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AnalyticalMethodsAdvancing Methods and Applications

1759-9660(2010)2:4;1-7

Volume 2 | N

umber 4 | 2010

Analytical M

ethods

Pages 301–416

www.rsc.org/methods Volume 2 | Number 4 | April 2010 | Pages 301–416

CRITICAL REVIEWClarkeGlucosinolates, structures and analysis in food

PAPERSchazmann et al.A wearable electrochemical sensor for the real-time measurement of sweat sodium concentration

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 (cis-

N-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 chemi-

cally induced decomposition of glucosinolates,5 the biology and

biochemistry,6 their role in insect-plant relationships,7 their

The Food and Environment Research Agency (Fera), Sand Hutton, York,YO41 1LZ, UK. E-mail: [email protected]; Fax: +044-1904-462133; Tel: +044-1904-462000

† Electronic supplementary information (ESI) available: A database ofstructures, formulae and accurate masses of both the 200 known, anda further 180 predicted GLS, for use in mass spectrometry; Fig. S1.Further glucosinolates; Fig. S2. Screening for cinnamoyl and benzoylesters; and Table S1. Some sources of Brassicale seeds in the UK. SeeDOI: 10.1039/b9ay00280d

Don Brian Clarke

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 environ-

mental contaminants in food,

natural toxicants and beneficial

plant constituents.

310 | Anal. Methods, 2010, 2, 310–325

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.9–13 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 chemotaxo-

nomic classifications, the recognition of new GLS structures and

analytical methods for their determination.

2. Glucosinolate structures

Glucosinolates are characterized by a core sulfated iso-

thiocyanate 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 Bel-

lostas 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 glucosi-

nolate, the majority contain 2–5, while 34 individual glucosino-

lates 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

This journal is ª The Royal Society of Chemistry 2010

Fig. 1 Reported glucosinolates structures by chemical class.

This journal is ª The Royal Society of Chemistry 2010 Anal. Methods, 2010, 2, 310–325 | 311

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.

312 | Anal. Methods, 2010, 2, 310–325 This journal is ª The Royal Society of Chemistry 2010

C1–C12 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-butyl-

GLS (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 1–120 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 ¼ thio,

3MTP ¼ 3-methylthiopropyl, 4MOI3M ¼ 4-methoxyindol-3-

ylmethyl 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-hydroxyl-

phenyl)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 elonga-

tions (2-benzoyloxy-3-butene-GLS),27 5-benzylsulfonyl-4-pen-

tenyl-GLS28 and benzyl substitutions of both the thioglucose

(60-O-benzoyloxy-glucoerucin) and apiose sugars (glucohesma-

trolalin) 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-(gluco-

disulfanyl)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-mercapto-

butyl-GLS.33 Completing the homologous series (with e.g.

C1–C10 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 ‘‘cross-

bearing’’, because the four petals of the flowers are reminiscent of

a cross. Historically there has been confusion over the


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 mustard-

oil-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 methyl-

GLS (glucocapparin) found in capers, and one of the more

complex (rhamnopyranosyloxy)benzyl-GLS (glucomoringin)

found in Moringa species.15,22,35 The traditional taxonomic clas-

sification 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.36–39 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 prod-

ucts 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 (Bras-

sicales) and one outlier Drypetes,41 bringing this back into doubt.

Drypetes genus was traditionally placed in the sub-family Phyl-

lanthoideae in Euphorbiaceae, but is now in the family Putran-

jivaceae. Many botanists are now adopting the Angiosperm

Phylogeny Group classification (APG) for the orders and fami-

lies 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 Angio-

sperms. This clade contains the order Brassicales, which in this

Anal. Methods, 2010, 2, 310–325 | 313

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 Setch-

ellanthaceae 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

Fig. 2 Summarized taxonomy of the order Brassicales indicating the current r

the approximate numbers of genus and or species in each branch. These numbe

is exemplified by a species used either in previous phylogenetics reclassificatio

314 | Anal. Methods, 2010, 2, 310–325

lineage with many small but distinct clades and a large Brassi-

caceae 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

elationship of all the glucosinolate producing families.36–39,42 Numbers are

rs are in flux and question marks denote a lack of clear data. Each branch

ns, or as a food crop.


Table 1 Summary of the commonest edible Brassica species

Genera, species, group and formaCommonname

Brassica carinata Ethiopian mustardBrassica juncea Indian mustardBrassica juncea var. crispifolia Chinese mustardBrassica juncea var. integlifolia Red giant mustardBrassica juncea rugosa Wrapped heart mustard

cabbageBrassica hirta Yellow mustardBrassica napus Canola/rape seedsBrassica napus var. pabularia Siberian kaleBrassica napobrassica Rutabaga (swede)Brassica nigra Black mustardBrassica oleracea var. acephala KaleBrassica oleracea var. alboglabra Kai-lan (Chinese broccoli)Brassica oleracea var. botrytis CauliflowerBrassica oleracea var. botrytis f.

romanescoRomanesco broccoli

Brassica oleracea var. capitata f.alba

White cabbage (drum)

Brassica oleracea var. capitata f.conica

Pointed cabbage

Brassica oleracea var. capitata f.ruba

Red cabbage

Brassica oleracea var. capitata f.sabauda

Savoy cabbage

Brassica oleracea var. gemmifera Brussels sproutsBrassica oleracea var. gongylodes Kohl rabiBrassica oleracea var. italica BroccoliBrassica oleracea var. italica �

botrytisBroccoflower

Brassica oleracea var. komatsuna KomatsunaBrassica oleracea var. viridis Collard greensBrassica rapa Mustard spinachBrassica rapa var. chinensis Pak Choi (Cantonese)Brassica rapa var. narinosa Broad beak mustardBrassica rapa var. japonica MibunaBrassica rapa var. parachinensis Choy Sum (false Pak Choi)Brassica rapa var. pekinensis Chinese cabbageBrassica rapa var. perviridis KomatsunaBrassica rapa var. perviridis �

pekinensisSenposai

Brassica rapa var. purpuraria Purple stem mustardBrassica rapa var. rapifera TurnipBrassica rapa var. rosularis Tatsoi (rosette Pak Choi)Brassica rapa var. ruvo 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 Co

Broccoli 743 6.4 14.Head cabbage 737 5.9 13.Cauliflower 767 5.6 12.Brussels sprouts 212 1.9 15.Kohl rabi 0 — —Chinese cabbage 26 0.2 10.Totals 1341 19.9

a Reproduced and adapted from: Henderson L., Gregory J. Swan G. Nationalquantities of foods consumed, The Stationery Office 2002.43


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 avail-

able 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

10–15 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

4. Glucosinolate content of plants

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 glucosi-

nolate content (sinalbin/gluconapin-herbaceous; sinigrin-

pungency), 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 ‘Super-

broccoli’ has been produced by traditional plant breeding

including wild Sicilian broccoli, to produce a cross with a glu-

coraphanin content 3–4 times higher than that of normal varie-

ties. 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 vari-

able, 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-benzyl-

GLS in the seeds of Moringa oleifera.22 The young leaves and

nsumer meanConsumermax Brassica species

7 80.7 Brassica oleracea var. italica7 112.1 Brassica oleracea var. capitata f. alba6 158.7 Brassica oleracea var. botrytis9 72.7 Brassica oleracea var. gemmifera

— Brassica oleracea var. gongylodes1 66.4 Brassica rapa var. pekinensis

158.7

Diet and Nutrition Survey: adults aged 19–64 years. Volume 1: types and

Anal. Methods, 2010, 2, 310–325 | 315

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 10–40 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 gluco-

sinolates. 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 degra-

dation.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 consump-

tion 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 65–100 �C,

close to the solvent or mixtures boiling point, on the assumption

that the overarching concern should be the inactivation of

myrosinase.55–59 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 vali-

dation data available on the effects of avoiding high tempera-

tures 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, 310–325

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 degra-

dation 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

14–16% 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 methanol-

water (7 : 3) are recommended for freeze-dried green tissues.20

Water or phosphate buffer (20 mM, pH¼ 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 gluco-

sinolates from oilseed protein products), methanol-water-

ammonia 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 10–15 min quiescent period being

sufficient to lower GLS content to below the limit of detec-

tion.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 dm�3 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 concen-

trations or less complex mixtures of GLS.15,20,50 Aqueous

extraction may also include Pb(OAc)2 and Ba(OAc)2 to precip-

itate 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 evapora-

tion 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


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 chroma-

tography 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 chromatographi-

cally similar glucosinolates. Using a propanol-acetonitrile satu-

rated aqueous ammonium sulfate-water, biphasic system, with

the organic phase as the mobile phase and the aqueous as the

stationary phase, the homologues glucoraphanin [methyl-

sulfinylbutyl-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 non-

destructive totals, and as individual components by chromato-

graphic separation and detection. The collection of degradative

Fig. 3 Chemical workflow for degradati


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

majority of these: thiourea,78 thymol,79 benzenedithiol cyclo-

condensation,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 hexo-

kinase coupled to NADH production and glucose oxidase and

peroxidase coupled to various coloured dyes such as quinonei-

mine 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 cyclo-

condensation of isothiocyanates and benzenedithiol gives

a single chromatographically stable product 1,3-benzodithiole-2-

thione for any GLS, and with a 3-fold increase in molar extinc-

tion to 3365 23,000 M�1 cm�1 it is well suited for the more sensitive

HPLC-UV detection format.80 Biosensing is a second such

ve methods of glucosinolate analysis.

Anal. Methods, 2010, 2, 310–325 | 317

example, where the enzymatic cascade from GLS through iso-

thiocyanate to glucose and on to hydrogen peroxide has also

been automated. Biosensing using amperometric enzyme elec-

trodes based on glucose oxidase and tyrosinase were utilized after

conversion to glucose and thiourea.87 Biosensing with a myrosi-

nase-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-con-

ducting binding material is also under development, but is as yet

unpublished.

Near infrared reflectance spectroscopy (NIRS) is a validated

non-destructive technique, in which O–H, C–H and N–H 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

Table 3 UV relative response factorsa

Chainlength Trivial name R Side chain

Buchner1987102

E1

C1 Glucocapparin MethylC3 Sinigrin 2-Propenyl 1.00 1

Glucoibervirin 3-MethylthiopropylGlucoiberin 3-Methylsulfinylpropyl 1.07 1Glucocheirolin 3-MethylsulfonylpropylGlucoputranjivin 1-MethylethylGlucosisymbrin 2-Hydroxy-1-methylethylGlucoerysimumhieracifolium 3-Hydroxypropyl

C4 Gluconapin 3-Butenyl 1.00 1Progoitrin (2R)-2-Hydroxy-3-butenyl 1.09 1Epiprogoitrin (2S)-2-Hydroxy-3-butenyl 1Glucoerucin 4-MethylthiobutylGlucoraphasatin 4-Methylthio-3-butenylGlucoraphanin 4-Methylsulfinylbutyl 1Glucoraphenin 4-Methylsulfinyl-3-butenylGlucoarabidopsithalianain 4-HydroxylbutylGlucoconringiin 2-Hydroxy-2-methylpropyl

C5 Glucoalyssin 5-Methylsulfinylpentyl 1Glucobrassicanapin Pent-4-enylGluconapoleiferin 2-Hydroxy-pent-4-enyl 1Glucocleomin 2-Hydroxy-2-methylbutyl

C6 Glucolesquerellin 6-MethylthiohexylGlucohesperin 6-Methylsulfinylhexyl

C7 Glucoarabishirsutain 7-MethylthioheptylC8 Glucoarabishirsuin 8-Methylthiooctyl

Glucohirsutin 8-MethylsulfinyloctylInd Glucobrassicin 3-Indolylmethyl 0.29 0

4-Hydroxyglucobrassicin 4-Hydroxy-3-indolylmethyl 0.28 04-Methoxyglucobrassicin 4-Methoxy-3-indolylmethyl 0Neoglucobrassicin N-Methoxy-3-indolylmethyl 0.20 0

Ar Glucotropaeolin Benzyl 0.95 0Glucosinalbin p-HydroxybenzylGluconasturtiin 2-Phenethyl 0Glucobarbarin (2S)-2-Hydroxy-2-phenethylGlucomalcomiin 3-Benzoyloxypropyl— 4-Benzoyloxybutyl

a Thymol-sulfuric acid assay derived UV response factors [relative proportio1864/90 ¼ 1990.103 ND ¼ retention time provided without a response fact5-methylthiopentyl, 7-methylsulfinylheptyl, (2R)-2-hydroxy-2-phenethyl.

318 | Anal. Methods, 2010, 2, 310–325

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

1–92 (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 (epithioalkane-

nitriles, 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

C990103

Haughn19912

ISO199257

AOCS199859

Brown200350

Vinjam2004104

Wathelet200420

Recommendedvalue

1.0 1.25 1.25.00 1.00 1.00 1.0 1.05 1.00 1.00

0.8 0.8.07 1.07 1.07 1.2 1.13 1.07 1.07

0.9 1.26 1.261.0 1.0

1.32 1.322.1 2.1

.11 1.11 1.11 1.0 1.17 1.11 1.11

.09 1.09 1.09 1.15 1.09 1.09

.09 1.09 1.09 1.15 1.09 1.091.0 0.9 1.04 1.04

0.40 0.40.07 1.07 1.07 0.9 1.13 1.07 1.07

0.9 0.91.48 1.4 1.4

1.00 1.00.07 1.07 1.07 0.9 1.13 1.07 1.07

1.15 1.15 1.15 1.15.00 1.00 1.00 1.05 1.00 1.00

1.07 1.071.0 1.01.0 1.01.0 1.0

1.0 1.1 1.11.1 1.1

.29 0.25 0.29 0.29 0.31 0.29 0.29

.28 0.28 0.28 0.29 0.28 0.28

.25 0.25 0.25 0.26 0.25 0.25

.20 0.20 0.20 0.21 0.20 0.20

.95 0.95 0.95 0.8 1.00 0.95 0.950.4 0.50 0.50

.95 0.95 0.95 1.0 1.00 0.95 0.951.09 1.09

0.4 0.40.41 0.3 0.3

nality factors (RPF)] for desulfoglucosinolates. EC 1990 ¼ method EECor. No data available for: ethyl, propyl, butyl, 4-methylsulfonylbutyl,


desulfation process has been miniaturized and adapted to a 96-

well 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 speci-

ficity 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

(ds–GLS). These are calculated from individual purified stan-

dards 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

Table 4 Individual glucosinolates expected in UK foods

No Chain length Trivial name

1 C1 Glucocapparin2 C2 Glucolepidiin3 C3 —4 Glucoputranjivin5 Sinigrin6 Glucoiberin7 Glucoibervirin8 Glucocheirolin9 C4 Glucocapparisflexuosain10 Gluconapin11 Progoitrin12 Epiprogoitrin13 Glucoerucin14 Glucoraphanin15 Glucoerysolin16 Dehydroerucin17 Glucoraphenin18 C5 Glucobrassicanapin19 Glucoberteroin20 Glucoalyssin21 Gluconapoleiferin22 C7 Glucosiberin23 C8 Glucohirsutin24 Ind 4-Hydroxyglucobrassicin25 Glucobrassicin26 4-Methoxyglucobrassicin27 Neoglucobrassicin28 Ar Glucotropaeolin29 Glucosinalbin30 Gluconasturtiin31 Glucobarbarin32 Glucosibarin


9167-1 1992,57 Ak 1–92 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 (0.2–2.1), 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 flow-

injection-analysis approach, a departure from the thymol-

sulfuric acid assay.50 Data for minor components ethyl, propyl,

isopropyl, 3-methylthiopropyl, butyl, 4-methylsulfonylbutyl,

5-methylthiopentyl, 7-methylsulfinylheptyl and (2R)-2-hydroxy-

2-phenethyl-GLS are still required to encompass all of the indi-

vidual 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

R Side chain Food source

Methyl CapersEthyl RadishPropyl CabbageIsopropyl Radish2-Propenyl Cabbage3-Methylsulfinylpropyl Cabbage3-Methylthiopropyl Cabbage3-Methylsulfonylpropyl Cow’s milkButyl Cabbage3-Butenyl Cabbage(2R)-2-Hydroxy-3-butenyl Cabbage(2S)-2-Hydroxy-3-butenyl Sea kale4-Methylthiobutyl Cabbage4-Methylsulfinylbutyl Broccoli4-Methylsulfonylbutyl Cabbage4-Methylthiobut-3-enyl Daikon radish4-Methylsulfinylbut-3-enyl Radish4-Pentenyl Chinese cabbage5-Methylthiopentyl Cabbage5-Methylsulfinylpentyl Rocket2-Hydroxy-pent-4-enyl Swede7-Methylsulfinylheptyl Watercress8-Methylsulfinyloctyl Watercress4-Hydroxy-3-indolylmethyl Cabbage3-Indolylmethyl Cabbage4-Methoxy-3-indolylmethyl CabbageN-Methoxy-3-indolylmethyl CabbageBenzyl Cabbagep-Hydroxybenzyl Mustard2-Phenethyl Cabbage(2S)-2-Hydroxy-2-phenethyl Land cress(2R)-2-Hydroxy-2-phenethyl White mustard

Anal. Methods, 2010, 2, 310–325 | 319

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 ds-

GLS 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 spec-

trometry (MS) detection has been a natural progression, the most

important change is arguably in the choice of the chromato-

graphic stationary phase. Novel approaches, such as super-

critical fluid chromatography,108 micellar electrokinetic109,110 and

capillary zone electrophoresis have found use.111 Hydrophilic

interaction liquid chromatography (HILIC) has been investi-

gated,112 employing second-generation HILIC phases based on

silica zwitterions, which are reportedly more robust and repro-

ducible 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 (0.1–0.5% TFA) still

finds regular use as buffer, despite clear incompatibility issues

with MS/MS detection.100,114,116–118 These TFA based chro-

matographic 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, 310–325

separations are now directly comparable with the earlier sepa-

rations; 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 func-

tion 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 bombard-

ment (FAB)122 and matrix assisted laser desorption ionization

time-of-flight mass spectrometry (MALDI-TOF),123,124 atmo-

spheric 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) instru-

ments118 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 20 0, 300 and 40 0-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 moni-

toring (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


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


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

7–8 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-hydroxygluco-

brassicin. 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 stan-

dards 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 M�1 cm�1.

Indole, aryl and alkenyl side chains contribute absorbances with

relatively lower intensity at 250–280 nm.112 The determination of

definitive literature extinction values relies heavily on achieving

Anal. Methods, 2010, 2, 310–325 | 321

consensus. Extinction coefficient measurements are generally of

high accuracy and precision. An interlaboratory trial (n ¼ 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 concen-

trations (n ¼ 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) M�1 cm�1.77,109,117,128�131 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 M�1 cm�1)

are available.81,117 A much earlier value 3230.5¼ 6,720 M�1 cm�1 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 M�1 cm�1 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 ¼ 6,872], has a more pronounced decrease in extinction

coefficient, with 3238 of just 910 M�1 cm�1.77,133

In a single study, the 3227 values for purified sinigrin ranged

from 7,140–7,662 M�1 cm�1, (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-hydroxygluco-

brassicin.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-dipheny-

lethyl-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

Fig. 5 Isotopic purity determination of 13C6-labelled sinigrin analytical stand

0.1% formic acid. The labelled form has no measurable isotopic impurities fro

trace of [12C113C5]-glucose]sinigrin. The unlabelled form has no isotopic con

respective measurement channels (grey boxes), indicating suitability for use i

322 | Anal. Methods, 2010, 2, 310–325

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 diphenylethyl-

ITC and 5,008 to 4,649 for (biphenyl)methyl-ITC).134

It appears that robust consensus values for extinction coeffi-

cient values of GLS have not yet been achieved, hence consid-

erable 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 con-

ducted as 3227 in water at ambient (20–25 �C) temperature, using

6-point calibration lines with absorbance <1 (e.g. 5–60 mmol/l

sinigrin A ¼ 0.1–1.0), with r2 > 0.995, and where a plot of

absorbance vs. molar concentration (M.L�1) has a slope equal to

the extinction coefficient. For best accuracy this protocol should

be conducted with replicate (n¼ 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 ([Sinigrin-

H]�$K+$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

ard, as full scan mass spectra by infusion of 10 mg/ml solutions in aqueous

m M–H to M + 4 (grey box), and is >99% isotopically pure with a <1%

tribution form M + 3 to M + 7 (grey box). There is no overlap in the

n isotope dilution standardisation.


infrequently.21,55,56,58,75 Indicative values of individual glucosi-

nolate 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 stan-

dards, stable isotope labelled glucosinolates are required for use

as internal standards (IS) in mass spectrometric determination by

isotope dilution mass spectrometry (IDMS). To reduce inter-

ference 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 deuter-

ated 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 gluco-

sinolate. In a greatly simplified approach, specifically to produce

IDMS internal standards, our collaborators have prepared the13C6-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 mono-

hydrated-GLS is required before any meaningful purity correc-

tion factors can be applied. Further work is needed on the

optimisation of extraction conditions with the view to doc-

umenting enhanced chemical and thermal stability by processes

such as microwave inactivation of enzymes prior to room

temperature extractions. Chromatographic separation tech-

niques and mass spectrometric assays continue to improve and it


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 tech-

nologies 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 chro-

matographic condition for the separation of intact glucosino-

lates. 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 bench-

marking values continues to be the single largest issue in quan-

tification, 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 effi-

ciencies, and most importantly, the analysis of reference mate-

rials. 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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