Collision-induced dissociation mass spectra of glucosinolate anions

Article abstract of DOI:10.1002/jms.1711

Collision-induced dissociation (CID)mass spectra of differently substituted glucosinolates were investigated under negative-ion mode. Data obtained from several glucosinolates and their isotopologues (³⁴S and ²H) revealed that many p

Full text of DOI:10.1002/jms.1711

Research Article
Received: 22 September 2009
Accepted: 19 November 2009
Published online in Wiley Interscience: 23 December 2009
(
www.interscience.com) DOI 10.1002/jms.1711
Collision-induced dissociation mass spectra
of glucosinolate anions
a
a†
a
b
Jason B. Bialecki, Josef Ruzicka, Carl S. Weisbecker, Meena Haribal
a∗
and Athula B. Attygalle
Collision-induceddissociation(CID)massspectraofdifferentlysubstitutedglucosinolateswereinvestigatedundernegative-ion
mode. Data obtained from several glucosinolates and their isotopologues (34S and H) revealed that many peaks observed are
independent of the nature of the substituent group. For example, all investigated glucosinolate anions fragment to produce
a product ion observed at m/z 195 for the thioglucose anion, which further dissociates via an ion/neutral complex to give
two peaks at m/z 75 and 119. The other product ions observed at m/z 80, 96 and 97 are characteristic for the sulfate moiety.
The peaks at m/z 259 and 275 have been attributed previously to glucose 1-sulfate anion and 1-thioglucose 2-sulfate anion,
respectively. However, based on our tandem mass spectrometric experiments, we propose that the peak at m/z 275 represents
2
the glucose 1-thiosulfate anion. In addition to the common peaks, the spectrum of phenyl glucosinolate (β-D-Glucopyranose,
−
1
-thio-, 1-[N-(sulfooxy)benzenecarboximidate] shows a substituent-group-specific peak at m/z 152 for C6H5-C( NOH)S , the
CID spectrum of which was indistinguishable from that of the anion of synthetic benzothiohydroxamic acid. Similarly, the m/z
−
2
Ltd.
01 peak in the spectrum of phenyl glucosinolate was attributed to C6H5-C( S)OSO2 . Copyright ꢀc 2009 John Wiley & Sons,
Supporting information may be found in the online version of this article.
Keywords: glucosinolates; deuterium-labeled compounds; isotopologues; negative ions; even-electron ions; collision-induced
dissociation; fragmentation
Introduction
Several mass spectrometric techniques have been used for
qualitative and quantitative determination of glucosinolates.
Recently, it has been shown that glucosinolates upon negative-
mode ESI produce abundant ions, which fragment under collision-
induced dissociation (CID) conditions to form several product ions.
One of the dominant fragment ions derived from glucosinolates is
Glucosinolates are an important group of natural products
isolated predominately from plants of the Brassicaeae family.
Enzymatic action on glucosinolates during food preparation,
cooking, and chewing releases the characteristic flavors, or off-
flavors, associated with many cruciferous vegetables such as
cabbage, broccoli, and cauliflower. These off-flavors are attributed
primarily to the formation of isothiocyanates, which have been
demonstrated to have anti-cancer effects.[ Glucosinolates also
play an important role in the ecology of Brassicaceae vegetables
because many pest insects take cues from these compounds
to locate host plants. Moreover, the biosynthetic pathways
of glucosinolates are widely investigated to understand plant
genetics and genomics. Consequently, a significant research
effort has been devoted to developing chromatographic and
spectroscopic procedures for analyzing of glucosinolates found in
biological and food samples often as complex mixtures.[
−
[11]
that observed at m/z 97 for the HSO4 anion. Most quantitative
determinations of glucosinolates have been performed by LC-MS
multiple reaction monitoring (MRM) procedures using the parent
[10]
[11]
glucosinolate anion, and the product ion m/z 97, as the key
1,2]
ions. Two recent papers have reported quantitative LC/MS–MS
analysis of glucosinolates from plant extracts using this MRM
[15,16]
technique.
The advantages of LC-MS/MS procedure is that no
[
3]
desulfation or derivatization of the glucosinolates are necessary.
Although most MRM analyses have used the product ion at m/z 97,
thisioniscommonformostorganicsulfatecompounds. Therefore,
[
4]
[
17]
the use of more specific product ions would be more beneficial.
5–8]
Chemically, glucosinolates are a group of substituted cis-
hydroxyimine sulfate β-D-thioglucopyranosides (Fig. 1). Over 120
different glucosinolates have been identified from natural sources.
Several ionization methods have been utilized for generating
gaseous ions from glucosinolates for mass spectrometry.[9–12]
For earlier studies, fast-atom bombardment (FAB) ionization has
∗
Correspondence to: Athula B. Attygalle, Department of Chemistry, Chemical
Biology and Biomedical Engineering, Center for Mass Spectrometry, Stevens
Institute of Technology, Hoboken, NJ 07030, USA.
E-mail: athula.attygalle@stevens.edu
†
Current address: Josef Ruzicka, Scientific Instruments Division, Thermo Fisher
Scientific, 265 Davidson Avenue, Suite 101, Somerset, NJ 08873, USA.
[
13]
been the most informative.
For more recent investigations
a Department of Chemistry, Chemical Biology and Biomedical Engineering,
negative-ion electrospray ionization (ESI) has been employed,
however, direct ESI has been considered to generate very little
diagnosticallyusefulfragmentionsbecausespectraaredominated
by the glucosinolate anion derived from the glucosinolate salt.[
Center for Mass Spectrometry, Stevens Institute of Technology, Hoboken, NJ
07030, USA
14]
b Boyce Thompson Institute, Tower Road, Ithaca, NY 14853, USA
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.

CID mass spectra of glucosinolate anions
◦
masspeaks.Thesourcetemperaturewasheldat80 C.Thecapillary
voltage was held at 3000 V. Pressure of argon gas in the collision
−
5
cell was held at 5.1 × 10 Pa.
For deuterium exchange experiments, samples were dissolved
in 1 ml of 90 : 10 (v/v) acetonitrile: D2O (99% isotopic purity, Sigma-
Aldrich)and0.05 mlof1.0%(v/v)ND4OD(99atom%D;Cambridge
Isotope Laboratories, Andover, MA) in D2O.
Figure 1. General structure of a glucosinolate.
In order to properly identify more selective product ion(s) suitable
for MRM quantification of glucosinolates, a better understanding
of their fragmentation pathways is necessary.
Extraction
Glucosinolates were extracted from seeds based on a previously
described procedure.
Glucosinolates can be categorized into several chemical classes
[20]
Seeds (0.1 g) of a specific variety were
[
18]
based on the chemistry of the R-substituent.
In our quest for
placed in a closed vial and submerged in boiling water for
5 min to deactivate the enzyme myrosinase. The seeds were then
pulverizedandextractedthreetimeswithboilingwater–methanol
19 : 1). After each hot water–methanol extraction, samples were
understanding intricate rearrangement mechanisms involved in
the fragmentation processes of organic anions, we investigated
spectra of glucosinolates bearing allylic, aromatic, and sulfur-
containing R-substituent (Fig. 2), and we present here our results.
1
(
allowed to cool and were centrifuged at 11 000 rpm for 2–3 min.
The supernatant layers were separated, combined and passed
through a DSC-18 plug (Supelco). The eluent was concentrated
under reduced pressure to yield a crude mixture of natural
glucosinolates. This concentrated extract was taken up in 0.5 ml
of water and centrifuged at 11 000 rpm for 2 min to give the final
seed extract as the supernatant.
Experimental
Materials
All materials and reagents, including sinigrin (1) as the potassium
salt, were obtained from Sigma-Aldrich Chemical Company (St
Louis, MO) unless otherwise stated. Glucotropaeolin (5) and
gluconasturtiin (6) were purchased as potassium salts from
Carl Roth GmbH (Karlsruhe, Germany). Glucoerucin (7) and
glucoiberin (8)were isolated and from Waltham-29 broccoli seeds,
and progoitrin was separated from Red Russian kale seeds
Chromatography
The preparative high performance liquid chromatography (HPLC)
was conducted on an HP1090 (Hewlett–Packard) liquid chro-
matograph. The method used was based on a previously reported
(Organica Seed, Wilbraham, MA). The synthesis of phenyl-d5
[21]
procedure.
The final seed extract was injected (50 µl) onto an
desulfoglucosinolate(11)andbenzothiohydroxamicacid(theacid
of 21) are reported below. The synthesis of phenyl glucosinolate
Aqua C18 RP column (150 × 4.60-mm i.d., 3-µm particle size) from
Phenomenex (Torrance, CA). The column was eluted at a flow rate
(
3), which is reported below, was based on a previously reported
−1
of 1 ml min with mobile phase A (water with 0.1% HCOOH) to
[
19]
synthesis for phenyl-d5 glucosinolate (4).
All synthesized
which mobile phase B (acetonitrile with 0.1% HCOOH) was added
by a linear gradient (initially 2% B for 5 min, and then increased
to 40% at 20 min, to 100% at 25 min), and then kept isocratic for
products were detected as one spot when subjected to thin
layer chromatographic analysis (precoated 0.25-mm silica plates
from Fisher Scientific, Pittsburgh, PA).
5
min. The column was maintained at ambient temperature and
the peaks were monitored by an ultraviolet (UV) detector set at
35 nm. Progoitrin (2) was collected (multiple times) at retention
2
NMR spectrometry
time 3–4 min from the final Red Russian kale seed extract. Glu-
coiberin (8) and glucoeurcin (7) were collected (multiple times) at
retention times 3–4 min and 16–17 min, respectively, from the
final Waltham-29 broccoli seed extract. Solvents were removed
under reduced pressure to yield off-white solids, which appeared
pure under negative-ion electrospray MS.
All nuclear magnetic resonance (NMR) spectra were recorded on
a Varian INOVA 400 spectrometer as CDCl3 or D2O solutions.
Chemical shifts for H NMR are reported in ppm relative to
1
the internal reference TMS or DMSO. All coupling constants (J
1
3
values) are reported in Hertz (Hz). All C NMR spectra are proton
decoupled and all chemical shifts are reported relative to the
central peak of CDCl3 or DMSO.
Synthetic reactions
Mass spectrometry
Phenyl-d5 desulfoglucosinolate (11)
ꢁ
ꢁ
ꢁ
ꢁ
2
The CID mass spectra were recorded on a Micromass Quattro I
tandem mass spectrometer equipped with an electrospray ion
source. Samples were infused as acetonitrile–water–ammonia
S-2 ,3 ,4 ,6 -Tetra-O-acetyl-β-D-glucopyranosyl
[2,3,4,5,6- H5]
phenylthiohydroxamate (0.20 g, 0.36 mmol), which was pre-
viously prepared,[
methanol (1.0 ml, 7 M, Aldrich) and allowed to stand for 48 h at
19]
was dissolved in anhydrous ammonia in
−
1
(
9 : 1 : 0.00001, v/v/v) solutions at a flow rate of 6 µl min . The
◦
◦
source temperature was held at 80 C. The capillary voltage
was held at 4000 V. The argon gas pressure in the collision cell
was adjusted to attenuate precursor transmission by 30–50%.
Accurate mass measurements were performed at high resolution
−10 C. The solvent was removed under reduced pressure to
afford a white, solid residue as the final product (11, 0.10 g, 76%).
δH (400 MHz; CDCl3) δ 2.58 (1H, m), 3.12 (1H, t, J = 9.2 Hz), 3.31 (2H,
m), 3.58 (1H, dd, J = 12.4 Hz, J = 5.2 Hz), 3.70 (1H, dd, J = 12.4 Hz,
J = 2.4 Hz), 4.20 (1H, d, J = 10.0 Hz), 7.46 (3H, m), 7.59 (2H, t,
J = 7.2 Hz); δC (400 MHz; CDCl3) 62.4, 70.9, 74.1, 79.9, 82.2, 85.3,
129.8, 131.1, 135.3, 155.2; m/z (negative-ion electrospray) [M −
(
R ≈ 15000) on a Waters-Micromass Q-ToF API-US spectrometer
equipped with a nanoelectrospray ion source. Signals were
acquired in the W-mode operation. Ion series generated by water
clusters charged by the bicarbonate anion were used as reference
−
H] required for C21H19D5NO9 319.1018, found 319.1031.
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

J. B. Bialecki et al.
Figure 2. Structures of the anions of the investigated glucosinolate salts. Allyl class: sinigrin (1), progoitrin (2). Aromatic class: phenyl glucosinolate (3),
phenyl-d5 glucosinolate (4), glucotropaeolin (5), gluconasturtiin (6). Sulfur class: glucoerucin (7), glucoiberin (8).
Benzothiohydroxamic acid (N-hydroxybenzenecarbothioamide, the
free acid of anion 21)
mixture was stirred for 2 h at RT. The organic layer was separated
and washed with H O (10 ml), dried over Na SO and evaporated
2
2
4
to give the product (PhC(Cl) NOH, 0.53 g, 89%), which was used
in the next step without further purification. δH (400 MHz; CDCl3)
Magnesium turnings (0.32 g, 13.3 mmol) and a crystal of iodine
were heated until purple vapors formed, and a solution of
bromobenzene (2.0 g, 12.7 mmol) in Et2O (8 ml) was added.[
7
1
.52 (5H, m), 10.5 (1H, s); δC (400 MHz; CDCl3) 126.8, 127.6, 130.3,
37.03, 144.2; m/z (positive-ion electrospray) 137 ([M − HCl +
22]
The mixture was refluxed for 30 min, cooled in an ice bath to
+
+
◦
NH4] , 100%), 120 (20, [M − HCl + H] ), 104 (30), 77 (25).
0
C, and carbon disulfide (1.1 ml, 18.4 mmol) was added. After
refluxing for 1 h, solvents were removed, and the dark residue was
taken up in KOH (6 ml, 2 M) and filtered over Celite to give a
clear, reddish solution. A solution of hydroxylamine hydrochloride
ꢁ
ꢁ
ꢁ
ꢁ
S-2 ,3 ,4 ,6 -Tetra-O-acetyl-β-D-glucopyranosyl phenylthiohydroxa-
mate
(
0.9 g, 13.0 mmol)inwater(4 ml)wasaddedtothereddishsolution
A solution of Et3N (0.2 ml) and Et2O (1.0 ml) was added to a stirred
mixture of 1–thio-β-D-glucose tetraacetate (0.4 g, 1.1 mmol) and
phenylhydroxamic chloride (0.21 g, 1.4 mmol) in dry Et2O (4.0 ml)
andDCM(2.0 ml). Aprecipitateoftriethylaminehydrochloridewas
formed immediately. The reaction mixture was stirred for 2 h at RT,
and washed with aq. HCl (10 ml, 1 M) and H2O (10 ml). The organic
layerwasseparated,driedoverNa2SO4,andevaporated.Thecrude
product was recrystallized from ethanol to afford the pure white
cis product (0.34 g, 62%). δH (400 MHz; CDCl3) 2.03 (3H, s), 2.04 (3H,
s), 2.09 (3H, s), 2.12 (3H, s), 3.04 (1H, s), 3.97 (1H, d, J = 12.4), 4.09
and the mixture was stirred overnight. The aqueous mixture was
extracted with Et2O (3 × 5 ml); the Et2O layer was separated, dried
over MgSO4 and evaporated to yield the crude product which was
dissolved in MeOH, filtered hot over charcoal and evaporated to
give the final product as a yellowish solid (the acid of anion 21,
0
.20 g, 66%). δH (400 MHz; CDCl3) δ 7.40 (2H, t, J = 7.4 Hz), 7.46
(
1
1H, t, J = 7.6 Hz), 7.65 (2H, d, J = 7.2 Hz); δC (400 MHz; CDCl3)
26.7, 128.8, 131.2, 135.1; m/z (negative-ion electrospray) [M −
−
H] required for C7H6NSO 152.0176, found 152.0181.
(
1H, dd, J = 12.4 Hz, J = 4.8 Hz), 4.45 (1H, d, J = 9.6 Hz), 5.01 (2H,
d, J = 8.4 Hz), 5.08 (1H, td, J = 7.2 Hz, J = 2.8 Hz), 7.46 (3H, m),
Benzaldehyde oxime
7
2
1
.52, (2H, d, J = 7.4 Hz), 8.94 (1H, s); δC (400 MHz; CDCl3) 20. 4, 20.5,
A solution of sodium carbonate (0.28 g, 2.7 mmol in 5 ml of H2O)
was slowly added to a solution of hydroxylamine hydrochloride
0.6, 20.7, 61.7, 67.8, 69.8, 73.71, 75.7, 81.3, 128.5, 129.0, 130.1,
32.3, 169.2, 170.2 and 170.6; m/z (positive-ion electrospray) [M −
(0.35 g, 4.9 mmol in 5 ml of H2O) and benzaldehyde (0.52 g,
−
H] required for C21H24NO10S 482.1126, found 482.1134.
4
.8 mmol), and the mixture was stirred at RT for 2 h. The reaction
mixture was extracted with Et2O (15 ml); the Et2O layer was
separated, dried over Na2SO4 and evaporated to give the product,
a colorless oil, as mixture of E and Z isomers. (0.57 g, 94%). δH
ꢁ
ꢁ
ꢁ
ꢁ
Pyridinium S–2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosyl
phenylthiohydroxamic N-sulfate
ꢁ ꢁ ꢁ ꢁ
(
1
400 MHz; CDCl3) 7.36 (3H, m), 7.56 (2H, d, J = 7.4), 8.18 (1H, s),
A
solution of S-2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosyl
phenylthiohydroxamate (0.60 g, 1.26 mmol in 4.0 ml of DCM) and
dry pyridine (5 ml) was cooled to 0 C and a cold solution of
chlorosulfonic acid (1.6 ml, 24.2 mmol in 4.0 ml of dry Et2O) was
slowly added. After the addition, the ice bath was removed and
the mixture was stirred overnight at RT. All solvents were removed
underreducedpressureat60 C. TheresiduewasdissolvedinDCM
10 ml) and washed with H2SO4 (20 ml, 1 M) and H2O (10 ml). The
0.20 (1H, s); δC (400 MHz; CDCl3) 127.0, 128.7, 130.1, 131.97, 150.4;
◦
+
m/z (EI) 121 (M , 100%), 120 (20), 103 (15), 94 (40), 78 (80), 51 (70),
9 (20).
3
Phenylhydroxamic chloride (N-hydroxybenzenecarboximidoyl chlo-
ride)
◦
(
To a solution of benzaldehyde oximes from the previous step
organiclayerwasseparatedanddriedoverMgSO4 andevaporated
to afford the white product (0.36 g, 44%). δH (400 MHz; CDCl3) 1.96,
1.97, 2.06 and 2.10, 3.09 (1H, s), 4.01 (1H, d, J = 11.6 Hz), 4.09 (1H,
dd, J = 11.6 Hz, J = 4.8 Hz), 4.58 (1H, d, J = 7.6 Hz), 5.03 (3H,
(
0
0.45 g, 3.7 mmol in 5 ml of DCM) and pyridine (0.1 ml) cooled to
◦
C, N-chlorosuccinimide (0.49 g, 3.7 mmol) was slowly added.[23]
Upon completion, the ice bath was removed and the reaction
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 272–283

CID mass spectra of glucosinolate anions
m), 7.38 (2H, t, J = 7.2 Hz), 7.46 (1H, t, J = 7.2 Hz), 7.50 (2H, d,
J = 7.6 Hz), 7.87 (2H, t, J = 6.4 Hz), 8.39 (1H, t, J = 6.4 Hz), 8.86
154.9; m/z positive-ion electrospray) [M − NH4]− required for
C13H16NO9S2 394.0272, found 394.0275.
(
2H, d, J = 5.2 Hz); δC (400 MHz; CDCl3) 20.4, 20.6, 20.7, 61.5, 67.6,
6
1
−
9.4, 73.6, 75.7, 81.5, 126.9, 128.4, 129.2, 131.2, 142.5, 145.5, 157.0,
Computational methods
69.2, 169.3, 170.0 and 170.6; m/z (negative-ion electrospray) [M
−
C6H6N] required for C21H24NO13S2 562.0695, found 562.0704.
All QM calculations were performed using the Gaussian 03 W
program package. Computations were performed using the
restricted Hartree–Fock method as described in the review article
Ammonium β-D-glucopyranosyl phenylthiohydroxamic N-sulfate
ammonium salt of phenyl glucosinolate, 3) (β-D-Glucopyranose,
by Mercero et al.,[ using the 6-31G(d) basis set.
24]
[25]
Structures
(
depicted in the results section were energy minimized to obtain a
stationary state. Calculated relative energies include a correction
1
-thio-, 1-[N-(sulfooxy)benzenecarboximidate ammonium salt)
ꢁ ꢁ ꢁ ꢁ
S–2 ,3 ,4 ,6 -tetra-O-acetyl-β-D-glucopyranosyl
Pyridinium
for the zero-point energy of each structure (scaled by a factor of
26]
phenylthiohydroxamic N-sulfate (0.35 g, 0.55 mmol) was dis-
0
.9135).[
solved in anhydrous ammonia in methanol (1.5 ml, 7 M, Aldrich)
and allowed to stand for 48 h at −10 C. The solvent was removed
In addition, structures were also energy minimized using the
◦
B3LYP exchange-correlation functional and the 6-311++G(d,p)
basis set. Calculated relative energies include a correction for the
zero-point energy scaled by a factor of 0.9877.[ Energies of
optimized structures were also calculated using the MP2 method
with the 6-311++G(d,p) basis set.
under reduced pressure to afford a crude product. The crude
product was dissolved in water (2 ml), mixed with activated
charcoal, heated and filtered to give a colorless solution. The
solution was evaporated, and the residue was subjected to
repeated dissolution in methanol and evaporation. The final
residue was dissolved in a minimal amount of methanol (0.1 ml),
and triturated with THF (0.5 ml) followed by Et2O (1.5 ml) to form
a white precipitate. The solvent was decanted and the white
solid was washed with Et2O and dried to afford the final product
as a slightly off-white amorphous solid (3, 0.17 g, 65%). Reverse
phase-HPLC with UV detection at 235 nm showed that the purity
of the product was >99%. δH (400 MHz; D2O) 2.47 (1H, m), 2.89
27]
Results and Discussion
Glucosinolates are ideal candidates for negative-ion electrospray
ionizationmassspectrometrybecauseofthesulfatemoietyintheir
molecular structure. Thus, the anions derived from glucosinolates
have been widely used for their qualitative and quantitative
(
1H, t, J = 8.6 Hz), 3.06 (2H, td, J = 8.4 Hz, J = 4.2 Hz), 3.35 (1H,
determinations. The product ion spectra of all glucosinolate
anions[
16]
are known to show an intense signal at m/z 97,
dd, J = 12.2 Hz, J = 5.6 Hz), 3.51 (1H, d, J = 9.6 Hz), 3.95 (1H, d,
J = 7.6 Hz), 7.46 (3H, m), 7.53 (2H, d, J = 7.4 Hz); δC (400 MHz;
D2O) 54.9, 60.4, 69.2, 72.3, 78.2, 81.1, 128.1, 129.0, 129.5, 132.4,
−
representing a bisulfate anion (HSO4 ). For example, the low-
energy CID spectrum of m/z 394 of phenyl glucosinolate (3), which
Figure 3. Product ion spectra of ions derived from phenyl glucosinolate (3, m/z 394) (A), phenyl glucosinolate (3) in D2O (m/z 398) (B), phenyl-d5
∗
glucosinolate (4, m/z 399) (C) and phenyl-d5 glucosinolate (4) in D2O (m/z 403) (D). Peaks for ions containing the R-substituent are denoted with an ‘ ’.
Laboratory-frame collision energy was set at 25 eV.
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

J. B. Bialecki et al.
was synthesized in our laboratory, showed a significant peak at
m/z 97 (Fig. 3A). Similar results were obtained from several other
glucosinolate anions (Fig. 4). However, the m/z 97 product ion
is not specific for glucosinolates because most organic sulfate
anions form this anion via a six-membered, cyclic syn-elimination
mechanism as depicted in Scheme 1.
However, a six-membered transition state for a similar hy-
drogen transfer (from R substituent) cannot be attained from
glucosinolates because the N-sulfated thiohydroximate moiety of
glucosinolates bears a (Z)-configuration.[ For some molecules,
particularly for those without a hydrogen atom at the C-2 posi-
tion, an alternative mechanism in which two neutral molecules
29]
are eliminated via an eight-membered transition state has been
[
28]
[28]
proposed.
In fact, this mechanism entails properties of het-
[
30]
erolytic fragmentations described by Grob and Schiess.
H/D
exchange studies carried out in solution with phenyl glucosino-
Figure 4. Product ion spectra of ions derived from sinigrin (1, m/z 358) (A), progoitrin (2, m/z 388) (B), glucotropaeolin (5, m/z 408) (C), glucoerucin (7,
m/z 420) (D), gluconasturtiin (6, m/z 422) (E) and glucoiberin (8, m/z 422) (F). Note: the m/z 195 in spectrum (B) represents two different ions. Peaks for
∗
ions containing the R-substituent are denoted with an ‘ ’, and the m/z values of the ions originating from a dissociation within the substituent are given
in brackets. Laboratory-frame collision energy was set at 25 eV.
Scheme 1. Production m/z 97 ion from most organic sulfates.[25]
.
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 272–283

CID mass spectra of glucosinolate anions
Scheme 2. Formation of m/z 97 ion from glucosinolate anions.
Scheme 3. Fragmentation of phenyl glucosinolate anion (3, m/z 394).
late (3) and its deuteriated analog 4 (Fig. 3B, D) confirmed that
thiohexoseanion(9).[5,29] Theoriginofthem/z195fragmention(9)
from the 1-thiohexose moiety of glucosinolates can be attributed
to a charge-mediated multiple bond cleavage mechanism similar
to that proposed for phenyl glucosinolate in Scheme 4.
−
the major source of the hydrogen atom in the HSO4 is a non-
exchangeable hydrogen atom from the sugar moiety. The most
plausible atom for this transfer is the hydrogen atom attached
to the C-1 carbon. Thus, we propose a mechanism similar to the
Although a mechanism of eliminating SO3 and phenylnitrile
oxide (10) by a stepwise pathway can be considered, a peak
for a SO3 loss was not observed in the spectrum of phenyl-d5
glucosinolate (Fig. 5A), or any other glucosinolate. On the other
hand, the anion of synthetic phenyl-d5 desulfoglucosinolate, m/z
319 (11), which was synthesized in our laboratory, dissociated
under very low collision energy to produce an intense signal at
m/z 195 (Fig. 5B). It appears that without the sulfate group to
stabilize the negative charge, the desulfo species of m/z 319,
readily transfers the charge from the oxime oxygen to the thio
function and eliminates phenyl-d5-nitrile oxide (12) (Scheme 5).
Moreover, a comparison of the product ion spectrum of m/z 399
ion from phenyl-d5 glucosinolate (4) with that from the m/z 319
ion from phenyl-d5 desulfoglucosinolate (11) (Fig. 5), established
the identity of product ions that still contain the sulfur atom from
the sulfate moiety. The product ions observed at 75, 119, 157
and 195 are common to both spectra (Fig. 5). Because phenyl-d5
[
28]
reported eight-member transition mechanism,
to rationalize
−
the formation of m/z 97 (HSO4 ) from glucosinolate ions (Scheme
2
).
The product ion spectra of all investigated glucosinolates (Figs 3
and 4) also show peaks for two odd-electron fragment ions at m/z
−•
0 and m/z 96. The formation of an m/z 80 (SO3 ) product ion
8
−
−
from both m/z 97 (HSO4 ) and 98 (DSO4 ) ions, by loss of a
hydroxyl or deuteroxyl radical, respectively (Fig. 3A, B), confirmed
that the bisulfate anion fragments by a homolytic fission of the
[
28]
S–OH(D)bond. Basedonthereportedfragmentationoforganic
N-sulfate anions,[ the facile formation of m/z 96 (SO4 ) from
glucosinolate anions occur through a direct homolytic cleavage of
the N–O bond of the N-sulfate function (Scheme 3).
31]
−•
The characteristic fragment peak at m/z 195, observed in all
recorded glucosinolate spectra (Figs 3A, C, 4 and 5), is analogous
to that observed under negative-ion FAB conditions for the 1-
Figure 5. Product ion spectra of m/z 399 ion derived from phenyl-d5 glucosinolate at collision energy setting of 30 eV (A), and the m/z 319 ion derived
from phenyl-d5 desulfoglucosinolate at collision energy setting of 15 eV (B). The inset in Figure B shows the product ion spectrum of in-source generated
m/z 195 ion (cone voltage 25 V). Laboratory-frame collision energy was set at 25 eV.
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

J. B. Bialecki et al.
Scheme 4. Formation of m/z 195 ion from phenyl glucosinolate anion.
Scheme 5. Fragmentation of m/z 319 anion of phenyl-d5 desulfoglucosinolate (11).
desulfoglucosinolate (11) contains no sulfate residue, it was clear
that these ions do not contain the sulfur atom from the sulfate
moiety. In contrast, the product ions observed at m/z 80, 96, 97,
between them which contained one imaginary vibrational mode.
Presumably, after the initial transfer of the acidic proton, the
carbon–carbon bond between C-2 and C-3 positions elongates
to form a transition state which eventually breaks down to form
an ion/neutral complex (15) between thioglycal and tetrose anion
both of which remain closely associated as depicted in Scheme 6.
2
06, 259, and 275 all contain the sulfur atom from the sulfate
moiety (Fig. 5A).
A tandem mass spectrometric experiment conducted with the
thioglucose anion of m/z 195 (9) indicated its fragmentation to
two product ions observed at m/z 75 and 119 (see inset in Fig. 5B).
For the formation of these two ions, the negative charge on
the sulfur atom of thioglucose anion should be transferred to
the hydroxy group on position 3 by a proton transfer via a six-
membered transition state (Scheme 6). Computations carried out
atHartree–Focklevelwitha6-31G(d)basisset, andB3LYPandMP3
levelswitha6-311++G(d,p)setshowednotonlythatthetransition
state energy barrier for proposed flip from a chair conformation (9)
isrelativelysmall(about6 kcal/mol)butalsotheboatconformation
The formation of such ion/neutral complexes have been noted
in several other anion fragmentation mechanisms.[
32,33]
A simple
dissociation of the complex leads to the ion observed at m/z 119
(17) (Scheme 6). Alternatively the complex can break via a proton
transfer[ to give a peak m/z 75 (16). This fragmentation pathway
is considered to be the more favorable pathway in the product
ion spectra of glucosinolates (Figs 3 and 4). The structural identity
of m/z 119 was established to be a deprotonated tetrose by a
comparison of its product ion spectrum with that derived from
deprotonated D-(−)-erythrose (m/z 119). Both spectra were nearly
identical (Fig. S2). Evidently, the formation m/z 75 and m/z 119
ions appear to be characteristic for all glucosinolate anions.
Alternatively, a ring-opening mechanism similar to that pro-
posed for the fragmentation of deprotonated glucose can be
considered.[ Although calculations at the HF/6-31G(d) level for
34]
1
3 (about −2 kcal/mol) that is formed is relatively more stable
than the chair form 9 (0.00 kcal/mol) (Supplementary Fig. S1).
Moreover, when the stationary states were verified by a frequency
analysis, it confirmed that only the chair and boat conformations
bear real vibrational modes, as opposed to the transition state
35]
Scheme 6. Proposed Fragmentation pathway for thioglucose anion (m/z 195).
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 272–283

CID mass spectra of glucosinolate anions
Scheme 7. Formation of m/z 259 ion from glucosinolates.
Scheme 8. Proposed rearrangement to form glucose 1-thiosulfate anion (19).
the glucose anion identified a ring-opened intermediate structure
with a terminal aldehyde group, when the aldehyde oxygen of this
intermediate was replaced with a sulfur atom and the structure
was reoptimized, the structure failed to remain open anymore.
Instead, the thioglucose anion from a ring-opened starting point
optimized to a ring-closed ground state. This computational result
suggested that ring opening of thioglucose anion is energetically
lessfavorablethantheringopeningofglucoseanion, anddoesnot
lead to an open intermediate structure. Furthermore, the general
appearance of the CID spectrum of glucose anion is very different
to that obtained from the thioglucose anion (Fig. S3).
Further support to the proposed mechanism in Scheme 6 was
obtained by H/D exchange experiments conducted in solution.
When glucosinolates without any exchangeable hydrogens in
the R group were dissolved in D2O and spectra were recorded,
the peak for the glucosinolate anion underwent a shift of 4 m/z
units indicating the exchange of the four hydroxyl hydrogen
atoms in the glucose ring with deuterium (Fig. 3A, B). Under these
conditions, the peak for the 1-thiohexose product ion, (m/z 195),
underwent a shift to m/z 199. This result confirmed that the four
exchangeable hydroxyl hydrogen atoms were still intact in the
glucose ring after the fragmentation. Upon fragmentation of the
m/z 199 ion, the m/z 75 peak shifted to m/z 76 indicating the
presence of one exchangeable hydrogen atom in the thioglycal
anion. Similarly, the peak at m/z 119 was shifted to m/z 121
indicating the presence of two exchangeable hydrogen atoms in
the tetrose anion (Fig. 3A, B).
anion (19). Because the S–S bond in m/z 275 would cleave
more easily than the S–O bond in m/z 259 (18), an SO3 loss can be
expected from a thiosulfate but not from a sulfate (a sulfate moiety
loses SO3 only when it is attached directly to a phenyl ring or a
double bond,[
28,31]
or when another acidic group is present in the
[
38]
molecule which can accommodate the negative charge ). Thus,
the thioglucose 2-sulfate structure, in which the sulfate group is
linked directly to the C-2 position on the sugar ring, suggested by
Kokkonen et al.,[ Bojen and Larsen, and Fabre et al., for the
5]
[36]
[39]
m/z 275 ion appears to be erroneous.
Product ion experiments conducted with samples under H/D
exchangeconditions(Fig. 2B, D)revealedashiftofm/z 259and275
peaks to m/z 263 and m/z 279, respectively. This result indicated
that each ion bears four exchangeable hydrogen atoms, and the
glucoseringremainsintactduringtherearrangementasillustrated
in Scheme 8.
Two other significant fragment ion peaks observed in glucosi-
nolate product ion spectra are denoted by asterisks in Figs 3 and 4.
Unlike the product ions discussed thus far, these ions contain the
R-substituent. Even though the m/z values of these two fragment
ions vary according to the mass of the R-substituent of the glu-
cosinolate, the mechanisms for their formation appear to follow
a generalized pathway. For example, the CID spectrum of phenyl
glucosinolate(3)showtwopeaksatm/z 152and201,andthatfrom
the phenyl-d5 glucosinolate (4) show the corresponding peaks at
m/z 157 and 206 (Fig. 3A, C). This observation confirmed that these
peaks are attributable to ions bearing the phenyl substituent. Deu-
terium/hydrogen exchange experiments (Fig. 3B, D) showed that
fragment ions m/z 152 and 157 bear only one exchangeable hy-
drogen. An accurate mass determination experiment established
The CID spectra of glucosinolates (Figs 3A, C and 4) also
show two minor peaks at m/z 259 and 275. Both these ions
have been previously reported from negative-ion FAB spectra of
glucosinolates.[
5,36]
Thus, we inferred that the m/z 259 fragment
the molecular composition of the m/z 152 ion to be C7H6NSO
−
ion (Fig. 3) represents the glucose 1-sulfate anion (18) which
presumably originates by an elimination of phenyl isothiocyanate
from the precursor anion (Scheme 7). An open-ring structure has
been previously proposed for the m/z 259 ion by Rochfort et al.[
A product ion experiment conducted with the m/z 259 ion,
(Supplementary Table S1). Moreover, the product ion spectrum of
m/z 152 was indistinguishable from that obtained under identi-
cal conditions from the anion of benzothiohydroxamic acid (21),
which was synthesized in our laboratory. Both m/z 152 and 157
ions fragmented to give a single intense product ion peak at
m/z 33 (HS ), which becomes m/z 34 (DS ) under H/D exchange
conditions. The fragmentation occurs by a loss of a phenylnitrile
oxide molecule from the m/z 152 ion (21). Although the structure
of the m/z 152 product ion (21) formed from phenyl glucosinolate
(3) is confirmed, the exact pathway of its formation is less certain.
37]
revealed that it does not undergo a direct loss of SO3[
31,37]
In
−
−
contrast, the m/z 275 ion underwent a direct loss of SO3, upon
CID. Because glucose sulfates are not known to eliminate SO3, we
propose that the m/z 394 anion undergoes a fragmentation to
eliminate phenyl cyanate (20) and produce a glucose 1-thiosulfate
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

J. B. Bialecki et al.
Scheme 9. Proposed pathway for the formation of m/z 152 ion from phenyl glucosinolate anion.
Scheme 10. Formation of m/z 201 ion (24) from phenyl glucosinolate anion.
A proposed pathway based on available experimental details is
illustrated in Scheme 9.
anhydroglucose molecule (22) and a nitrosyl hydride molecule
(23) (Scheme 10).
Anotherintriguingpeakinthespectrumofphenylglucosinolate
3) is that observed at m/z 201. Unlike the ions discussed above,
The m/z 201 ion (24) represents the deprotonated anhydride of
thiobenzoicacidandsulfurousacid(Fig. 6).Theproposedstructure
is supported by the results obtained with phenyl-d5 glucosinolate
(4), which showed a peak at m/z 206 in its product ion spectrum
(Fig. 3C). Upon CID, both m/z 201 and 206 ions underwent a facile
loss of SO2 confirming the proposed sulfite anhydride structure
(Fig. 6). The thiobenzoate anion (m/z 137 or 142) formed in this
way, loses COS to form the phenyl anion (m/z 77 or 82), or loses
CO by a rearrangement to give the thiophenoxide anion (m/z 109
or 114).
(
H/D exchange experiments revealed that the m/z 201 ion (24)
contains no exchangeable hydrogen atoms (Fig. 3). An accurate
mass determination experiment of the m/z 201 ion confirmed its
molecular formula to be C7H5O3S2 (Table S1). In other words, the
precursor m/z 394 ion should undergo an unprecedented skeletal
rearrangement and then fragment to eliminate the nitrogen atom
while retaining both sulfur atoms. To rationalize the observations,
we propose that upon collisional activation the m/z 394 ion
rearranges to a nitroso derivative, which undergoes a charge
remote fragmentation via six-membered system to eliminate an
To obtain further support for the proposed structures of the
fragment ions of glucosinolates, we recorded and compared
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 272–283

CID mass spectra of glucosinolate anions
Figure 6. Product ion spectra of in-source generated fragment ions of m/z 201 from phenyl glucosinolate (A), and that of m/z 206 from phenyl-d5
glucosinolate (B). (Laboratory-frame collision energy was set at 25 eV, and cone voltage 30 V).
Figure 7. Product ion spectra derived from m/z 358 of [32S2]sinigrin (A), and m/z 360 of [34S132S1]sinigrin (B). Laboratory-frame collision energy was set
at 25 eV.
the product ion spectra of 32S2 and 32S134S1 isotopologues of
Gluconasturtiin (6) and glucoiberin (8), two common glucosino-
latesinBrassicaseeds,areconsideredisobariccompoundsbecause
they have the same nominal mass. A comparison of their product
ion spectra revealed that they can be differentiated directly by
MS/MS. In addition to the usual fragment ions, the CID spectrum
of glucoiberin displays two other significant peaks because of the
distinctive chemistry of its R-substituent (peaks denoted by brack-
ets in Fig. 4). Both gluconasturtiin (Fig. 4E) and glucoiberin (Fig. 4F)
show essentially the same peaks, with the notable exception of
m/z 358 and 407 peaks in the spectrum of glucoiberin, which is
a unique three-sulfur-atom glucosinolate with a sulfoxide moiety.
The two peaks at m/z 358 and 407 represent neutral losses of
methyl sulfoxide (64 Da) and methyl radical (15 Da), from the m/z
sinigrin (Fig. 7). In the spectrum recorded from the m/z 360 ion
3
4
32
of [ S1 S1]sinigrin, peaks for all product ions bearing one sulfur
atom appeared as double peaks, whereas those bearing two sulfur
atoms showed only one peak at a value two m/z units higher
3
2
than that observed from the S2 isotopologue. For example, the
3
2
peak at m/z 275 for the 1-phenyl[ S2]thiosulfate anion, increased
3
2
34
to m/z 277 for the 1-phenyl[ S1 S1]thiosulfate anion indicating
the presence of two sulfur atoms. The product ion at m/z 259 on
the other hand, changed to a double peak at m/z 259 and 261
indicating the presence of one sulfur atom. Under this experiment,
product ions m/z 75, 80, 96, 97 and 195 all behaved in the same
fashion as that observed from the m/z 259 ions confirming that
each of them contain a single sulfur atom. The product ion at m/z
4
22 ion of glucoiberin, respectively.
1
19 for the tetrose (17) on the other hand remained unchanged
in this experiment because it is not a sulfur-containing ion. The
productionCH2 CH-CH2-C( NOH)S ion(m/z 116)fromsinigrin
Conclusions
−
(
1), which corresponds to the product ion at m/z 152 from phenyl
The fragmentation pathways of glucosinolate anions under CID
conditions are dictated by the chemistry of the sulfate, thiohy-
droxamate, and sugar moieties. Consequently, all glucosinolates
generate common ions that are diagnostically useful for the gen-
eral group. In the low-mass region, besides the peak at m/z 97,
intense peaks observed at m/z 75 and m/z 96 are also useful to
identify glucosinolates in a complex mixture. Although the m/z 97
glucosinolate (as shown in Table 1) changed to a double peak
at m/z 116 and 118 indicating the presence of one sulfur atom.
Finally, product ion CH2 CH-CH2-C( S)OSO2 (m/z 165) from
sinigrin, which corresponds to the product ion m/z 201 from
phenyl glucosinolate (as shown in Table 1) changed to a single
peak at m/z 167 indicating the presence of two sulfur atoms.
−
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

J. B. Bialecki et al.
Table 1. A list of peaks observed in negative-mode CID fragmentation of [M − X]− ions of some glucosinolatesa
Fragment ions specific to each
R-substituent (m/z)
Fragment ions common to all
−
[
M − X]
(m/z)
R-substituent and its
nominal mass (Da)
glucosinolates
−
−
Glucosinolate
(m/z)
R-C( NOH)S
R-C(S)OSO2
Sinigrin (1)
358
388
394
399
408
422
420
422
-CH2CH CH2 (41)
-CH2CH(OH)CH CH2 (71)
-C6H5 (77)
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
75, 80, 96, 97, 119, 195, 259, 275
116
146
152
157
166
180
178
180
165
195
201
206
215
229
227
229
Progoitrin (2)
Phenyl glucosinolate (3)
Phenyl-d5 glucosinolate (4)
Glucotropaeolin (5)
Gluconasturtiin (6)
Glucoerucin (7)
-C6D5 (82)
-CH2C6H5 (91)
-CH2-CH2-C6H5 (105)
-(CH2)4SCH3 (103)
-(CH2)3SOCH3 (105)
Glucoiberin (8)
a
X = K or NH4.
ion is widely used for qualitative and quantitative investigations
of glucosinolates, the thioglycosyl ion at m/z 75, which is specific
for thioglucosyl moiety, appears to be a better and more specific
choice for MRM determinations.
[11] F. A. Mellon, R. N. Bennett, B. Holst, G. Williamson. Intact
glucosinolate analysis in plant extracts by programmed cone
voltage electrospray LC/MS: performance and comparison with
LC/MS/MS methods. Anal. Biochem. 2002, 306, 83.
[
[
[
12] B. Matthaus, H. Luftmann. Glucosinolates in members of the family
brassicaceae: separation and identification by LC/ESI-MS–MS.
J. Agric. Food Chem. 2000, 48, 2234.
13] G. R. Fenwick, J. Eagles, R. Self. The fast atom bombardment spectra
of glucosinolates and glucosinolate mixtures. Org. Mass Spectrom.
Acknowledgements
ThisresearchwassupportedbyfundsprovidedbyStevensInstitute
of Technology, NJ. J. B. acknowledges the support received by a
Robert Crooks Stanley Fellowship.
1
982, 17, 544.
14] C. L. Zrybko, E. K. Fukuda, R. T. Rosen. Determination of
glucosinolates in domestic and wild mustard by high-performance
liquid chromatography with confirmation by electrospray mass
spectrometry and photodiode-array detection. J. Chromatogr. A
Supporting information
1
997, 767, 43.
Supporting information may be found in the online version of this
article.
[15] Q. Tian, R. A. Rosselot, S. J. Schwartz. Quantitative determination of
intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts,
and cauliflower by high-performance liquid chromatography-
electrospray ionization-tandem mass spectrometry. Anal. Biochem.
2
005, 343, 93.
References
[16] L. Song, J. J. Morrison, N. P. Botting, P. J. Thornalley. Analysis of
glucosinolates,isothiocyanates,andaminedegradationproductsin
vegetable extracts and blood plasma by LC-MS/MS. Anal. Biochem.
[
[
[
[
[
1] S. S. Hecht. Chemoprevention by isothiocyanates. J. Cell. Biochem.
995, 22, 195.
2] S. S. Hecht. Inhibition of carcinogenesis by isothiocyanates. Drug
Metab. Rev. 2000, 32, 395.
1
2
005, 347, 234.
[
17] S. Mill a´ n, M. C. Sampedro, P. Gallejones, A. Castell o´ n, M. L. Ibargoitia,
M. A. Goicolea, R. J. Barrio. Identification and quantification of
glucosinolates in rapeseed using liquid chromatography-ion trap
mass spectrometry. Anal. Bioanal. Chem. 2009, 394, 1661.
18] J. W. Fahey, P. Talalay, A. T. Zalcmann. The chemical diversity and
distribution of glucosinolates and isothiocyanates among plants.
Phytochemistry 2001, 56, 50.
3] B. A. Halkier, J. Gershenzon. Biology and biochemistry of
glucosinolates. Ann. Rev. Plant Biol. 2006, 57, 303.
4] J. Gershenzon, C. Mueller. Phytochemistry reviews special issue on
glucosinolates. Phytochem. Rev. 2009, 8, 1.
[
5] P. S. Kokkonen, J. van der Greef, W. M. A. Niessen, U. R. Tjaden,
G. J. ten Hove, G. van der Werken. Identification of intact
glucosinolates using direct coupling of high-performance liquid
chromatography with continuous-flow frit fast atom bombardment
tandem mass spectrometry. Biol. Mass Spectrom. 1991, 20, 259.
6] G. R. Fenwick, J. Eagles, R. Gmelin, R. Rakow. The mass spectra
of glucosinolates and desulphoglucosinolates. A useful aid for
structural analysis. Biomed. Mass Spectrom. 1981, 9, 410.
[
19] J. B. Bialecki, J. Ruzicka, A. B. Attygalle. Synthesis of d5-phenyl
glucosinolate. J. Labelled Comp. Rad. 2007, 50, 711.
20] L. Xian, M. H. Kushad. Correlation of glucosinolate to myrosinase
activity in horseradish (Armoracia rusticana). J. Agric. Food Chem.
[
[
[
2
004, 52, 6950.
[
[
[
[
21] L. West, I. Tsui, G. Haas. Single column approach for the liquid
chromatographic separation of polar and non-polar glucosinolates
from broccoli sprouts and seeds. J Chromatogr. A 2002, 966, 227.
22] A. J. Mitchell, K. S. Murray, P. J. Newman, P. E. Clark. Thiohydroxam-
ato complexes of iron(III). Synthesis, magnetism and Moessbauer
spectra. Aust. J. Chem. 1977, 30, 2439.
23] A. F. Hegarty, M. Mullane. Large stereoelectronic effect in 1,3-
dehydrohalogenation to form a 1,3-dipole. J. Chem. Soc. Chem.
Commun. 1984, 4, 229.
7] T. Prestera, J. W. Fahey, J. L. Kachinski, P. Talalay. Comprehensive
chromatographic and spectroscopic methods for the separation
and identification of intact glucosinolates. Anal. Biochem. 1996,
239, 168.
[
[
8] R. N. Bennett, F. A. Mellon, P. A. Kroon. Screening crucifer seeds
as sources of specific intact glucosinolate using ion-pair high-
performance liquid chromatography negative ion electrospray
mass spectrometry. J. Agric. Food Chem. 2004, 52, 428.
9] J. Eagles,G. R. Fenwick,R. Gmelin,R. Rakow.Thechemicalionization
mass spectra of glucosinolates (mustard oil glycosides) and
desulphoglucosinolates. A useful aid for structural analysis. Biomed.
Mass Spectrom. 1981, 8, 265.
24] J. M. Mercero, J. M. Matxain, X. Lopez, D. M. York, A. Largo, L. A.
Eriksson,J.M.Ugalde.Theoreticalmethodsthathelpunderstanding
the structure and reactivity of gas phase ions. Int. J. Mass Spectrom.
2
005, 240, 37.
[
25] R. Ditchfield, W. J. Hehre, J. A. Pople. Self-consistent molecular-
orbital methods. IX. An extended gaussian-type basis for molecular-
orbital studies of organic molecules. J. Chem. Phys. 1971, 54, 720.
[
10] C. H. Botting, N. E. Davidson, D. W. Griffiths, R. N. Bennett,
N. P. Botting. Analysis of intact glucosinolates by MALDI-TOF mass
spectrometry. J. Agric. Food Chem. 2002, 50, 983.
www.interscience.wiley.com/journal/jms
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 272–283

CID mass spectra of glucosinolate anions
[
26] A. P. Scott, L. Radom. Harmonic vibrational frequencies: an
evaluation of Hartree-Fock, Møller-Plesset, quadratic configuration
interaction, density functional theory, and semiempirical scale
factors. J. Phys. Chem. 1996, 100, 16502.
27] M. P. Andersson,P. Uvdal.Newscalefactorsforharmonicvibrational
frequencies using the B3LYP density functional method with the
triple-ζ basis set 6-311+G(d,p). J. Phys. Chem. A 2005, 109, 2937.
28] A. B. Attygalle, S. García-Rubio, J Ta, J. Meinwald. Collisionally-
induced dissociation mass spectra of organic sulfate anions.
J. Chem. Soc. Perkin Trans. 2 2001, 4, 498.
[34] M. L. Bandu, T. Grubbs, M. Kater, H. Desaire. Collision induced
dissociation of alpha hydroxy acids: evidence of an ion-neutral
complex intermediate. Int. J. Mass Spectrom. 2006, 251, 40.
[35] J. A. Carrol, D. Willard, C. B. Lebrilla. Energetics of cross-ring
cleavages and their relevance to the linkage determination of
oligosaccharides. Anal. Chim. Acta 1995, 307, 431.
[
[
[
[36] G. Bojesen, E. Larsen. Characterization of five glucosinolates by fast
atom bombardment mass spectrometry and collision activation of
−1
[M − H] . Biol. Mass Spectrom. 1991, 20, 286.
[37] S. J. Rochfort, V. C. Trenerry, M. Imsic, J. Panozzo, R. Jones. Class
targeted metabolomics: ESI ion trap screening methods for
glucosinolates based on MS fragmentation. Phytochemistry 2008,
69, 1671.
29] M. Mavratzotis, V. Dourtoglou, C. Lorin, P. Rollin. Glucosinolate
chemistry. First synthesis of glucosinolates bearing an external
thio-function. Tetrahedron Lett. 1996, 37, 5699.
[
30] C. A. Grob, P. W. Schiess. Heterolytic fragmentation. A class of
organic reactions. Angew. Chem. Int. Ed. Engl. 1967, 6, 1.
[38] Y. Go’mez-Gonza’lez, J. D. Surratt, F. Cuyckens, R. Szmigielski,
R. Vermeylen,
M. Jaoui,
M. Lewandowski,
J. H. Offenberg,
[
31] L. Yi, J. Dratter, C. Wang, J. A. Tunge, H. Desaire. Identification of
sulfation sites of metabolites and prediction of the compounds’
biological effects. Anal. Bioanal. Chem. 2006, 386, 666.
32] R. A. J. O’Hair, J. H. Bowie, R. N. Hayes. Formation of the bicarbonate
anion from deprotonated oxalic acid. Rapid Commun. Mass
Spectrom. 1988, 2, 275.
T. E. Kleindienst, E. O. Edney, F. Blockhuys, C. Van Alsenoy,
W. Maenhaut, M. Claeys. Characterization of organosulfates from
the photooxidation of isoprene and unsaturated fatty acids in
ambient aerosol using liquid chromatography/(−) electrospray
ionization mass spectrometry. J. Mass. Spectrom. 2008, 43, 371.
[39] N. Fabre, V. Poinsot, L. Debrauwer, C. Vigor, J. Tulliez, I. Fourast e´ ,
C. Moulis. Characterisation of glucosinolates using electrospray ion
trapandelectrosprayquadrupoletime-of-flightmassspectrometry.
Phytochem. Anal. 2007, 18, 306.
[
[
33] J. B. Bialecki, F. U. Frank Axe, A. B. Attygalle. Hydroxycarbonyl anion
(
m/z 45), a diagnostic marker for α-hydroxy carboxylic acids. J.Mass.
Spectrom. 2009, 44, 252.
J. Mass. Spectrom. 2010, 45, 272–283
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms