HPLC-ESI-MS/MS analysis of sulfated flavor compounds in plants

Article abstract of DOI:10.1016/S0031-9422(98)00526-3

Synthesis of six new flavor sulfates (benzyl sulfate, 2-phenylethyl sulfate, 2,5-dimethyl-4-hydroxy-3(2H)-furanone sulfate, α-ionol sulfate, vomifoliol sulfate, linalyl sulfate) was performed in order to screen for these compounds in plants. Structural elucidation was performed by NMR spectroscopy and a screening method developed by using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC- ESI-MS/MS). The results obtained with various plant tissues indicate that sulfation of flavor compounds is a common pathway in plant metabolism.

Full text of DOI:10.1016/S0031-9422(98)00526-3

PERGAMON
Phytochemistry 50 (1999) 219±225
HPLC±ESI±MS/MS analysis of sulfated ¯avor compounds in
plants
Barbara Boss, Elke Richling, Markus Herderich, Peter Schreier *
Lehrstuhl fur Lebensmittelchemie, Universitat Wurzburg, Am Hubland, D-97074 Wurzburg, Germany
È
È
È
È
Revised 1 July 1998
Abstract
Synthesis of six new ¯avor sulfates (benzyl sulfate, 2-phenylethyl sulfate, 2,5-dimethyl-4-hydroxy-3(2H)-furanone sulfate, a-
ionol sulfate, vomifoliol sulfate, linalyl sulfate) was performed in order to screen for these compounds in plants. Structural
elucidation was performed by NMR spectroscopy and a screening method developed by using high performance liquid
chromatography±electrospray ionization tandem mass spectrometry (HPLC±ESI±MS/MS). The results obtained with various
plant tissues indicate that sulfation of ¯avor compounds is a common pathway in plant metabolism. # 1998 Elsevier
Science Ltd. All rights reserved.
Keywords: Sulfate esters; Flavor; Benzyl sulfate; 2-Phenylethyl sulfate; 2,5-Dimethyl-4-hydroxy-3(2H)-furanone sulfate; a-Ionol sulfate; Vomifo-
liol sulfate; Linalyl sulfate; HPLC±ESI±MS/MS; Electrospray ionization (ESI)
1. Introduction
cosides (Spencer & Seigler, 1985) and anthocyanins
(Toki et al., 1994).
In 1937, Kawaguchi and Kim (1937) reported for
the ®rst time a sulfate ester of a ¯avonol, namely iso-
rhamnetin 3-sulfate, as naturally occurring in
Persicaria hydropiper. Up to the seventies, when
Harborne (1975) reviewed this new class of sulfur com-
pounds in plants, little attention has been paid to these
polar ¯avonoid derivatives. Later there has been an
increasing number of reports dealing with these conju-
gates, summarized by Barron, Varin, Ibrahim,
Harborne, and Williams (1988). The data show the
common occurrence of sulfate esters, mostly deriva-
tives of hydroxy¯avones and -¯avonols or their methyl
ethers and, less widely distributed, the corresponding
glycosylated derivatives in a variety of plants. Besides,
other reports describe the occurrence of sulfated de-
rivatives of hydroxycinnamic acids (Imperato, 1982),
coumarins (Lemmich & Shabana, 1984), anthraqui-
nones (Harborne & Mokhtari, 1977), cyanogenic gly-
In contrast to the sulfation mechanism in animal tis-
sues leading to changes of solubility and metabolic ac-
tivity of phenols, steroids, xenobiotics, or ¯avor
compounds (Mulder, 1981), the physiological role of
these plant constituents has not been clari®ed to date.
Nothing is known about the sulfation of ¯avor com-
pounds in plants leading to the question about a poss-
ible co-occurrence of sulfates besides well-known
¯avor precursors like glycosides (Winterhalter
&
Schreier, 1994) or phosphates (Ney, Jager, Herderich,
Schreier, & Schwab, 1996). Previously, for ¯avonoids a
co-occurrence of sulfates and glycosides has been
described (Varin, Barron, & Ibrahim, 1986).
In order to screen for sulfated ¯avor precursors, syn-
thesis of sulfate esters 1±6 of di€erent chemical classes
(benzyl alcohol, 2-phenylethanol, 2,5-dimethyl-4-
hydroxy-3(2H)-furanone, a-ionol, vomifoliol, and lina-
lol) was performed and a screening method developed
by HPLC±ESI±MS/MS. The present paper describes
the results obtained by screening a widespread vari-
ation of plant tissues for these compounds.
* Corresponding author. E-mail: schreier@pzlc.uni-wuerzburg.de.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00526-3

220
B. Boss et al. / Phytochemistry 50 (1999) 219±225
2. Results and discussion
experiments of these precursor ions produced charac-
teristic product ion spectra. Sulfate ester 3 was the
only compound under study which showed an abun-
dant fragment m/z 127 corresponding to the aglycone
moiety [aglycone H] . This observation is in good
agreement with MS/MS studies using 2,5-dimethyl-4-
hydroxy-3(2H)-furanone derivatives in positive mode,
where the aglycone fragment was the main fragment in
the product ion spectrum (Roscher, Herderich, Ste€en,
Schrier, & Schwab, 1996). The other compounds pro-
duced base peaks with m/z 97 (for 2, 4, and 5) corre-
sponding to [HSO4] or m/z 96 (for 1 and 6). In
accordance to data recorded for steroid sulfates
(Weidolf, Lee, & Henion, 1988) the fragment m/z 96
corresponds to a lack of the aglycone moiety leading
possibly to [SO4] Á. By increasing the collision induced
dissociation (CID) o€set voltage the fragmentation
pattern could be shifted to the fragment m/z 80 corre-
sponding to [SO3] (cf. Fig. 1). The detection of m/z
80 was less sensitive in comparison to m/z 96/97/127,
as it was not possible to shift the fragmentation
pattern completely to m/z 80. To distinguish between
phosphates and sulfates, it is imperative to have a
For the screening of sulfated ¯avor compounds, syn-
thesis of a representative selection of such conjugates
was performed by standard methods (Lieberman,
Hariton, & Fukushima, 1948; Piasecki, 1992). The pur-
i®ed compounds were obtained in overall yields ran-
1
ging from 20% to 90% and characterized by H and
13C NMR spectroscopy (cf. Tables 1 and 2 and
Section 3). Distinct down®eld shifts (1±4 ppm) of the
a-carbons and slightly up®eld shifts of the b-carbons
observed in comparison with their corresponding alco-
hols were consistent with data reported for coumarin
sulfates (Lemmich & Shabana, 1984) as well as carbo-
hydrate and steroid sulfates (Lillard & Seib, 1978;
Goto, Kato, Hasegawa, & Nambara, 1979). In ad-
1
dition, the H NMR data con®rmed the observations
in the 13C NMR spectra by slightly down®eld shifts of
the hydrogens at the a-carbons.
Using HPLC±ESI±MS analysis in negative mode,
an abundant pseudomolecular ion [M] corresponding
to the molecular mass of the anion was observed for
each sulfated ¯avor compound under study. MS/MS

B. Boss et al. / Phytochemistry 50 (1999) 219±225
221
Table 1
1H NMR data of 4 and the corresponding alcohol (400 MHz; d ppm
relative to solvent signal, coupling constants in Hz)
Table 2
13C NMR data of 4 and the corresponding alcohol (100 MHz, d
ppm relative to solvent signal)
4 (DMSO-d6)
Alcohola (CDCl3)
4 (DMSO-d6)
Alcohola (CDCl3)
H-2a/3a
H-2b
H-3b
H-4
1.11±1.14
1.31±1.45
1.98
2H, m
1H, m
1.13±1.19
1.38±1.45
1.98
2H, m
1H, m
C-1
C-2
32.3
31.3/31.4
23.7
32.0
31.6
1H, br s
mb
1H, br s
mb
C-3
C-4
23.6
5.28±5.49
1.89
5.39±5.53
2.07
119.6/119.7
136.4
48.9
121.0
134.0
54.0
H-6
H-7/8
H-9
1H, d, 8.4
mb
1H, m
1H, d, 9.2
mb
C-5
C-6
5.28±5.49
4.25±4.30
1.19
5.39±5.53
4.22
1.26
1H, quin, 6.2
3H, d, 6.4
3H, s
C-7
C-8
132.0
132.5
72.0/72.7
20.0
131.2
136.0
68.8/68.9
23.1
H-10
H-11c
H-12c
H-13
3H, d, 6.2
3H, 2Â s
3H, 2Â s
3H, 2Â s
1.01/1.03
0.90/0.96
1.71
0.88
0.80/0.82
1.57
C-9
3H, 2Â s
3H, 2Â d, 1.5
C-10
C-11b
C-12b
C-13
28.8
27.6
27.4/27.5
27.0
22.8
aSee Pabst, Barron, Semon, & Schreier (1992). bSignals overlapped
c
(3H). Interchangeable values.
22.6
aSee Pabst et al. (1992). bAssignments may be reversed.
second detectable product ion speci®c for sulfates,
because both conjugates exhibit as abundant fragment
in the ESI negative mode m/z 97 corresponding to
[H2PO4] and [HSO4] , respectively (Ney et al., 1996;
Feurle, Jomaa, Wilhelm, Gutsche, & Herderich, 1998).
In contrast, the second fragment for sulfates m/z 80
corresponding to [SO3] is absent in the spectra of
phosphates exhibiting m/z 79 corresponding to [PO3]
(Ney et al., 1996; Feurle et al., 1998). With these data
in hand an unambiguous assignment of the sulfates is
possible.
With a test-mixture of 1±6, a screening method for
these new conjugates in plants was developed. To sep-
arate the reference compounds from each other, RP-18
HPLC separation using a gradient with ammonium
acetate in water and methanol, respectively, was cho-
sen. In order to increase the sensitivity of the analysis
and to avoid co-elutions of compounds 2 and 5, separ-
ate runs for compounds 1±3 (group 1) and 4±6 (group
2) were performed. For each group two di€erent time-
dependent selected reaction monitoring (SRM) exper-
iments, corresponding to the di€erent fragmentation
pattern at various CID o€set voltages, were selected to
screen plant extracts: (i) SRM 1 and 3 yielding the
product ions m/z 96/97/127, respectively; and (ii) SRM
2 and 4 yielding the product ion m/z 80 for all com-
pounds under study. SRM increased the sensitivity
and was highly selective because of excluding matrix
e€ects by ®ltration of the precursor ion [M] and its
speci®c product ion.
In leaves, the aromatic compounds were detectable in
most of the samples, while the norisoprenoid deriva-
tives were less widely distributed. The terpene deriva-
tive could not be detected in any of the samples,
probably due to the observed instability of the com-
pound under extraction and storage conditions. The
furanone conjugate was also not detectable in any of
the samples. This ®nding is in accordance to literature
data; the alcohol and its derivatives occur only in
fruits but not in leaves (Schwab & Roscher, 1998).
In fruits (blackcurrant, guava and passionfruit),
detection of the compounds could not be achieved,
probably due to too low concentrations of the conju-
gates. This observation is in good accordance with
Harborne's previously published data, showing the
occurrence of ¯avonoid sulfates mainly in leaves
(Harborne, 1975). For this reason, further attempts
with fruits were not performed.
In summary, we identi®ed for the ®rst time sulfated
¯avor conjugates in plant tissues. HPLC±ESI±MS/MS
o€ers the possibility to detect selectively these polar
minor compounds. In order to distinguish between
phosphates and sulfates exhibiting the same precursor
and product ions, an additional SRM experiment
speci®c for sulfates was developed to con®rm their
occurrence. These new ¯avor conjugates seem to be
common constituents of secondary plant metabolism
being overlooked in the last years.
With this method in hand a wide variety of plant tis-
sues was screened for compounds 1±6. Extracts from
fresh and dried leaves and fruits were obtained by
XAD-2 solid phase extraction. In order to further pur-
ify the extracts a second XAD-2 column was eluted
with 30% methanol sucient for elution of these polar
conjugates. A representative example from our study is
shown in Fig. 2. The results are summarized in Table 3.
3. Experimental
3.1. Plant material
Guava, blackcurrant, passionfruit, cress and dried
leaves were available from a local market. Vine leaves
cv. muscat and shiraz originated from INRA,

222
B. Boss et al. / Phytochemistry 50 (1999) 219±225
Fig. 1. Product ion mass spectra of 2 obtained by collision induced dissociation (CID) with di€erent o€set voltages (presursor ion: m/z 201.1
[M] ): (A) o€set voltage 21 eV; (B) o€set voltage 57 eV.
Montpellier. All other fresh leaves were collected in
summer 1997 on the campus of the University of
Wurzburg.
obtained by centrifugation (3000g, 20 min). To extract
leaves the method was modi®ed as follows: after
mixing of 200 g of leaves with 1000 ml MeOH and
macerizing the mixture at ambient temp. overnight, a
clear extract was obtained by ®ltration. MeOH was
removed under reduced pressure. The aq. residue was
extracted three times with 100 ml of pentane to remove
chlorophyll. The clear extract was then applied to an
Amberlite XAD-2-column (4 Â 35 cm). After a rinse
with 2000 ml of distilled H2O and 500 ml of pentane±
Et2O (1 +1), the extract was obtained by eluting with
3.2. Plant extracts
The ®rst step corresponded to the method described
for the isolation of a glycosidic extract (Gunata,
Bayonove, Baumes, & Cordonnier, 1985). Thus, after
mixing of 1000 g of fruits with 1000 ml of 0.2 M
citrate±phosphate bu€er (pH 7.0), a clear extract was

B. Boss et al. / Phytochemistry 50 (1999) 219±225
223
Fig. 2. Time-dependent SRM experiments for compounds 1±6 in strawberry leaf extract. Chromatogram A shows the product ion traces of
group 1: 1±3 (SRM 1). The characteristic ion pairs are as follows: m/z 207.0/127.1 (3), 187.1/96.1 (1), 201.1/97.2 (2). Compound 3 (RRt 2.5 min)
was not detectable in this sample. Chromatogram B shows the product ion traces of group 2: 4±6 (SRM 3). The characteristic ion pairs are as
follows: m/z 303.3/97.2 (4), 273.3/97.2 (5), 233.2/96.2 (6). Compound 6 (RRt 10.2 min) was not detectable in this sample (ESI, negative mode,
RP-18; gradient: 10 mM NH4Ac in H2O±10 mM NH4Ac in 90% MeOH).
1000 ml of MeOH. The MeOH eluate was conc. under
reduced pressure to dryness (yields ranging from 0.5 g
to 2.0 g).
mmol, 350 mg) in 30 ml Et2O (Lieberman et al., 1948).
After stirring of the mixture for one hour at room
temp., Et2O was removed by distillation under reduced
pressure. Neutralization with 2 N NaOH yielded a
crystalline precipitate which was dried and used with-
out further puri®cation for structural elucidation by
NMR and HPLC±ESI±MS/MS. The sodium salts 1
and 2 were obtained in overall yields ranging from
83% to 90%, respectively (1: 2.5 mmol, 520 mg; 2: 2.7
mmol, 605 mg). In case of 3 the procedure was modi-
®ed as follows: before treatment of the alcohol (3
mmol, 385 mg) with chlorosulfonic acid (3 mmol, 350
mg) diethylamine (400 ml) had to be added to ensure
milder conditions. After one hour at room temp. the
solution was neutralized as described before. The resi-
due was dissolved in 5 ml H2O with 0.05% HCO2H
and puri®ed on Lichrospher 100-C18 material (3 Â 10
cm). Elution was performed with increasing amounts
of CH3CN in H2O with 0.05% HCO2H. Pure 3 was
A part of the extract (corresponding to 25 g of dried
leaves, 50 g of fresh leaves or 250 g of fruits) was
redissolved in 10 ml 0.2 M citrate±phosphate bu€er
(pH 3.5) and applied to a second Amberlite XAD-2-
column (1.5 Â15 cm). After a rinse with 50 ml H2O,
elution was performed with 50 ml 30% MeOH in H2O
to yield the desired fraction. The solution was concen-
trated under reduced pressure to dryness and redis-
solved in 1 ml distilled H2O for the analysis of sulfated
conjugates by HPLC±ESI±MS/MS.
3.3. Reference compounds
The synthesis of 1 and 2 was performed by treat-
ment of the commercially available alcohols (3 mmol,
1: 320 mg; 2: 370 mg) with chlorosulfonic acid (3

224
B. Boss et al. / Phytochemistry 50 (1999) 219±225
Table 3
Screening results for compounds 1±6 in various plant tissues
MHz, DMSO-d6): d 19.0 (C-13), 22.3 (C-10), 23.1 (C-
11 ), 24.1 (C-12 ), 40.3 (C-1), 49.5 (C-2), 71.5/72.3 (C-
9), 77.8 (C-6), 125.7 (C-4), 131.5 (C-7), 133.1 (C-8),
163.8 (C-5), 197.0 (C-3) ( signals interchangeable).
6: 1H NMR (400 MHz, DMSO-d6): d 1.55 (3H, s,
H-8 ), 1.64 (3H, s, H-9 ), 1.93 (3H, s, H-10), 2.26 (4H,
m, H-4, H-5), 5.04 (1H, t, J= 7.0 Hz, H-6), 5.60 (1H,
d, J= 17.6 Hz, H-1a), 5.68 (1H, d, J =10.6 Hz, H-
1b), 6.35 (1H, dd, J =10.7 Hz, 17.5 Hz, H-2) ( signals
interchangeable); 13C NMR (100 MHz, DMSO-d6):
d 17.5 (C-9), 22.2 (C-5), 23.9 (C-10), 25.4 (C-8), 38.7
(C-4), 74.7 (C-3), 119.5 (C-1), 122.4 (C-6), 132.2 (C-7),
138.9 (C-2).
Leaves
1
2
3
4
5
6
Blackberrya
Blackcurrant
Blueberrya
Cress
[
[
[
[
[
[
[
[
[
[
Elder
Plum
Raspberrya
Redcurrant
Rosehip
Strawberrya
Sweet cherry
Vine cv. muscat
Vine cv. shiraz
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
3.5. HPLC±ESI±MS/MS analysis
aDried leaves.
Analysis of sulfated conjugates 1±6 was performed
on a triple stage quadrupole TSQ 7000 LC±MS/MS
system (Finnigan MAT, Bremen). Data acquisition
and data evaluation were carried out on a Personal
obtained with an overall yield of 50% (1.5 mmol, 340
mg). Synthesis of 4±6 was performed by the method of
Piasecki (1992). To a well stirred and cooled solution
of 10 mmol of the appropriate alcohol (4: 1.94 g; 5:
2.24 g; 6: 1.54 g) in 15 ml dry CCl4 and 500 ml dry
pyridine 11 mmol (1.75 g) of sulfur trioxideÁpyridine
complex was added slowly in small portions. After 2±3
h of stirring in an ice bath the mixture was left at
room temp. to the next day. Then, the solution was
evaporated under reduced pressure and a suspension
of 12 mmol Na2CO3 (Na2CO3 Â 12 H2O: 3.43 g) in 20
ml EtOH and 7.5 ml H2O was added slowly under vig-
orous stirring in an ice bath. After four hours the reac-
tion mixture was evaporated, the residue redissolved in
5 ml H2O with 0.05% HCO2H and puri®ed on
Lichrospher 100-C18 material as described before.
Yields ranged from 20% to 30% (4: 660 mg; 5: 945
mg; 6: 560 mg).
DEC
station
5000/33
(Digital
Equipment,
Unterfohring) and ICIS 8.1 software (Finnigan MAT,
Bremen). For HPLC an Applied Biosystems dual syr-
inge pump model 140B (bai, Bensheim) was used.
HPLC separation was carried out on an Eurospher
100-C18 (2 Â 100 mm, 5 mm, Knauer, Berlin) using a
linear gradient at a ¯ow rate of 200 ml/min. The
HPLC gradient was as follows: solvent A (10 mM
NH4Ac in H2O), solvent B (10 mM NH4Ac in 90%
MeOH); 0±10 min 10±90% B, 10±11 min 90±100% B,
11±15 min 100% B. For injection a Spark Holland
Triathlon autosampler (SunChrom, Friedrichsdorf)
was used, the injection volume was 10 ml using the ml
pick-up mode. Electrospray ionization (ESI) in nega-
tive mode was used. The temp. of the heated capillary
was set to 2308C and the capillary voltage to 3.4 kV.
Nitrogen served both as sheath (60 psi) and auxiliary
gas (10 l/min).
3.4. NMR data
The product ion spectra were available by collision
induced dissociation (CID) (2.5 mTorr argon; 20±60
eV). From the characteristic fragmentation pattern the
most abundant product ion was selected for selected
reaction monitoring (SRM) experiments.
1
1: H NMR cf. White, Li, and Lu (1992); 13C NMR
(100 MHz, DMSO-d6): d 67.6 (C-1), 127.7 (C-3, C-7),
128.3 (C-5), 128.8 (C-4, C-6), 138.1 (C-2).
1
2: H NMR cf. White et al. (1992); 13C NMR (100
MHz, DMSO-d6): d 35.5 (C-2), 66.4 (C-1), 126.1 (C-6),
128.3 (C-5, C-7), 128.9 (C-4, C-8), 139.0 (C-3).
1
3: H NMR (400 MHz, DMSO-d6): d 1.36 (3H, d,
3.6. HPLC±ESI±MS/MS data
J= 7.0 Hz, H-6), 2.28 (3H, s, H-1), 4.66 (1H, q,
J= 7.0 Hz, H-5); 13C NMR (100 MHz, DMSO-d6): d
14.4 (C-1), 16.4 (C-6), 80.0 (C-5), 130.6 (C-3), 181.0
(C-2), 196.8 (C-4).
1: product ions of m/z 187 [M] : m/z 96
[M C7H7] , 80 [SO3] ; 2: cf. Fig. 1; 3: product ions
of m/z 207 [M] : m/z 127 [aglycone H] , 80 [SO3] ;
4: product ions of m/z 273 [M] : m/z 97 [HSO4] , 80
[SO3] ; 5: product ions of m/z 303 [M] : m/z 97
[HSO4] , 80 [SO3] ; 6: product ions of m/z 233 [M] :
m/z 96 [M C10H17] , 80 [SO3] . Depending on the
o€set voltage of the CID the intensity of the product
ions is shifted to smaller fragments.
4: cf. Tables 1 and 2.
5: 1H NMR (400 MHz, DMSO-d6): d 0.98 (3H, s,
H-11 ), 1.00 (3H, s, H-12 ), 1.19 (3H, m, H-10), 1.87
(3H, m, H-13), 2.17±2.32 (2H, m, H-2), 4.52 (1H, m,
H-9), 5.67 (1H, m, H-4), 5.77 (1H, m, H-7), 5.86 (1H,
m, H-8) ( signals interchangeable); 13C NMR (100

B. Boss et al. / Phytochemistry 50 (1999) 219±225
225
For improved sensitivity the six compounds were
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and two di€erent MS/MS experiments were performed
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1±3: (i) SRM 1, time-dependent: 0±4 min m/z 207.0/
127.1 (20 eV) for 3; 4±6 min m/z 187.1/96.1 (30 eV)
for 1; 6±15 min m/z 201.1/97.2 (21 eV) for 2; scan-time
1 s; (ii) SRM 2, time-dependent: 0±4 min m/z 207.0/
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S. Sauer for performing a part of the HPLC±ESI±MS/
MS analyses. We also thank Y. Gunata (INRA,
Montpellier, France) for providing vine leaves and the
European Community for ®nancial support (AIR3-
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