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Analytical
Methods
Advancing Methods and Applications
www.rsc.org/methods

Volume 2 | Number 4 | April 2010 | Pages 301416

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

www.rsc.org/methods | Analytical Methods

Glucosinolates, structures and analysis in food

Don Brian Clarke*
Received 1st December 2009, Accepted 18th January 2010
First published as an Advance Article on the web 22nd February 2010
DOI: 10.1039/b9ay00280d
Glucosinolates (GLS) are sulfur rich, anionic secondary metabolites found principally in the plant
order Brassicales. This review focuses on identifying the range of GLS structures identified to date and
summarises the current state of taxonomic reclassifications of GLS producing plants. Those
Brassica species that are available to growers in the UK are highlighted and progress in the aspects of
analytical chemistry relevant to conducting accurate determinations of GLS content of foods is
reviewed. The degradation and derivatisation workflows that have been utilized for conducting
glucosinolate analysis are summarized. A review is made of aspects of extraction, isolation,
determination of purity, ultraviolet (UV) and mass spectrometry (MS) parameters, extinction
coefficients, UV response factors, quantification procedures, and the availability of stable isotope
labeled internal standards, and certified reference materials. An electronic database of structures,
formulae and accurate masses of both the 200 known, and a further 180 predicted GLS, is provided for
use in mass spectrometry.

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

bioavailability,8,9 bio-protective effects10 and significance for

human health.9,11 A large body of epidemiological evidence
indicates that the chemoprotective effects of Brassica vegetables
against initiation of tumours caused by chemical carcinogens
may be due to glucosinolates and their metabolic products.913 A
special issue of Phytochemistry (Issue 8, 2009, 21 papers) has
reviewed the progress made in many areas of glucosinolate
research. There are however obvious omissions in chemotaxonomic classifications, the recognition of new GLS structures and
analytical methods for their determination.

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

Dr Don Brian Clarke is a senior

analytical chemist in the
contaminants and authenticity
programme of the Food and
Environment Research Agency
(Fera). Research interests lie
within the areas of analytical
chemistry and clinical trials,
covering emerging environmental contaminants in food,
natural toxicants and beneficial
plant constituents.
Don Brian Clarke

310 | Anal. Methods, 2010, 2, 310325

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 Reported glucosinolates structures by chemical class.

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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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C1C12 alkyl side-chains. A number of recently discovered new

glucosinolates merely fill the final gaps in existing homologous
sequences. Moreover, since it is difficult to verify many of the
older reports, the references herein are relatively recent, but are
not necessarily the first report of a new GLS, e.g. 2-ethyl-butylGLS (iso-hexyl) does not appear in either review,14,16 but a recent
occurrence18 is referenced back to 1963.19 In this work the
numbering system of Fahey is retained for structures 1120 and
simply extends as each new structure was added to our database.
The database provided as supplemental information, contains
both the full, and trivial names, formulae and masses. While
various abbreviated naming acronyms have been suggested,
some GLS have no assigned trivial name. Moreover the three
letter code used by Wathelet20 is insufficient for >100 structures,
thus the only viable system is one which can combine various
letter codes for the chemistry of the R-group e.g. T thio,
3MTP 3-methylthiopropyl, 4MOI3M 4-methoxyindol-3ylmethyl which is not limited in size and does not rely on the
existence of trivial names.
Illustrative examples of newly characterized GLS are; alkyl
(hexyl),21 iso-alkyl (t-butyl [1,1-dimethyl-ethyl]),22 alkene (oct-7-ene,
non-8-ene, dec-9-ene),23 sulfur-chains (methylsulfonyldodecyl),
benzoates (7-benzoyloxyheptyl),17 and substitution sites,
5-hydroxyindole,21 7-methoxyindole24 2-hydroxy-2-(4-hydroxylphenyl)ethyl.25 There is a range of cinnamic esters (Fig. 1) and
while it is noted that these are widespread, they are not readily
hydrolyzed by sulfatase and are difficult to chromatograph.
These GLS derivatives have not been systematically studied to
date.26 Other novel structures include benzyl-branched elongations (2-benzoyloxy-3-butene-GLS),27 5-benzylsulfonyl-4-pentenyl-GLS28 and benzyl substitutions of both the thioglucose
(60 -O-benzoyloxy-glucoerucin) and apiose sugars (glucohesmatrolalin) and a variety of new indole esters (glucoisatsin).29
Furthermore, GLS structures such as 4-(cysteine-S-yl)butyl
(glucorucolamine),30 dimeric 4-mercaptobutyl and 4-(glucodisulfanyl)butyl21,31 are truly unique and indicate that additional
GLS with hitherto unknown chemistries will continue to be
isolated. Similar structures, but as the cysteine and glutathione
disulfides, have been reported by Bennett as oxidative artifacts.
These can be reverted to the parent GLS by reaction with the
reducing agent TCEP (tris-2-carboxyethyl phosphine).32 The
major GLS in salad rocket was then identified as 4-mercaptobutyl-GLS.33 Completing the homologous series (with e.g.
C1C10 R-group) for all these newly identified structures allows
screening for a further 180 theoretical structures (Supplemental
material Fig. S1). The LC-TOF screening procedure for benzyl
and cinnamoyl esters is also described (Supplemental material
Fig. S2).

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

classification of this plant family, which contains about 3,700

species (Fig. 2). Glucosinolate synthesis is under enzymatic
control and the structures are derived from both protein and
non-protein L-amino acids.2,34 The defining feature of mustardoil-producing plants is the system of compartmentalization of
glucosinolates and the presence of myrosin cells containing the
hydrolase enzyme myrosinase, which when released by e.g.
herbivore damage, converts glucosinolates to biologically active
isothiocyanates (mustard-oils), as part of the plants defence
system (the mustard-oil bomb). Specific glucosinolates are
often restricted within plant families, with the simplest methylGLS (glucocapparin) found in capers, and one of the more
complex (rhamnopyranosyloxy)benzyl-GLS (glucomoringin)
found in Moringa species.15,22,35 The traditional taxonomic classification placed the glucosinolate containing families in
a number of different orders, implying multiple origins of the
glucosinolate-myrosinase system. Since many unrelated plants
were placed in the Capparaceae family, the taxonomy has been
under review since 1975, with the view to placing all mustard-oil
taxa within a single major clade.3639 Naming of individual
species is also ill-defined, but beyond the scope of this review.
Species are simply referred to herein by the most recent name,
e.g. Sinapis alba refers to the species with common name white
or yellow mustard which is also referred to as Sinapsis alba,
Brassica hirta and Brassica alba.
Early work based on the identification of degradation products such as the release of the thiocyanate ion (SCN) as proof of
the presence of unstable isothiocyanates derived from
4-hydroxybenzyl and indol GLS40 has led to much of the early
work being queried by subsequent workers. This brings into
question the reliability of historical reports of glucosinolate
content and the classifications of plants based on them.
Following the lead of previous reviewers this review discounts
reports of glucosinolates in mushroom, plantain and coca, and
the reclassification of Pittosporaceae (e.g. Bursaria spinosa var.
incana) and Phytolaccaceae (e.g. Phytolacca americana) from
Capparales into Apiales and Caryophyllales, respectively. The
2001 review14 and earlier work (1991)40 would also appear to be
in error when reporting 4-hydroxybenzyl GLS in Bursaria
spinosa var. incana and in Phytolacca americana where the
original work reported no detectable glucosinolates in either.14,40
The genus Drypetes had five reports of glucosinolates (1975
1991) and this had been regarded as reliable to date. Genetic
sequencing has demonstrated a major mustard-oil clade (Brassicales) and one outlier Drypetes,41 bringing this back into doubt.
Drypetes genus was traditionally placed in the sub-family Phyllanthoideae in Euphorbiaceae, but is now in the family Putranjivaceae. Many botanists are now adopting the Angiosperm
Phylogeny Group classification (APG) for the orders and families of flowering plants. The APG II system42 has been adopted in
whole, or in part in a number of recent major works. There is
some disagreement on the relative merits of the traditional
morphological approach against chemotaxonomy and molecular
phylogenetics. The system is rather controversial at the family
level, splitting a number of long-established families and
submerging a number of other families, as it does with the
Brassicaceae. Under this system, the Brassicales are an order of
flowering plants, belonging to the eurosids II clade of Angiosperms. This clade contains the order Brassicales, which in this
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system (APG II) includes families classified under Capparales in

previous classifications. The sub-families Capparaceae and
Cleomaceae, and a number of monotypic genera, are now
elevated to familial status, and with the demotion of Setchellanthaceae there are now 16 glucosinolate containing families
in the order Brassicales. To date the full classification of the
Brassicales families is still in flux and no consensus has yet been
reached. Such emerging views are summarised in Fig. 2 with the
relationships between the chemotaxonomic marker species.
The detailed positioning of genera and species is therefore not
well defined and varies by source and classification system. In
overview, the Brassicales order is a biogeographical dispersed

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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Table 1 Summary of the commonest edible Brassica species

Genera, species, group and forma

Brassica carinata
Brassica juncea
Brassica juncea var. crispifolia
Brassica juncea var. integlifolia
Brassica juncea rugosa
Brassica hirta
Brassica napus
Brassica napus var. pabularia
Brassica napobrassica
Brassica nigra
Brassica oleracea var. acephala
Brassica oleracea var. alboglabra
Brassica oleracea var. botrytis
Brassica oleracea var. botrytis f.
romanesco
Brassica oleracea var. capitata f.
alba
Brassica oleracea var. capitata f.
conica
Brassica oleracea var. capitata f.
ruba
Brassica oleracea var. capitata f.
sabauda
Brassica oleracea var. gemmifera
Brassica oleracea var. gongylodes
Brassica oleracea var. italica
Brassica oleracea var. italica 
botrytis
Brassica oleracea var. komatsuna
Brassica oleracea var. viridis
Brassica rapa
Brassica rapa var. chinensis
Brassica rapa var. narinosa
Brassica rapa var. japonica
Brassica rapa var. parachinensis
Brassica rapa var. pekinensis
Brassica rapa var. perviridis
Brassica rapa var. perviridis 
pekinensis
Brassica rapa var. purpuraria
Brassica rapa var. rapifera
Brassica rapa var. rosularis
Brassica rapa var. ruvo

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

producing the three tetraploid species Brassica juncea (AABB),

Brassica napus (AACC) and Brassica carinata (BBCC). These
have further interbred, or been hybridized, producing the wide
range of species and cultivars that form a large part of our dietary
intake of vegetables (Table 1). Some major food uses are: roots
(swedes B. neobrassica and turnips B. rapa), stems (Kohl rabi
B. oleracea var. gongylodes), leaves (cabbages B. oleracea var.
capitata, Brussels sprouts B. oleracea var. gemmifera), flowers
(broccoli calabrese Brassica oleracea var. italica, cauliflower
B. oleracea var. botrytis), seeds (mustards Sinapis alba) and oil
(oil seed rape B. napus). In the UK the consumption of the
individual varieties is somewhat limited (Supplemental material
Table S1), where by definition varieties must be readily available to both commercial producers and domestic gardeners for
there to be any appreciable population based intake. In 2002 the
reported mean consumption of six key Brassica vegetables was
1015 g/day of each vegetable for persons that consume these
foods (consumer mean), with an overall population mean intake
of 20 g Brassicas/day/person (Table 2) and a maximum intake of
160 g/day.43

Savoy cabbage

4. Glucosinolate content of plants

Brussels sprouts
Kohl rabi
Broccoli
Broccoflower

Historically a sense-mediated adaptive mechanism to avoid

consumption of poisons has selected for low-glucosinolate
content in vegetables.45 Recent sensory trials have shown typical
rocket salad flavour and pungency are perceived as positive
sensory traits, while bitter notes, characterized by high glucosinolate content (sinalbin/gluconapin-herbaceous; sinigrinpungency), were much less acceptable.44 The reverse is true in
condiments such as Sinapis alba seeds (condiment mustard)
which were bred for piquancy and now contain one of the highest
reported concentrations (250 mmol/g sinalbin).15,45 Superbroccoli has been produced by traditional plant breeding
including wild Sicilian broccoli, to produce a cross with a glucoraphanin content 34 times higher than that of normal varieties. This has been shown to elevate plasma sulforaphane the
putative anticancer active principle and metabolites 3-fold.46
Since this is a larger non-volatile glucosinolate the acceptability
of the flavour of the broccoli is not adversely affected.
Glucosinolate concentrations in plants, although highly variable, are around 1% dry weight in some Brassica vegetables.47
There are a number of reports of amounts exceeding 10% in the
seeds of some species15,45 and as high as 26% of rhamnose-benzylGLS in the seeds of Moringa oleifera.22 The young leaves and

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)

contained within the Brassiceae tribe of Clade 2. The Brassica

genus evolved from three ancestral Brassica species with diploid
genomes (with 10, 9 and 8) chromosomes (Brassica rapa AA,
Brassica nigra BB, Brassica oleracea CC). These then interbred
Table 2 UK Brassica consumption data for 2002 (g/person/day)a

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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buds of the desert cabbage Schouwia purpurea contain unusually

high levels of gluconapin, up to 10% dry weight.48 Glucosinolates
are very stable water-soluble precursors of isothiocyanates and
some fresh plants have been show to contain almost exclusively
glucosinolates and no isothiocyanates. Glucosinolates are
therefore considered the storage form of their biologically active
aglycones (isothiocyanates). Reproductive tissues (florets,
flowers and seeds) often contain as much as 1040 times higher
concentrations of GLS than is found in vegetative tissues.15,22,49,50
Seeds are therefore the best bulk source of quantities of glucosinolates. Roots can often be an equally high source, but are
a less practical material to harvest. There are many practical
advantages in using seeds rather than plant tissues in preparing
a glucosinolate extract. Seeds are stable and can be readily
stored. The three rapeseed certified reference materials (CRMs)
have now been in use an astonishing 20 years, without degradation.51 Because of the low moisture content, ground seed-meal
does not activate the moisture sensitive myrosinases, thereby
preventing conversion to isothiocyanates and other degradates.
One of the key drivers for food-based analytical method
development is generating human exposure data, in this case the
dietary intake of glucosinolates obtained through the consumption of [cruciferous] vegetables.13 A summary of the available
glucosinolate content data collated from 18 studies was used to
produce single values for the total glucosinolate content of each
food.52 A further database has been prepared of 26 glucosinolates
in 18 vegetables and a dietary intake of 14 mg/day estimated in
Germany,53 an estimate of 6 mg/day has been calculated for
Spain.54

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

storage of seed-sprouts for 3-weeks at 4  C suggested that of the

species studied only rocket showed decline in glucoerucin and
glucoraphenin content.64 Gluconasturtiin has been shown to
undergo a non-enzymatic, iron-dependent degradation to
a simple nitrile. On heating the seeds to 120  C, thermal degradation of this heat-labile glucosinolate increased simple nitrile
levels many fold.65
Myrosinase can be deactivated in wet tissue by microwaving,
then cooling on ice.55 Microwave inactivation does not work as
well on dry materials, and an optimum moisture content of
1416% was needed in Crambe abyssinica seed.66 Canola seed at
the lowest moisture content, 6%, required 485 s at 1500 W for
deactivation.67 Microwaving resulted in an increase in the
extracted GLS content relative to the uncooked cabbage.62
5.2 Extraction
Extraction of GLS from plant material is best achieved using
protic solvents. This has been largely restricted to the use of
methanol-water.57,59 Both ethanol-water (1 : 1), or methanolwater (7 : 3) are recommended for freeze-dried green tissues.20
Water or phosphate buffer (20 mM, pH 7, 20 min, 100  C) was
more effective than alcoholic solvents for extracting sinigrin from
black mustard and horseradish.68 Phosphoric acid (2%) has been
use to extract GLS from Brussels sprouts.69 Since methanol
ruptures cell walls (where the aim was simply to remove glucosinolates from oilseed protein products), methanol-waterammonia has been successfully employed; 10% ammonia in
methanol containing 5% water, at a solvent-to-seed ratio of
6.7 and 2 min of blending with 1015 min quiescent period being
sufficient to lower GLS content to below the limit of detection.70,71 Large scale solvent based extractions are disfavored by
time, energy and safety concerns and have therefore not become
established. The noxious weed Cardaria draba is reported to give
optimum extractability of glucoraphanin into 20% ethanol in
water at 70  C, 50 g dm3 and pH 3 over 20 min.72 Given the
diversity of structures and the range of seed and plant matrices, it
is recommended that 70% methanol is used as the default
extractant unless validation proves alternative solvents are
required.
5.3 Isolation
Analytical standards of the individual GLS are isolated from
specific plants, preferably those containing either high concentrations or less complex mixtures of GLS.15,20,50 Aqueous
extraction may also include Pb(OAc)2 and Ba(OAc)2 to precipitate protein and free sulfate, respectively.50 After centrifugation,
purification of GLS generally involves an anion exchange step, in
most cases on the same DEAE-Sephadex A-25 resin used for the
later enzymatic desulfation and analysis prescribed in method
ISO9167-1.73 A typical procedure for intact GLS extraction is
elution from the resin with 0.5 M K2SO4, followed by evaporation and dissolution in hot methanol to leave insoluble salts, then
recrystallisation from cold ethanol and drying over P2O5.20
Florisil solid phase extraction is used to clean up intact GLS,
with application in methanol/dichloromethane/hexane, washing
with dichloromethane/hexane and elution with methanol/ethyl
acetate.74,75 Anion exchange on a styrene-divinylbenzene
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copolymeric anion exchanger gives an additional dimension for

separation, with high selectivity and elution in order of GLS
hydrophobicity, using the inorganic anions SO42, NO3, ClO3
and ClO4 in sequence.76 High-speed counter-current chromatography relies solely on the partition coefficient of the solute
between the stationary and mobile phases and can be used to
separate gram quantities of structurally and chromatographically similar glucosinolates. Using a propanol-acetonitrile saturated aqueous ammonium sulfate-water, biphasic system, with
the organic phase as the mobile phase and the aqueous as the
stationary phase, the homologues glucoraphanin [methylsulfinylbutyl-GLS] and glucoiberin [methylsulfinylpropyl-GLS]
are separated by their partition coefficients of 0.63 and 1.03.77
5.4 Detection methods
The detection of glucosinolates may be considered in three parts:
as degradative totals by colourimetric techniques, as nondestructive totals, and as individual components by chromatographic separation and detection. The collection of degradative

methods and chemical workflows are summarized in Fig. 3. The

majority of these: thiourea,78 thymol,79 benzenedithiol cyclocondensation,80 palladium chloride,81 ferricyanide assays82,83 and
sulfate ion release84 continue to be used without modification.
Various glucose assays85 are now available, primarily as hexokinase coupled to NADH production and glucose oxidase and
peroxidase coupled to various coloured dyes such as quinoneimine and dianisidine. An official method for glucosinolate
content following glucose release has recently been published
(ISO 9167-3 2007).86 All colourimetric methods have strengths
and weaknesses and it has been recommended not to rely on any
single method, but to conduct a number of assays to ensure
a consensus is reached for a given sample type in order to avoid
bias. The only technical advancement away from colourimetric
detection has been in automation and technology. The cyclocondensation of isothiocyanates and benzenedithiol gives
a single chromatographically stable product 1,3-benzodithiole-2thione for any GLS, and with a 3-fold increase in molar extinction to 3365 23,000 M1 cm1 it is well suited for the more sensitive
HPLC-UV detection format.80 Biosensing is a second such

Fig. 3 Chemical workflow for degradative methods of glucosinolate analysis.

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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

In the past, analysis of the intact glucosinolates was not

possible. This was overcome by using hydrolysis to produce more
chromatographically amenable forms. The original work was
published in 1982,94 describing the conversion via sulfatase to
desulfo-glucosinolates (ds-GLS) and was harmonized into an EU
official method ISO 9167-1 (1992)57 and an AOCS method Ak
192 (AOCS 1998).59 These remain in widespread use,95,96 with
suggested improvements,20,97,98 but have not yet been issued as
official methods. The guidelines for analysis of green tissues used
for biofumigation is the best attempt at a generic method for
plant tissues e.g. food.20 In parallel, conversion via myrosinase
and GC-MS of the more volatile isothiocyanates (epithioalkanenitriles, nitriles, oxazolidine-2-thiones etc.) or derivatisation of
the desulfo-GLS to trimethylsilyl (TMS)-ethers and GC-MS
have now largely been dropped, but remain in use for plant
volatiles and biofumigation work where the isothiocyanates
rather than the parent GLS are the key form. Limited data is
available for the myrosinase degradates, isothiocyanates, indoles
and oxazolidinethiones by HPLC-UV at 240 nm.99
Analytical determinations can now be undertaken on the
desulfo-GLS or intact-GLS forms, but it is uncommon to
perform UV quantification on the intact forms.100 The

Table 3 UV relative response factorsa

Chain
length Trivial name
C1
C3

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

9167-1 1992,57 Ak 192 AOCS59 1998). New data have been

published2,20,50 where the list of relative response factors, also
known as relative proportionality factors (RPF), has now grown
to 28 entries (Table 3). The source of response factors presented
by Vinjamoori104 is unreferenced and these are not normalized to
sinigrin (1.05) and therefore appear to have an upwards bias of
0.05. It is recommended that the values published by Brown,50
Wathelet20 and Haughn2 are used. While response factors may
vary up to 10-fold (0.22.1), the majority of determinations lie
within a very narrow range, indicating that chain elongation and
the sulfur oxidation state produce insignificant effects.
Hydroxylalkyl groups have the highest values, while indoles,
hydroxybenzyl and benzoate esters have the lowest. The value for
4-methylthio-3-butenyl ds-GLS is somewhat anomalous with
respect to other alkenes. The values reported by Brown appear to
have been obtained using an unspecified HPLC based flowinjection-analysis approach, a departure from the thymolsulfuric acid assay.50 Data for minor components ethyl, propyl,
isopropyl, 3-methylthiopropyl, butyl, 4-methylsulfonylbutyl,
5-methylthiopentyl, 7-methylsulfinylheptyl and (2R)-2-hydroxy2-phenethyl-GLS are still required to encompass all of the individual GLS that can be encountered in foods (Table 4). The
default of assigning the value of 1.00 to any unassigned
component has been shown to generate inaccuracies. Current
data generated with derived values for glucoraphasatin (0.40)
and glucosinalbin (0.50) will be a factor of 2-times higher than
data generated with the default values. A more rigorous
approach, identifying the structural features of ds-GLS and

Table 4 Individual glucosinolates expected in UK foods

No

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
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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

indicative values have been used to construct LC-MS calibration

curves to quantify unknowns.75 It is reported that ionisation is
significantly influenced by the vegetable matrix, and standard
addition and internal standardisation with isotopomers must be
used for accurate quantification.118 Accurate quantification in
LC-MS is completely reliant on having a pure standard of each
target analyte.
5.9 Purity of analytical standards: water content by NMR
The isolation of GLS and their elution from ion-exchange resin
with potassium sulfate produces potassium salts, which while
often presenting as white crystals, may not be pure. The organic
content is measured by various procedures, generally HPLC-UV
and acceptably high purities >95% are often quoted. Given the
highly hygroscopic nature of GLS salts, it is unclear how much
water of crystallisation is present in each standard. Water
content in standards can be assessed by quantitative proton
NMR (qHNMR). Trimethoxybenzene (0.333 mM) internal
standard in 10% d4-methanol in D2O was used to dissolve
1000  4 mg of solid glucosinolate (3 mM) in the NMR tube.60
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For non-aromatic GLS, the integral area of the anomeric

hydrogen at 4.5 ppm was compared to the area of nine methyl
protons of the trimethoxybenzene. For aromatic GLS the region
78 ppm was integrated and then divided by the number of
originating protons. True content ranged from 99% sinigrin/
glucotropaeolin, 77% gluconapin, to 17% for 4-hydroxyglucobrassicin. This appears to be the most appropriate and accurate
method for determining the absolute amount of a given GLS in
a hitherto chromatographically pure standard.60
5.10 Extinction coefficients
Molar extinction coefficients are a more accessible indication of
organic substances purity. One of the common correction
procedures used in quantification with natural products standards is calculation of actual purity from a reference extinction
coefficient. At the lmax of 224 nm, the N-hydroxysulfate moiety of
glucosinolates has an extinction 3224 of around 7,000 M1 cm1.
Indole, aryl and alkenyl side chains contribute absorbances with
relatively lower intensity at 250280 nm.112 The determination of
definitive literature extinction values relies heavily on achieving
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consensus. Extinction coefficient measurements are generally of

high accuracy and precision. An interlaboratory trial (n 5) for
the isoflavone genistein, with each participant independently
sourcing the standard produced an extinction coefficient with
a c.v. of 1.2%.127 The measurement precision 3235 for progoitrin
and glucotropaeolin with three determinations at three concentrations (n 9) was 3.2% c.v.117 For the most readily available
and best characterised GLS sinigrin, the reported extinction
coefficient values are 6,780 (3235), 6,784, 6,950, 7,273 (3227), 7,369
(3227) 7,800 (3227) and 8,000 (3227) M1 cm1.77,109,117,128131 The
average of these values is 7,279  483, with 6.6% c.v.
Extinction values (3235) for glucoiberin (6,234), glucoerucin
(6,531), progoitrin (4,130), glucotropaeolin (8,312, 8,870 M1 cm1)
are available.81,117 A much earlier value 3230.5 6,720 M1 cm1 is
available for glucoconringiin (1959).132 Note that these extinction
coefficients have no direct correlation to the desulfoglucosinolate
response factors from the thymol assay. A decrease in extinction
(3227) from 7,800 to 5,700 M1 cm1 for desulfosinigrin suggests
removal of the sulfate has a substantial effect on the central
chromophore.131 The isothiocyanate sulforaphane derived from
glucoraphanin [methylsulfinylbutyl-GLS
3235 6,872], has a more pronounced decrease in extinction
coefficient, with 3238 of just 910 M1 cm1.77,133
In a single study, the 3227 values for purified sinigrin ranged
from 7,1407,662 M1 cm1, (average 7,379  194, 2.6% c.v.).
The standard error associated with the individual values was
minimal (<0.4% c.v.), and the extraction solvent was considered
to have influenced the extinction outcome.68 This variation can
only be accounted for by the presence of impurities, such as
water, organic solvent residues and salt. Temperature and
solvent composition may also affect the accuracy of these
determinations. Wathelet reports that with poor HPLC column
temperature regulation, increasing the column temperature
above 30  C reduces the response of ds-4-hydroxyglucobrassicin.20 Linear regression calculations made at various
concentrations have been used to establish if the UV response is
linear in any given solvent or mixture. For synthetic 2,2-diphenylethyl-GLS 3227 decreased from 11,282 to 9,481 when
acetonitrile was added (1 : 1) to phosphate buffer (0.1 M, pH 7.4),
while 3227 of the isomer (biphenyl-2-yl)methyl-GLS remained

similar (14,922 to 14,632). The measurements failed in pure

acetonitrile. For the derived isothiocyanates the converse held and
measurements failed in the aqueous buffer, but were similar in the
1 : 1 mixture and pure acetonitrile (10,131 to 10,901 diphenylethylITC and 5,008 to 4,649 for (biphenyl)methyl-ITC).134
It appears that robust consensus values for extinction coefficient values of GLS have not yet been achieved, hence considerable further effort is required before these can be of use to the
analytical chemist in correcting purity. It is recommended
therefore that measurements are standardised as being conducted as 3227 in water at ambient (2025  C) temperature, using
6-point calibration lines with absorbance <1 (e.g. 560 mmol/l
sinigrin A 0.11.0), with r2 > 0.995, and where a plot of
absorbance vs. molar concentration (M.L1) has a slope equal to
the extinction coefficient. For best accuracy this protocol should
be conducted with replicate (n 6) weighings, each of 20 mg, into
wide mouthed vials to avoid static dispersion of the powder,
followed by quantitative transfer into 250 ml volumetric flasks,
giving 80 mg/ml. Individual solutions should be measured in
triplicate, with the spectrometer precision <0.5% c.v., and overall
measurement should achieve a precision of <1.0% c.v.
A (3227), value as the mono-hydrated potassium salt ([SinigrinH]$K+$H2O MW 415.48096) of 7,299  28 (0.4% c.v.) was
determined in this work. The 95% confidence interval of 7,271
7,327 overlaps with and is therefore statistically the same to the
literature average value.
5.11 Reference materials
Certified reference materials (CRM) with total glucosinolate
levels of 12.1  0.8 mmol/g (CRM 366), 25.5  0.9 mmol/g (CRM
190) and 102.4  3 mmol/g (CRM 367) were prepared between
1988 and 1991.51 As stocks of CRM 190 were depleted, a further
batch was prepared from the original refrigerated material and
the other two materials were re-certified as 11.9  1.3 mmol/g
(BCR-366R), 23  4 mmol/g (BCR-190R) and 99  9 mmol/g
(BCRM 367R).135 Now available as ERM-BC190, 366 and 367,
these materials remain in use for assessing method performance,
especially accuracy, some 20 years after preparation. Despite
their availability and a modest price, these are used surprising

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

is no longer essential to desulfate GLS prior to analysis,

providing that a robust quantification method is utilised. It is
essential to fully harmonise, document and validate the next
generation of glucosinolates methods and this has not yet been
attempted. A comprehensive intercomparison study that uses
these new approaches, including analysis of intact-GLC by
LC-MS/MS vs. desulfation and by HPLC-UV, NMR purity
measurement of standards, including the analysis of certified
reference materials is required to raise this field of analysis to an
acceptable standard.

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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