910 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Withopf et al.
Ta ble 4. Scr een in g Resu lts for Com p ou n d s 2a -e in
In summary, we have shown that HPLC-ESI-MS/
MS offers a highly selective method for the screening
for 6′-O-malonylated glucoconjugates in plants. These
first screening results indicate that 6′-O-malonyl â-D-
glucopyranosides occur nearly ubiquitous and are com-
mon in the composition of bound flavor precursors in
foodstuffs. Due to the already known instability ob-
served under traditional sample preparation conditions
(Koester et al., 1984; Horowitz and Asen, 1989), these
compounds can easily be overlooked as flavor precur-
sors.
Va r iou s P la n t Tissu es
2a
2b
2c
2d
2e
raspberry
strawberry
mountain papaya peel
guava
green tea
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
vine leaf (cv. Muscat)
(
MS/MS experiments of these “parent ions” produced
characteristic product ion spectra.
For all compounds under study nearly the same
fragmentation pattern was detectable (Table 3). While
the aglycon of 2e was easily ionized (Roscher et al.,
1996a) presumably due to the heteroatoms in the
moiety, the aglycons of 2a -d could hardly be ionized.
Consequently, ions of the aglycon moiety were less
intensive, except for 2e, whose LC-MS/MS spectrum
was dominated by the aglycon fragment.
ACKNOWLEDGMENT
We thank M. Schmitt and B. Pink for recording the
NMR spectra and D. Krajewski for preparing a part of
the glycosidic extracts. We also thank C. Duque (Uni-
versidad Nacional de Colombia, Bogota´, Colombia), Y.
Gunata (INRA, Montpellier, France), and K. S. Kim
(Chosun University, Kwanju, South Korea) for kindly
providing mountain papaya fruits, vine leaves, and
green tea, respectively.
With a test-mixture of 2a -e a screening method for
these compounds in plants was developed. The chosen
HPLC gradient with ammonium acetate in water and
methanol, respectively, was suitable to separate the
synthesized reference compounds 2a -e from each other
[relative retention times: (2a ) 5.0 min; (2b) 6.0 min;
(2c) 8.4 min; (2d ) 9.1 min; (2e) 3.2 min]. Three different
MS/MS experiments were performed to identify malo-
nylated glucoconjugates in plants: (i) detection of the
m/ z generating the specific product ion m/ z 249 by CID;
m/ z 249 was the most abundant product ion of 2a -d
and was assigned to be [M + H - aglycon]+; (ii)
detection of the m/ z forming the specific product ion
m/ z 129 by CID; due to the different fragmentation
pattern of 2e, m/ z 249 was not suitable for its detection,
so m/ z 129 [aglycon + H]+ was used for the screening
as most intensive fragment; and (iii) time-dependent
SRM. SRM increased the sensitivity and was highly
selective because of excluding matrix effects by filtration
of the “parent ion” ([M + NH4]+ and [M + H]+,
respectively) and its specific product ion (m/ z 249 and
129, respectively).
With this method, a widespread variety of plant
tissues was screened for compounds 2a -e. Glycosidic
extracts from fruits (guava; raspberry; strawberry),
leaves (green tea; vine), and mountain papaya (C.
pubescens) peel were obtained by XAD-2 solid phase
extraction and further purified by anion exchange
chromatography to separate charged glycoconjugates.
A representative example taken from our study on
strawberry is outlined in Figure 1. The results of the
screening are summarized in Table 4. While the
aromatic 6′-O-malonyl â-D-glucopyranosides were found
in all plants under study, the terpene derivatives were
less widely distributed; 2d was not present in any of
the samples. In addition to the already known occur-
rence of 2e in several fruits, i.e., strawberry, mango,
pineapple, and tomato (Roscher et al., 1996a), it was
also detectable in raspberry.
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These results were confirmed by enzymic hydrolysis
of the extracts with Rohapect D5L and subsequent
analysis of the liberated aglycons by HRGC-MS. It has
to be stressed, however, that for 2a -e about 10-fold
higher enzyme concentration was required than usually
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