Analysis of phenolic choline esters from seeds
Table 7. Relation between key ions and structural features of phenolic choline esters 1–31
Key ion
a
b
c1
c2
d
e
f and b2
g1 and g2 h1 and h2
Hexose
(4-O)
Monolignol
O-Acylated monolignol
(4-O-8ꢁ linkage)
Monolignol
Structural feature
Choline ester
(4-O-8ꢁ linkage)
(5-8ꢁ linkage)
Group I (1–9)
Group II (10–13)
Group III/4-O-8ꢁ (14 -24)
Group III/4-O-8ꢁ (25–27)
Group III/5-8ꢁ (28–31)
were clearly dominating over those derived from sinapyl alcohol
and syringin. Incorporation of vanilloyl- and syringoylcholine
leadingtocompounds22–24islesspronouncedincomparisonto
feruloyl- and sinapoylcholine. Interestingly, compounds 14–19,
21,and22appearaschromatographicallyseparatedisomericpairs
with undistinguishable mass spectral properties in B. napus but
not in A. thaliana seeds (Table 1). Since two chromatographically
separable isomers were also detectable in case of vanilloylcholine
4-O-8ꢁ cross-coupled to coniferyl alcohol (22), it can be assumed
that the relative configuration of the arylglycerol-β-arylether unit
ratherthantheconfigurationofthedoublebondcausesthiseffect.
However, a detailed analysis of this isomerism is beyond the scope
of this work. Compounds 14–24 show a consistent fragmentation
behavior resembling those of group II phenolic choline esters. The
a-type fragment underwent further decomposition by cleavage
of the monolignol moiety resulting in the formation of a d-
type ion (Scheme 4, Figure 2). Consecutive cleavage of C2H4O
yields a b-type ion and derived fragments indicative of the
phenolic acid ([b – CO], [b – MeOH], [b – MeOH – CO], Table 4).
In order to validate the structural assignments, elemental
compositions of 14–24 were confirmed by direct infusion ESI-
FTICRmassspectrometry(Table 1). Compound 14wasexemplarily
synthesized from the 4-O-8ꢁ cross-coupling product of ferulic acid
and coniferyl alcohol.[18]
is quite different compared with that of all as yet described
compounds. The a-type ion displays prominent losses of water
and formaldehyde resulting in g1- and g2-type ions, respectively
(Scheme 6, Table 6, Figure 2). Further fragmentation of these ions
requires higher collision energies and leads to complex product
ion spectra. Predominant fragmentation reactions include loss
of a methyl radical and methanol from g1-type ions, combined
loss of methanol and carbon monoxide from g2-type ions as well
as the formation of ions h1 and h2 being characteristic for the
monolignol moiety. The mass spectral behavior of compound 31,
the4ꢁ-O-hexosideof 28, ischaracterizedbyeliminationofahexose
moiety from ions a, g1, and g2. Interestingly, the characteristic loss
of C2H4O leading to a b-type ion is clearly absent in compounds
of group III possessing a 5-8ꢁ linkage. This phenomenon could
be explained by a ring opening reaction by cleavage of the
O4–C7ꢁ bond after initial elimination of trimethylamine leading
to a charge localization in the aromatic ring system of the
monolignol moiety. Thus, the formation of ions g1 and g2 is
preferred. For validation purposes compound 28 was prepared
from the 5-8ꢁ cross-coupling product of ferulic acid and coniferyl
alcohol. Elemental compositions of compounds 28–31 could be
unequivocally confirmed by direct infusion ESI-FTICR-MS (Table 1).
The set of 4-O-8ꢁ linked phenolic choline esters is supplemented
by three compounds (25–27) that were only observed in B. napus
seeds representing esterification products of compounds 14–16
andsinapicacid.Asobservedfornon-acylatedcompounds14–16,
the CID mass spectra of 25–27 display key ions of type a, d, and
b1 (Scheme 5, Table 5, Figure 2). The esterification by sinapic acid
causes an additional fragmentation reaction of ion a leading to
the formation of ion e, which is complementary to ions of type
d. A further fragment being characteristic for this group is ion f
originating from elimination of sinapic acid from ion e. The loss
of the monolignol moiety from ion e results in formation of a
sinapoyl cation b2 with its typical consecutively formed fragment
ions (Scheme 2). In the case of compound 27, ion b1 formally
corresponds to b2.
Conclusions
Capillary LC/ESI-QTOF-MS facilitates the detection of phenolic
choline esters via their molecular ions down to the mid-nanomolar
range. A characteristic loss of trimethylamine from the molecular
ion which is also inducible by source fragmentation can be
applied for systematic screening. Furthermore, accurate mass
measurements and high resolution CID mass spectra obtained
from molecular ions can be used for detailed structure elucidation.
It is noteworthy that following substance group separation by
SPE even low abundant phenolic choline esters are detectable
by direct infusion ESI-FTICR-MS facilitating the determination
of elemental compositions and allowing a rapid qualitative
monitoring of the phenolic choline ester pattern without detailed
identification of structures. LC/ESI-MS based profiling reveals a
high structural diversity of phenolic choline esters in A. thaliana
and B. napus seeds. About 30 phenolic choline esters could be
structurally characterized and relatively quantified. Their mass
spectral behavior under ESI CID conditions using different collision
energiesoffersthepossibilitytocharacterizeboththetypeandthe
substitution pattern of such compounds. The main correlations of
the key ions with respect to the structural features are summarized
in Table 7. Thus, the proposed analytical approach represents a
Identification and mass spectral behavior of group III phenolic
choline esters with 5-8ꢀ linkage
Besides the cross-coupling products possessing a 4-O-8ꢁ linkage
motif (14–27), compounds 28, 29, and 31 could be identified
as 5-8ꢁ cross-coupling products of feruloylcholine with coniferyl
alcohol, sinapyl alcohol, and coniferin, respectively. Compound
30 represents a 5-8ꢁ cross-coupling product of vanilloylcholine
and coniferyl alcohol. The fragmentation behavior of 28–31
c
J. Mass. Spectrom. 2009, 44, 466–476
Copyright ꢀ 2008 John Wiley & Sons, Ltd.