing and extraction.
Generally extractions are conducted at temperatures of 65100 C,
close to the solvent or mixtures boiling point, on the assumption
that the overarching concern should be the inactivation of
myrosinase.5559 The benefit of this assumption is brought into
question by data showing 80% degradation of glucobrassicin
(3-indolylmethyl-GLS) within 5 min at 100 C and 120 bar when
extracting into 70% methanol in water in a pressurized liquid
extractor. The optimal yield was obtained at 50 C.60 Processing
in the presence of a denaturing agent such as methanol should be
sufficient to ensure GLS are not hydrolysed by myrosinases.61
Current procedures to minimize degradation include, harvesting
into liquid nitrogen, microwave induced deactivation and freeze
drying before homogenisation.50,62 There is a lack of clear validation data available on the effects of avoiding high temperatures and the significance of myrosinase inactivation. Thermal
degradation has been studied in red cabbage, where cooking
reduced indole (38%) and alkyl (8%) content. Canning was the
most severe heat treatment studied (40 min, 120 C) and reduced
total-GLS by 73%.55 The vegetable matrix itself has an effect on
thermal stability, after microwave inactivation of myrosinase,
with the cellular environment of Brussels sprouts being one of the
least favourable with glucobrassicin content halving within
10 min at 100 C and gluconapin halving within 35 min.63 Cold
316 | Anal. Methods, 2010, 2, 310325
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example, where the enzymatic cascade from GLS through isothiocyanate to glucose and on to hydrogen peroxide has also
been automated. Biosensing using amperometric enzyme electrodes based on glucose oxidase and tyrosinase were utilized after
conversion to glucose and thiourea.87 Biosensing with a myrosinase-immobilized eggshell membrane, with glucose oxidase
activity and an oxygen-sensitive optode membrane, measuring
depletion of dissolved oxygen has also been reported.88,89 The
first amperometric flow analyzer based on the biosensor concept
was described in 2003, using myrosinase and glucose oxidase.90
A gold nanoparticle-carbon nanotube composite electrode using
myrosinase and glucose oxidase with Teflon as the non-conducting binding material is also under development, but is as yet
unpublished.
Near infrared reflectance spectroscopy (NIRS) is a validated
non-destructive technique, in which OH, CH and NH groups
are associated with total glucosinolate content.91 This simple
method continues in use, since protein and oil content are also
determined simultaneously along with the GLS content.92 The
official X-ray fluorescence method determines total sulfur
(ISO 9167-2 1994).93 ELISA assays have been investigated, but
recent reports are lacking.69
C4
C5
C6
C7
C8
Ind
Ar
Glucocapparin
Sinigrin
Glucoibervirin
Glucoiberin
Glucocheirolin
Glucoputranjivin
Glucosisymbrin
Glucoerysimumhieracifolium
Gluconapin
Progoitrin
Epiprogoitrin
Glucoerucin
Glucoraphasatin
Glucoraphanin
Glucoraphenin
Glucoarabidopsithalianain
Glucoconringiin
Glucoalyssin
Glucobrassicanapin
Gluconapoleiferin
Glucocleomin
Glucolesquerellin
Glucohesperin
Glucoarabishirsutain
Glucoarabishirsuin
Glucohirsutin
Glucobrassicin
4-Hydroxyglucobrassicin
4-Methoxyglucobrassicin
Neoglucobrassicin
Glucotropaeolin
Glucosinalbin
Gluconasturtiin
Glucobarbarin
Glucomalcomiin
R Side chain
Methyl
2-Propenyl
3-Methylthiopropyl
3-Methylsulfinylpropyl
3-Methylsulfonylpropyl
1-Methylethyl
2-Hydroxy-1-methylethyl
3-Hydroxypropyl
3-Butenyl
(2R)-2-Hydroxy-3-butenyl
(2S)-2-Hydroxy-3-butenyl
4-Methylthiobutyl
4-Methylthio-3-butenyl
4-Methylsulfinylbutyl
4-Methylsulfinyl-3-butenyl
4-Hydroxylbutyl
2-Hydroxy-2-methylpropyl
5-Methylsulfinylpentyl
Pent-4-enyl
2-Hydroxy-pent-4-enyl
2-Hydroxy-2-methylbutyl
6-Methylthiohexyl
6-Methylsulfinylhexyl
7-Methylthioheptyl
8-Methylthiooctyl
8-Methylsulfinyloctyl
3-Indolylmethyl
4-Hydroxy-3-indolylmethyl
4-Methoxy-3-indolylmethyl
N-Methoxy-3-indolylmethyl
Benzyl
p-Hydroxybenzyl
2-Phenethyl
(2S)-2-Hydroxy-2-phenethyl
3-Benzoyloxypropyl
4-Benzoyloxybutyl
Buchner EC
Haughn ISO AOCS Brown Vinjam Wathelet Recommended
1987102 1990103 19912
199257 199859 200350 2004104 200420
value
1.00
1.00
1.00
1.00
1.07
1.07
1.07
1.07
1.0
1.0
0.8
1.2
0.9
1.0
1.05
1.13
1.25
1.00
1.07
1.26
1.32
1.00
1.09
1.11
1.09
1.09
1.11
1.09
1.09
1.11
1.09
1.09
1.0
1.07
1.00
1.07
1.07
0.9
0.9
1.4
1.13
1.07
1.15
1.00
1.07
1.15
1.00
0.9
1.13
0.20
0.95
0.29
0.28
0.25
0.20
0.95
0.25
0.95
0.41
1.05
1.11
1.09
1.09
1.04
0.40
1.07
1.00
1.07
1.15
1.00
1.07
1.0
1.0
1.0
1.1
1.1
1.0
0.29
0.28
1.17
1.15
1.15
0.9
1.48
1.07
2.1
1.0
0.29
0.28
0.25
0.20
0.95
0.29
0.28
0.25
0.20
0.95
0.95
0.95
0.8
0.4
1.0
0.4
0.3
0.31
0.29
0.26
0.21
1.00
1.00
0.29
0.28
0.25
0.20
0.95
0.50
0.95
1.09
1.25
1.00
0.8
1.07
1.26
1.0
1.32
2.1
1.11
1.09
1.09
1.04
0.40
1.07
0.9
1.4
1.00
1.07
1.15
1.00
1.07
1.0
1.0
1.0
1.1
1.1
0.29
0.28
0.25
0.20
0.95
0.50
0.95
1.09
0.4
0.3
Thymol-sulfuric acid assay derived UV response factors [relative proportionality factors (RPF)] for desulfoglucosinolates. EC 1990 method EEC
1864/90 1990.103 ND retention time provided without a response factor. No data available for: ethyl, propyl, butyl, 4-methylsulfonylbutyl,
5-methylthiopentyl, 7-methylsulfinylheptyl, (2R)-2-hydroxy-2-phenethyl.
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desulfation process has been miniaturized and adapted to a 96well filter plate format for 5 mg seed and 10 mg of leaf samples.17
The desulfation process typically acts as the combined sample
extraction and clean-up step.
Rationalization of detection of glucosinolate degradates as
a means of verifying the (implied) presence of the parent GLS is
still in general use,18 but there are clear limitations to the specificity and accuracy of these degradative approaches. In this
review it is accepted that the presence of an isothiocyanate or
a desulfo-GLS is proof of the parent GLS.
The HPLC-UV and GC-FID (flame ionisation detector)
methods of measuring desulfo-glucosinolates were rigorously
validated with no reported difference between the two detection
techniques, when measuring eleven analytes in rapeseed by
HPLC and seven by GC.101 GC-based techniques are fraught
with difficulties and hence are mostly considered unsuitable for
identification and quantification.61
5.5 Response factors for desulfated glucosinolates
The accuracy of the HPLC-UV desulfo-method clearly rests on
a correct approach to numerical quantification, i.e. it relies on
relative response factors (RRF) of the desulfo-glucosinolates
(dsGLS). These are calculated from individual purified standards relative to the response of sinigrin in the thymol-sulfuric
acid UV assay. The majority of response factors have been
continuously transcribed from the original work,102 from which
three official methods were derived (EEC 1864/90 1990,103 ISO
Chain length
Trivial name
R Side chain
Food source
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
C1
C2
C3
Glucocapparin
Glucolepidiin
Glucoputranjivin
Sinigrin
Glucoiberin
Glucoibervirin
Glucocheirolin
Glucocapparisflexuosain
Gluconapin
Progoitrin
Epiprogoitrin
Glucoerucin
Glucoraphanin
Glucoerysolin
Dehydroerucin
Glucoraphenin
Glucobrassicanapin
Glucoberteroin
Glucoalyssin
Gluconapoleiferin
Glucosiberin
Glucohirsutin
4-Hydroxyglucobrassicin
Glucobrassicin
4-Methoxyglucobrassicin
Neoglucobrassicin
Glucotropaeolin
Glucosinalbin
Gluconasturtiin
Glucobarbarin
Glucosibarin
Methyl
Ethyl
Propyl
Isopropyl
2-Propenyl
3-Methylsulfinylpropyl
3-Methylthiopropyl
3-Methylsulfonylpropyl
Butyl
3-Butenyl
(2R)-2-Hydroxy-3-butenyl
(2S)-2-Hydroxy-3-butenyl
4-Methylthiobutyl
4-Methylsulfinylbutyl
4-Methylsulfonylbutyl
4-Methylthiobut-3-enyl
4-Methylsulfinylbut-3-enyl
4-Pentenyl
5-Methylthiopentyl
5-Methylsulfinylpentyl
2-Hydroxy-pent-4-enyl
7-Methylsulfinylheptyl
8-Methylsulfinyloctyl
4-Hydroxy-3-indolylmethyl
3-Indolylmethyl
4-Methoxy-3-indolylmethyl
N-Methoxy-3-indolylmethyl
Benzyl
p-Hydroxybenzyl
2-Phenethyl
(2S)-2-Hydroxy-2-phenethyl
(2R)-2-Hydroxy-2-phenethyl
Capers
Radish
Cabbage
Radish
Cabbage
Cabbage
Cabbage
Cows milk
Cabbage
Cabbage
Cabbage
Sea kale
Cabbage
Broccoli
Cabbage
Daikon radish
Radish
Chinese cabbage
Cabbage
Rocket
Swede
Watercress
Watercress
Cabbage
Cabbage
Cabbage
Cabbage
Cabbage
Mustard
Cabbage
Land cress
White mustard
C4
C5
C7
C8
Ind
Ar
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assigning a class specific value e.g. 0.3 for indoles, 0.4 for benzoyl
ester is recommended. It is therefore crucial to ensure that
updated values are used when comparing data. A more rigorous
approach to the documentation of experimental procedures is
needed, including the listing of all RPF values used in each work.
It is unacceptable to refer to the official methods and the limited
range of factors therein. All individual ds-GLS components in
a sample should be separated chromatographically and then
integrated down to the 1% level. This process however then relies
on the attainment of precisely reproducible retention times.
Assigning the correct name-peak combinations in each new plant
material requires careful comparison or the use of LC-UV-MS.
While all researchers quote the official methods, a major problem
in accuracy and cross comparison is the failure to indicate exactly
which factor from Table 3 is used for each analyte. It is noted
that the desulfation protocol was optimised for the analysis of
gluconapin, epi- and progoitrin in rapeseed, and that velocity of
desulfation and feedback inhibition were critical parameters.
Other GLS such as glucoiberin require removal of hydrolysed dsGLS and a second incubation.20 A flow through bioreactor with
nylon-immobilised sulfatase has been used for large scale
desulfation.105
5.6 Chromatography
The current state-of-the-art in the analytical measurement of
GLS is for HPLC-MS analysis of the intact glucosinolates
reconstituted in water.15,22,106,107 This approach has yet to be
cross-validated against any of the validated official methods.
While the change of detection systems from UV to mass spectrometry (MS) detection has been a natural progression, the most
important change is arguably in the choice of the chromatographic stationary phase. Novel approaches, such as supercritical fluid chromatography,108 micellar electrokinetic109,110 and
capillary zone electrophoresis have found use.111 Hydrophilic
interaction liquid chromatography (HILIC) has been investigated,112 employing second-generation HILIC phases based on
silica zwitterions, which are reportedly more robust and reproducible than the original polyhydroxylethyl aspartamide
columns.113 Earlier applications of anion exchange and porous
graphite phases have not progressed to date.114,115
The use of octadecyl (C18) reverse phase remains the preferred
chromatographic approach. When not constrained to a MS
compatible buffering system, ion-pairing chromatography with
5 mM tetraoctylammonium bromide,56 tetrapentylammonium
bromide and triethylamine/formate24,116 remain viable. The
strong acid modifier trifluoroacetic acid (0.10.5% TFA) still
finds regular use as buffer, despite clear incompatibility issues
with MS/MS detection.100,114,116118 These TFA based chromatographic separations however remain the benchmark for
analysis of intact glucosinolates.15,116 Separations of intact GLS
is difficult to achieve without ion-pair buffers, and the use of an
acetonitrile/water mixture without any buffer has been
reported,32 as well as the use of 30 mM ammonium acetate pH
5.0 (formic acid),75 10 mM ammonium formate (formic acid),60
and 5 mm NH4$acetate.107,119 The use of formic acid mobile
phase modifier coupled with 100% aqueous compatible columns,
shows great promise as a viable alternative without the
involvement of nonvolatile ion-pair agents or TFA, and modern
320 | Anal. Methods, 2010, 2, 310325
separations are now directly comparable with the earlier separations; e.g. water (0.1% HCOOH)/acetonitrile, with Luna C18
column,21 water/acetonitrile each with 0.1% formic acid.34,120,121
Early claims of simultaneous analysis of intact and desulfated
glucosinolates were unsubstantiated.114 Ion-pair reagents function by neutralizing the charge on the sulfate group and the most
appropriate modern stationary phases function by minimizing
this effect almost solely by hydrophobic interactions, hence
analysis of both intact and desulfo-glucosinolates can now be
readily achieved with surprisingly small retention time shifts in
the same chromatographic run without the need to change the
mobile phase. It is therefore recommended that this approach be
considered for assessing desulfation efficiency.
5.7 Mass spectrometry
The majority of the currently available mass spectrometry
ionization techniques and detector configurations have been
reported for GLS detection. This includes fast atom bombardment (FAB)122 and matrix assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF),123,124 atmospheric pressure chemical ionization (APCI) and electrospray
ionization (ESI). Ion traps,21,75,125 single quadrupole
(LC-MS)15,74 and tandem quadrupole (LC-MS/MS) instruments118 have all been utilised. Quantification of known target
analytes in single plant varieties is more commonly undertaken
using quadrupole instruments.15,117 The preferred configuration
for rapid identification of glucosinolates in crude plant extracts is
ESI-LC-TOF.34,60,74,120,125 However, LC-MS cannot discriminate
between the numerous GLS isomers. As an illustration, all three
of the possible isomers for the 200 , 300 and 400 -acetylation of
rhamnose-benzyl-GLS were readily observed, but the isomeric
positions could not be assigned.22
Precursor ion scanning can be used to locate all masses that
produce the ions m/z 75 [S]C]NOH], 80 [SO3H], 96 [SO4]
and 97 [SO4H],107 which is an advance over selected ion monitoring (SIM) for the same ions.15 Fragmentation patterns have
been studied (Fig. 4), and match well with those data acquired by
LC-MS/MS),119 and by Q-TOF.,125 whilst differing from those by
ion-trap,106 an extension of the fragment naming system of Fabre
is proposed (Fig. 4).
Regulation of the biosynthetic pathways to glucosinolate
production is central to Brassica metabolomics. Qualitative
identification has progressed to become a sensitive tool for
focused metabolomic analysis.106 One approach is based on
a tandem quadrupole mass spectrometry, by multiple reaction
monitoring (MRM) as the RIKEN database.121,126 A simpler
approach is LC-TOF.34,120
5.8 Quantification
The most modern mass spectrometry studies on GLS analysis are
able to report glucosinolate contents using semi-quantitative
methods, whereby concentrations of other GLS are calculated
using the response of sinigrin as a single calibration standard.126
While linear in response, the individual analyte calibration lines
have been shown to be offset in slope 3-fold. The variation in
absolute response makes semiquantitation (using one standard in
place of another) inaccurate.117 CRMs and the corresponding
This journal is The Royal Society of Chemistry 2010
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Fig. 4 MS/MS fragmentation pattern of glucosinolates. Illustrated with sinigrin (2-propenyl-glucosinolate). The glucosinolate molecule fragments
about the central isothiocyanate group, with cleavage of the alkyl, glucose and sulfate chains resulting in major ions for hydrogen sulfate m/z 97, sulfate
radical anion m/z 96 and N-hydroxy-isothiocyanate m/z 75. Minor ubiquitous ions based on cleavage of the thioglucose and transfer of the sulfate group
Glc1-5 m/z 195, 241, 259, 275 and 291 are present in most glucosinolate spectra. Other diagnostic ions are dependent on the R-group and are M-SO3
[M-80], M-glucose [M-162], M-thioglucose (+hydroxyl) [M-178] and M-thioglucose-SO3 [M-242].21,106,107,119,125
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Fig. 5 Isotopic purity determination of 13C6-labelled sinigrin analytical standard, as full scan mass spectra by infusion of 10 mg/ml solutions in aqueous
0.1% formic acid. The labelled form has no measurable isotopic impurities from MH to M + 4 (grey box), and is >99% isotopically pure with a <1%
trace of [12C113C5]-glucose]sinigrin. The unlabelled form has no isotopic contribution form M + 3 to M + 7 (grey box). There is no overlap in the
respective measurement channels (grey boxes), indicating suitability for use in isotope dilution standardisation.
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infrequently.21,55,56,58,75 Indicative values of individual glucosinolate contents are given and portions of these CRMs and the
corresponding indicative values have been used to construct
calibration curves to quantify these known GLS.75
5.12 Labelled internal standards
Concurrently with obtaining pure individual analytical standards, stable isotope labelled glucosinolates are required for use
as internal standards (IS) in mass spectrometric determination by
isotope dilution mass spectrometry (IDMS). To reduce interference due to the natural abundance isotopic distribution in the
mass spectrum, a suitable internal standard should have at least
a +3 mass unit difference from the native analyte. Labels must be
both stable and non-exchangeable and hence the use of deuterated standards is therefore discouraged. Most of the existing
methods for isotopic labelling of GLS have incorporated either
unstable deuterium136 or the isotopes into the side chain, which
are more appropriate for metabolism studies, for example,
[2,3,4,5,6-2H5]-phenylglucosinolate.137 The drawback of this
approach is that a separate synthesis is required for each glucosinolate. In a greatly simplified approach, specifically to produce
IDMS internal standards, our collaborators have prepared the
13
C6-labelled building block 2,3,4,6-tetra-O-acetyl-1-thio-b-D[13C6]glucopyranose. Coupling of this intermediate with various
hydroximoyl chlorides affords the synthesis of any GLS in
another three-step sequence. Three typical 13C6-labelled GLS
were synthesised [glucose-13C6]-gluconasturtiin, [glucose-13C6]sinigrin and [glucose-13C6]-glucoerucin.138 An isotopic purity
assessment of [13C6]-sinigrin relative to unlabelled sinigrin is
shown in Fig. 5 and these will be included in an IDMS-LC-MS
validation against the official methods in due course.
5.13 Workplan for future research
This review highlights the basic aspects of glucosinolates research
that are of importance to analytical chemists, and there is
considerable scope for further progress. Readers are initially
invited to add to the list of identified glucosinolates with any
omissions, or new structures and other data, such as response
factors and other relevant comments. An updated list would then
be published as a technical note. Availability of the required
range of individual analytical standards, together with their
purity measurements is still hindering progress, and perhaps this
will be the most difficult issue to resolve. Very little work has
been carried out on enhancing stability with stabilisers and
keepers and formal stability data are needed, especially in
aqueous solution. An integrated programme is required where
for example after ensuring the removal of ionic species such as
free sulfate by ion exchange SPE, the water and glucosinolate
ratios are established using quantitative NMR. A comprehensive
table of assigned consensus extinction coefficients of monohydrated-GLS is required before any meaningful purity correction factors can be applied. Further work is needed on the
optimisation of extraction conditions with the view to documenting enhanced chemical and thermal stability by processes
such as microwave inactivation of enzymes prior to room
temperature extractions. Chromatographic separation techniques and mass spectrometric assays continue to improve and it
This journal is The Royal Society of Chemistry 2010
6. Discussion
Glucosinolate research continues to progress, with new technologies being applied to many well established problems.
Enzymatic desulfation and the use of relative response factors
remains the favoured route to the quantification of GLS in more
complex samples. However, there remains a need for simple,
sensitive, and robust, automated methods for the determination
of intact glucosinolates that can be readily transferred into the
wider analytical community. A mobile phase gradient with
water/methanol and formic acid (0.1%) on a 100% aqueous
compatible reverse phase support provides the optimal chromatographic condition for the separation of intact glucosinolates. Whilst new chromatographic phases and mass
spectrometer configurations are facilitating the separation,
detection and identification issues, quantification still remains
the most problematic issue. Supply of analytical standards and
measurement of their absolute purity against literature benchmarking values continues to be the single largest issue in quantification, and is the one where least progress has been made.
Available official methods have been well validated, but are
specific to the small niche of rape seed analysis. All subsequent
work, such as on green-tissues, has not yet been validated.
The standard of quality control (QC) in GLS analysis is poor
relative to other areas of analytical chemistry. It is essential that
published work in the area provides expanded experimental
details and associated QC. Further data are needed on the
absolute purity of standards, especially with respect to water and
salt content, stability of standard mixtures in solution, extraction
recoveries, myrosinase deactivation, enzymatic desulfation efficiencies, and most importantly, the analysis of reference materials. Provision of within and between batch reproducibility and
precision measurements is requisite, as is thorough validation of
new methods and a benchmarking comparison to the official
methods is also required.
Acknowledgements
Financial support was provided by the UK Food Standards
Agency (FSA) under contract E01086. The conclusions and
opinions expressed are the views of the author alone.
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Published on 22 February 2010. Downloaded by Centro de Edafologia y Biologia Aplicada del Segura (CEBAS) on 31/05/2016 09:10:31.
Published on 22 February 2010. Downloaded by Centro de Edafologia y Biologia Aplicada del Segura (CEBAS) on 31/05/2016 09:10:31.