ISSN 1759-9660 Analytical Methods Advancing Methods and Applications 1759-9660(2010)2:4;1-7 Volume 2 | Number 4 | 2010 Analytical Methods Pages 301–416 www.rsc.org/methods Volume 2 | Number 4 | April 2010 | Pages 301–416 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
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ISSN 1759-9660
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
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,
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.
This journal is ª The Royal Society of Chemistry 2010
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’ 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
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
This journal is ª The Royal Society of Chemistry 2010
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
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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