Journal of Chromatography A, 1207 (2008) 101–109
Contents lists available at ScienceDirect
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Analysis of flavonol glycoside isomers from leaves of Maytenus ilicifolia
by offline and online high performance liquid
chromatography–electrospray mass spectrometry
Lauro M. de Souza, Thales R. Cipriani, Rodrigo V. Serrato, Denise E. da Costa,
Marcello Iacomini, Philip A.J. Gorin, Guilherme L. Sassaki ∗
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19046, CEP 81531-980, Curitiba-PR, Brazil
a r t i c l e
i n f o
Article history:
Received 16 April 2008
Received in revised form 30 July 2008
Accepted 11 August 2008
Available online 14 August 2008
Keywords:
Liquid chromatography–mass spectrometry
Maytenus ilicifolia
Flavonol glycosides isomers
Kaempferol
Quercetin
Radical aglycone
a b s t r a c t
Flavonol glycosides present in leaves of Maytenus ilicifolia, were examined after fractionation on silicagel column. Flavonol mono-, di-, tri-, and tetraglycosides, containing kaempferol, quercetin or myricetin
were identified by offline electrospray mass spectrometry. Increasing the cone energy induced to adducts
variation, from H+ to Na+ . Protonated ions were characteristically fragmented by sequentially removing
the monosaccharide residues, whereas in the sodiated ions, the aglycone was firstly removed. Online
high performance liquid chromatography–mass spectrometry, with simple gradients of water, acetonitrile and acetic acid indicated the presence of several isomers, which were further identified by gas
chromatography–mass spectrometry as containing galactose or glucose.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Maytenus ilicifolia Mart. ex Reissek (Celastraceae) is an important folk medicinal plant, largely found in Paraguay, Uruguay,
Argentina and Brazil. From the infusion of its leaves is made
a tea, which has several benefits to human health, from gastric protection to anti-ulcerogenic and analgesic activities [1–3].
Moreover, extracts of M. ilicifolia containing phenolic compounds, particularly flavonoids, induced to vascular relaxation
in rat aorta [4,5]. These compounds also have anti-oxidants,
anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer
properties [6–13].
Flavonoids include a class of compounds widely found in higher
plants, their chemical architecture is based in a three-ring structure
(A, B and C). Flavonoids may exist as free aglycones, and as O- or Cglycosides, however, other substitutions or chemical groups such
as acetyl or sulfate groups, may be attached to the rings [14–16].
According to their structural variability, it has been suggested that
the carbohydrate moieties are important for their biological activities [17,18]. Previous investigations showed the presence of several
∗ Corresponding author. Tel.: +55 41 3361 1577; fax: +55 41 3266 2042.
E-mail address: sassaki@ufpr.br (G.L. Sassaki).
0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.08.032
types of flavonol 3-O-glycosides in leaves of Maytenus spp, such
as rutin, quercitrin, hyperoside, and isoquercitrin, and other structures were characterized as kaempferol and quercetin di-, tri, and
tetraglycosides [19–22].
Mass spectrometry (MS) has been successfully applied on
the elucidation of structures of several flavonoid glycosides
[6–8,23–30]. More accurate results are usually obtained in
combination with chromatographic techniques, overcoming the
difficulties in identifying isomers in complex matrixes. Some techniques, such as metal complexation-mass spectrometry, have been
applied to distinguish some standard of isomeric glycosides [31].
However, this offline technique is not suitable to be applied on
complex mixtures, since nonspecific complexation may lead to misinterpretation.
The glycosylation site of flavonols from M. ilicifolia appears
exclusively at the 3-OH position. However, carbohydrate composition and interglycosidic linkages have been shown to be
of great variability. Thus, we now describe the structures of
flavonol glycosides, including several isomers, found in extracts
of M. ilicifolia leaves, using high performance liquid chromatography (HPLC), electrospray mass ionization (ESI-MS) and gas
chromatography–mass spectrometry (GC–MS). The different fragments obtained for protonated and sodiated precursor ions were
the basis for monosaccharide sequencing and for structural eluci-
102
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
dation of these flavonol glycosides, which included some previously
undescribed structures.
2. Experimental
2.1. Plant material
Leaves of M. ilicifolia were collected in Curitiba (Southern Brazil),
and donated by the “Central de Produção e Comercialização de Plantas
Medicinais, Aromáticas e Condimentares do Paraná Ltda”. The plant
was identified by Prof. Olavo Guimarães, Botany Department, Federal University of Paraná (UFPR), Curitiba, Brazil and it is deposited
in the Herbarium of UFPR as voucher no. 30842.
2.2. Extraction and chromatographic fractionation of components
from M. ilicifolia
Defatted leaves (100 g) of M. ilicifolia were extracted thrice
with water (500 ml) under reflux (100 ◦ C), for 3 h. The extracts
were combined, concentrated to lower volume (200 ml), and then
added to cold ethanol (3 vol.) in order to precipitate macromolecules, which were removed by centrifugation (10,000 rpm).
The ethanol-soluble compounds (ESC) were concentrated and then
freeze-dried.
A fraction of ESC (500 mg) was applied to a column
(50 cm × 5 cm) packed with silica-gel 60 G (Merck), and eluted
using a series of mixtures of CHCl3 –MeOH (ranging from 95:5,
v/v to 100% of MeOH). Each collected fraction (10 ml) was evaporated under nitrogen stream at room temperature (∼25 ◦ C) and
analyzed on thin layer chromatography (TLC) with silica-gel 60 G
(Merck), using n-PrOH–H2 O (7:3, v/v) as solvent. The plates were
general stained by 10% H2 SO4 in ethanol (v/v) at 100 ◦ C, or
with orcinol–H2 SO4 at 100 ◦ C, in order to identify carbohydratecontaining compounds [32]. Similar fractions were combined to
give rise to 10 further fractions, namely F1–F10.
2.3. HPLC conditions
HPLC analysis was performed in a Shimadzu apparatus,
equipped with an SPA-10 UV/Vis detector, the samples were
detected at 360 nm, or by online HPLC–ESI-MS. The oven temperature was held at 60 ◦ C, and the separation was carried out at a flow
rate of 1 ml min−1 on a Supelco LC-18 column, 250 mm × 4.6 mm
(4 m, particle size) as analytical and semi-preparative modes. Two
mobile phases using HPLC grade solvents (Tedia-USA) prepared in
deionized water, were: solvent A 1% of acetic acid in water (v/v), and
solvent B H2 O:CH3 CN:HOAc (49:50:1, v/v). The gradient I (used on
F3) was: 10–30% B in 5 min, 30–50% B in 25 min held for 5 min, back
to 10% B in 35 min held for more 10 min. The gradient II (used on
F4 and F5) was: 10–40% B in 15 min, held for 2 min, back to 10% in
20 min held for more 10 min.
The samples were dissolved in MeOH:H2 O (1:1, v/v), the concentration of the analytical run being 1 mg/ml, with 10 l injected.
For purification purposes, semi-preparative runs were carried out
using concentration of 10 mg/ml, with 50 l of injection volume.
Non-resolved peaks were collected together for further purifying
by preparative TLC, using n-PrOH–H2 O (7:3, v/v) as solvent, and
being detected at UV 366 nm.
2.4. Mass spectrometry
Mass spectrometry (MS) analysis was acquired in an ESI-MS
source, with a triple quadrupole, Quattro LC (Micromass-Waters), at
atmospheric pressure ionization (API). For detecting the negative
ions, the energy setup was: cone 25 V (low), 50 V (medium) and
100 V (high) and capillary 2.73 kV, and for positive ions was: cone
35 V (low), 63 V (medium), 85 V (high), and the capillary at 2.6 kV.
Nitrogen was used as nebuliser and desolvation gas, the source
block temperature was held at 80 ◦ C and desolvation gas at 200 ◦ C.
For tandem-MS experiments, each precursor ion was selected by
MS1 and fragmented by collision-induced dissociation (CID) with
argon as collision gas. The CID-energies were 20 eV (low), 40 eV
(medium) and 60–80 eV (high).
The samples (200 g/ml) were solubilized in MeOH–H2 O (1:1,
v/v), or supplemented with 5 mM of HCl or NaCl. Offline analysis was performed by direct injection into the ESI-MS source with
a syringe-infusion pump (KDScientific) at a constant flow rate of
10 l min−1 . Online analysis was carried out by splitting the HPLC
flux at the column exit, into ESI-MS. Data acquisition and processing
were performed using MassLynx 3.5 software, and all scans were
obtained in the continuum mode.
The glycosides fragments were analyzed based on the nomenclature proposed by Domon and Costello [33], adapted to denote the
observed product ions (Fig. 1). Considering that the fragmentation
occurred mainly in the glycosidic linkages, those ions containing
the aglycone moiety were designated as Yj , where j is the number
of glycosidic residues remaining linked to aglycone. Y0 meaning
that no sugar remained linked to the aglycone and Y* meaning the
rearrangement of monosaccharide linked to the aglycone. Product
ions containing only the carbohydrate moiety were designated as
Bi , where i indicates the number of monosaccharide units, counting from the non-reducing end. Bx indicates the product ion from a
monosaccharide residue linked to the aglycone. The charge of each
ion is shown as superscripts (Fig. 1).
Fig. 1. Fragmentation nomenclature for flavonoid glycosides.
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
103
Table 1
Flavonol glycosides detected in offline negative ESI-MS/MS
MS1
MS2
[M−H]
[Y0 −H]
417
433
447
463
579
593
593
609
625
625
739
755
771
771
871
887
901
284:285
300:301
284:285
300:301
300:301
284:285
300:301
300:301
300:301
316:317
284:285
300:301
300:301
316:317
284:285
300:301
284:285
−
•−
:Y0
−
NL
Main fraction
132a
132a
162a
162a
278b
308b
292b
308b
324b
308b
454c
454c
470c
454c
586d
586d
616d
F2
F2, F3
F2, F3
F2, F3
F2, F3
F2, F3
F2, F3
F2, F3
F3, F4
F3, F4
F3, F4
F4, F5
F4, F5
F4, F5
F4, F5
F5
F5
R
–
3.9
4.1
4.5
4.3
3.9
3.3
4.2
3.8
–
3.8
3.6
3.5
3.3
4.1
4.3
4.2
255
271
255
271
271
255
271
271
271
–
255
271
271
–
255
271
255
227
243
227
243
243
227
243
243
243
–
227
243
243
–
227
243
227
163
163
163
163
163
163
163
163
163
163
163
163
163
163
163
163
163
179
179
179
179
179
179
179
179
179
179
179
179
179
179
179
179
179
151
151
151
151
151
151
151
151
151
151
151
151
151
151
151
151
151
135
135
135
135
135
135
135
135
135
135
135
135
135
135
135
135
135
133
149
133
149
149
133
149
149
149
165
133
149
149
165
133
149
133
121
137
121
137
137
121
137
137
137
153
121
137
137
153
121
137
121
93
108
93
108
108
93
108
108
108
125
93
108
108
125
93
108
93
R: ratio between [Y0 −H]• − and Y0 − obtained in 40 eV CID-MS; NL: neutral losses—consistent with: a monosaccharide; b disaccharide; c trisaccharide; and d tetrasaccharide.
2.5. Derivatization and GC–MS analysis
The samples (∼500 g) were hydrolyzed in 1 M TFA (200 l) at
100 ◦ C for 14 h, and then dried under a nitrogen stream. The resulting monosaccharides were reduced to alditols with NaBH4 (Sigma),
and acetylated in 200 l of pyridine and acetic anhydride (1:1, v/v)
(Merck), at 100 ◦ C for 2 h. Ice was then added and the alditol acetates
formed were extracted with CHCl3 (Merck), and analyzed by GC–MS
on a capillary column DB-225MS (J&W-USA). The injector temperature was 250 ◦ C, with the column being initiated at 50 ◦ C, heated
at 40 ◦ C min−1 , and held at 220 ◦ C for 25 min.
For methylation analysis, the samples were dissolved in 200 l
DMSO (Merck), powdered NaOH (5 mg) and 200 l CH3 I (Merck)
[34]. The suspension was vigorously stirred for 30 min and left
overnight at room temperature. The O-methylated derivatives were
extracted with CHCl3 . The extract was washed with water several
times, until neutral pH, and then it was dried under nitrogen stream.
The resulting per-O-methylated compounds were hydrolyzed in
45% formic acid (v/v) at 100 ◦ C for 14 h. The samples were reduced
using NaBD4 (Sigma) and acetylated in 200 l of pyridine and
acetic anhydride (1:1, v/v). The resulting partially methylated
alditol acetates (PMAAs) were extracted in CHCl3 and analyzed
by GC–MS (Varian-USA, model Saturn 2000R), using a capillary
column DB-225MS. The injector temperature was 250 ◦ C, with
the column being initiated at 50 ◦ C, heated at 40 ◦ C min−1 , and
held at 210 ◦ C for 30 min. The EI-MS spectra were compared with
those obtained from the library for O-glycosidic linkage analysis
[35].
arising from isotopes of Cl− adducts, were observed. By increasing
the cone voltage to 50 V, both ions were formed, at m/z 755 [M−H]−
and at m/z 791 [M+Cl]− , with similar intensities, and with the cone
energy at 100 V the main ion observed was the deprotonated form,
at m/z 755.
Each fraction was analyzed by CID-MS, and the resulting fragments showed the formation of the deprotonated regular aglycone
ion [Y0 ]− , as well as the formation of the deprotonated radical aglycone ion ([Y0 −H]•− ) which is obtained by a hemolytic cleavage of
the glycosidic bond, between the aglycone and the monosaccharide
[26,36]. The main fragment ions observed for deprotonated radical
3. Results and discussion
3.1. Offline negative ESI-MS analysis
Fractions were collected from the silica-gel column and those
reactive to orcinol–H2 SO4 (F2–F5) were submitted to negative ESI
detection, which provided mixtures of flavonol glycosides, according to the pseudo-molecular ions observed (Fig. 2A–D, Table 1).
Fraction F5, which contained mainly compound with m/z 755
[M−H]− , was solubilized in MeOH–H2 O (1:1, v/v), and MeOH–H2 O
with 5 mM HCl or 5 mM NaCl, and analyzed in the negative ESI mode
using a capillary energy at 2.73 kV. It was observed that changes on
the cone energy affected the relative abundances of resulting ions.
Thus, at low cone energy (25 V) two main ions at m/z 791 and 793,
Fig. 2. Offline negative ESI-MS, in scan mode, of fractions F2–F5.
104
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
Fig. 3. Formation of radical aglycone ([Y0 −H]− • ) and regular aglycone (Y0 − ) ions in negative ESI mode (A), and negative CID-MS of flavonol triglycosides, showing the
formation of radical from kaempferol, quercetin and myricetin, respectively (B–D).
aglycones and regular deprotonated aglycone were m/z 284–285,
consistent with kaempferol, and m/z 300–301, consistent with
quercetin (Fig. 3A–C). These two aglycones had been previously
found in M. ilicifolia leaves [21,22]. However, at least 2 precursor
ions gave fragments at m/z 316 and 317, indicating that flavonol glycosides from M. ilicifolia can contain myricetin as aglycone (Fig. 3A
and D).
The formation of the [Y0 −H]•− ion is dependent on the glycosylation sites, and it is directly affected by the energy used in CID-MS.
Thus, different flavonoid glycosides may form both deprotonated
radical aglycone and deprotonated regular aglycone ions, with different ratios, providing useful information about the glycosylation
sites [26]. However, no significant variation in the ratio between
[Y0 −H]•− :Y0 − was observed, and both CID-energies used (20 and
40 eV) favored the formation of the radical aglycone. Although
the glycosylation site could not be inferred only by negative CID
Fig. 4. Influence of the cone energy on ionization of a flavonol triglycoside from F5.
Positive ionization with cone energy at: (A) 35, (B) 63 and (C) 85 V.
examination, Cuyckens and Claeys [26] observed that under certain
energy conditions, the radical aglycone had been formed in high
abundance, especially for flavonol 3-O-glycosides. Thus the pronounced [Y0 −H]•− ion observed in all CID-fragments (Fig. 3A–D)
suggests a possible glycosylation site on 3-OH. The other compounds exhibited similar results (Table 1, Figs. S-1–S-5).
In the negative ESI detection, the pseudo-molecular ions in MS1 ,
and the neutral losses (NL) observed in MS2 , showed that the
flavonols present in F2–F5 are mainly linked to mono-, di-, tri-, and
tetrasaccharides, respectively (Table 1). This separation according
to molecular weight is due to increments of MeOH in the solvent
used on silica-gel column fractionation, i.e. the higher the polarity
of the solvent, the better its ability to remove the glycosides with
higher degree of polymerization.
3.2. Offline positive ESI-MS analysis
At low cone energy, the main ions observed were those corresponding to protonated compounds, whereas at high energies the
sodiated compounds were predominant when the fractions were
dissolved in MeOH:H2 O (1:1, v/v). This typical behavior was also
tested with the fraction F5 dissolved in MeOH:H2 O (1:1, v/v) containing 5 mM of HCl or NaCl. With HCl, the ionization followed
a similar behavior, alternating adducts with changes on the cone
voltage. Setting the cone energy at 35 V, the main ion was at m/z
757 [M+H]+ , increasing the cone voltage to 63 V both species were
observed, at m/z 757 and 779 [M+Na]+ , and finally at 85 V the main
ion observed was the more stable form at m/z 779. In all cases the
capillary energy was 2.6 V (Fig. 4A–C). As expected, the solution
containing NaCl gave only Na+ adducts, with the main ion formed
at m/z 779, even changing the cone voltage.
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
105
Fig. 5. (A) Positive CID-MS of protonated ions of the major flavonol triglycosides present in F4. (B) Precursor ion at m/z 741 [M+H]+ and (C) at m/z 757.
3.3. Tandem-MS analysis of protonated compounds
The protonated ions were obtained using the cone energy at
35 V. The CID-fragmentation profiles fallowed the nomenclature
proposed by Domon and Costello [33] by the formation of Yj +
fragments, which were important to determine the sequence of
monosaccharide units linked to flavonols.
For example, the fraction F4, containing mainly the flavonol
triglycosides with m/z 741 and 757 [M+H]+ , produced CIDfragments correspondent to the sequential monosaccharide losses
(Fig. 5A–C). Thus, the precursor ion at m/z 741 gave rise to fragments Y2 + at m/z 595 [M−146]+ , Y1 + at m/z 449 [M−292]+ , and
Y0 + at m/z 287 [M−454]+ , which is compatible with the structure
previously found, kaempferol-3-O-˛-l-rhamnopyranosyl-(1 → 6)-
Fig. 6. (A) CID-MS of flavonol triglycosides from F4 as Na+ adducts, showing the Bi -type fragments of precursor ion at m/z 763 (B) and 779 (C). Its worth noting that the
formation of radical aglycone ([Y0 −H]+ • ) was intensified by increasing the CID energy.
106
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
Table 2
Offline positive ESI-MS/MS detection of sodiated flavonol glycosides
MS1
MS2
[M+Na]
Y0 • + :Y0 +
*R
BX +
B1 + (␣)
B1 + 
B2 + (␣)
B2 + 
B3 + (␣)
B3 + 
B4 +
441
457
471
487
603
617
617
633
649
649
763
779
795
795
895
895
911
911
925
308:309
324:325
308:309
324:325
324:325
308:309
324:325
324:325
324:325
340:341
308:309
324:325
324:325
340:341
308:309
308:309
324:325
324:325
308:309
–
5.1
4.9
4.5
4.8
4.5
4.9
4.8
5.2
–
3.8
3.4
3.6
3.8
3.4
3.4
3.3
3.3
3.4
155
155
185
185
169
185
169
185
185
185
185
185
185
185
185
169
185
169
185
–
–
–
–
155
169
169
169
185
169
169
169
169
169
169
169
169
169
163
–
–
–
–
–
–
–
–
–
–
169
169
185
169
155
185
155
185
185
–
–
–
–
301
331
315
331
347
331
331
331
331
331
331
315
331
315
331
–
–
–
–
–
–
–
–
–
–
331
331
347
331
301
317
301
317
331
–
–
–
–
–
–
–
–
–
–
477
477
493
477
477
447
477
447
477
–
–
–
–
–
–
–
–
–
–
–
–
–
–
463
463
463
463
493
–
–
–
–
–
–
–
–
–
–
609
609
609
609
639
+
R: ratio between [Y0 +Na]• + and [Y0 +Na]+ , *CID energy 60–80 eV.
O-[-˛-l-rhamnopyranosyl-(1 → 2)]-O-ˇ-d-galactopyranoside
[19,20]. The precursor ion at m/z 757 [M+H]+ gave rise
to fragments Y2 + at m/z 611 [M−146]+ , Y1 + at m/z 465
[M−292]+ , and Y0 + at m/z 303 [M−454]+ , compatible with the
quercetin-3-O-˛-l-rhamnopyranosyl-(1 → 6)-O-[-˛-l-rhamnopyranosyl-(1 → 2)]-O-ˇ-d-galactopyranoside [20,21]. Additionally,
both precursor ions had similar neutral losses corresponding
to sugar moieties, confirming that both structures have similar
oligosaccharides.
It is worth noting that in both compounds, the fragment-ion Y*
was formed, at m/z 433 (Fig. 5 B) and m/z 449 (Fig. 5C). Y* is an irregular ion that corresponds to the loss of the inner hexosyl residue,
and a rearrangement with a rhamnose residue, usually observed
at lower collision energies. Although the Y* ion may yield false
monosaccharide sequence, with appropriate instrumentation, the
ratio Y1 /Y* can provide useful information on the type of flavonoid,
and can also be used to infer differentiation between neohesperidose and rutinose, two common disaccharides found attached to
flavonoids [24].
3.4. Tandem-MS analysis of sodiated compounds
When a higher energy was applied to the cone (above 85 V),
the formation of more stable ions from flavonoid glycosides was
favored, which were found as Na+ adducts. Since the sodiated compounds are more stable, they need more CID energy to produce
fragments, rather than protonated compounds. Interestingly, the
sodiated flavonol glycosides gave rise to fragment-ions different
from those obtained in the protonated form, yielding mainly the
Bi + -type fragments, evidencing the glycan moieties, and confirming those neutral losses observed in negative detection. Typically,
the Bi + fragments are formed as a consequence of a breakdown on
glycosidic linkages, referred to as A1 -type cleavage, that generates
an oxonium ion, due to the formation of a third bond to the oxygen from the glycosidic ring. On the other hand, in the formation of
Yj + fragments, the positive charge is retained in the portion of the
molecule that contains the aglycone group, releasing the adjacent
sugar residues as neutral species, with a double bond between C-1
and C-2 (see Fig. 1), being referred to as ˇ-cleavage [33,37]. Interestingly, in our examinations these neutral carbohydrate-species,
produced by ˇ-cleavage, were observed as sodiated ions. For example, fraction F4 gave rise to 2 main ions at m/z 763 [M+Na]+ and m/z
779 [M+Na]+ , both producing similar CID-fragments, characteristic of glycosyl moieties at m/z 477 [B3 +Na]+ , confirming that these
flavonols are attached by a trisaccharide group and not by monosaccharides at different sites on the aglycone. It were also observed
fragments at m/z 331 [B2 +Na]+ , and others at m/z 169 [B1 +Na]+ , and
185 [Bx +Na]+ (Fig. 6A–C, see Figs. S-1–S-8 for other compounds).
Table 3
GC–MS analysis of flavonol glycosides as PMAAs derivatives
PMAAs
2,3-Me2 -Ara
2,3,4-Me3 -Ara
2,4-Me2 -Rha
2,3,4-Me3 -Rha
3,4-Me2 -Glc
2,3,4-Me3 -Glc
3,4,6-Me3 -Glc
2,3,4,6-Me4 -Glc
3,4-Me2 -Gal
2,3,4-Me3 -Gal
3,4,6-Me3 -Gal
2,3,4,6-Me4 -Gal
Peaks
2
3
4
5
6
7
8
9
11
12
13
14
15
17/19
18/20
23/25
27
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
1
–
–
–
–
1
–
–
1
–
–
–
–
–
–
–
–
1
–
–
1
–
–
–
–
–
–
–
–
–
–
–
2
–
–
1
–
–
–
1
–
–
–
–
1
–
–
–
–
–
–
1
–
–
–
–
1
–
–
–
–
–
1
–
–
–
–
–
1
–
–
1
–
–
–
–
–
–
–
–
1
–
1
–
–
–
–
–
–
–
–
–
–
–
1
1
4
–
1
1
–
–
–
–
2
–
–
–
–
1
–
–
–
–
–
–
2
1
–
–
–
–
–
–
–
–
1
1
1
–
–
–
–
1
–
–
–
–
–
1
1
–
–
–
1
1
–
–
–
The values are indicated as molar ratios. The peaks 1, 10, 16, 21, 22, 24, 26, 28 were not detected in GC–MS analysis.
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
107
Fig. 7. HPLC-UV at 360 nm of fractions F3 (A1), F4 (B1), F5 (B2), and HPLC–ESI-MS of selected ions from F3 (A2–A8) and from F5 (B3–B8).
As observed for deprotonated compounds on CID-MS, the Na+
adducts also produced the fragments of regular aglycone and
radical aglycone ions, and the ratio of [Y0 −H]•+ /Y0 + was dependent of the energies used. Cuyckens and Claeys [26] discussed its
application to determine the glycosylation sites of flavonoid monoglycosides, employing the ratio of [Y0 −H]•+ /Y0 + ions, observed at
high CID-energy. In our examination, the collision energy at 40 eV
produced similar ratios between [Y0 −H]•+ and Y0 + . On the other
hand, using CID at 60–80 eV the formation of radical aglycone
was ∼3–4-fold higher than the regular aglycone ion. The higher
[Y0 –H]•+ to Y0 + ratio observed (Fig. 6 B and C) might be an evidence of flavonoid 3-O-glycosides, since it is distinct from ratios
observed for flavonoid 7-O-glycosides, that exhibit a ratio value
of ∼1. However, only the ratio between [Y0 −H]+• and Y0 + , did not
provide enough information to differentiate between flavonoid 3O-glycosides and flavonoid 4′ -O-glycosides, but characteristically
flavonoid 4′ -O-glycosides must form fragments correspondent of Bring containing the carbohydrate moieties [26], which were absent
in our spectra. These results strongly suggested that the flavonols
studied herein are 3-O-glycosilated. The CID-MS from other sodiated compounds are shown in Table 2.
3.5. Online HPLC–ESI-MS analysis
For the online analysis, the cone energy at 50 V in the ESIMS, and the acidic solvent used in the HPLC separation, favored
the protonation of compounds, observed in the positive ESI detection. Furthermore, under these conditions, the ionization promoted
the in-source fragmentation, providing similar fragments to those
obtained in offline CID-MS from protonated precursor ions. The insource fragmentation was useful for monosaccharide sequencing,
however it could lead to a misinterpretation of the chromatograms.
108
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
Table 4
Online positive ESI-MS detection of protonated flavonol glycosides
Peak
Rt
MS1 [M+H]+
MS2
Y0
1a
2a
3
4
5
6
7a
8a
9
10a
11
12a
13a
14
15a
16a
17
18a
19
20a
21a
22a
23
24a
25
26a
27
28a
14.34
14.90
15.39
17.04
12.63
13.19
14.06
14.48
12.91
13.50
11.09
11.40
12.14
12.59
10.00
11.23
15.15
15.50
13.68
13.96
12.24
12.52
15.04
15.53
13.68
14.10
14.06
14.80
435
435
449
449
465
465
581
581
595
595
611
611
611
611
627
627
741
741
757
757
773
773
873
873
889
889
903
903
+
303
303
287
287
303
303
303
303
287
303
303
303
303
303
303
319
287
287
303
303
303
319
287
287
303
303
287
–
Compound identification
Y1
+
–
–
–
–
–
–
435
435
449
449
465
465
465
465
465
481
449
449
465
465
465
481
449
433
465
449
449
–
Y2
+
␣
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
595
595
611
611
611
627
595
565
611
581
595
–
Y2
+

–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
595
595
611
611
627
627
595
577
611
595
595
–
Y3
+
␣
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
741
711b
757
727
741b
–
Y3
+

–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
727
727
743
743
757
–
Ara-(1 → )-Quer
Araf-(1 → 3)-Quer
Galp-(1 → 3)-Kaemp
Glcp-(1 → 3)-Kaemp
Galp-(1 → 3)-Quer (hyperoside)
Glcp-(1 → 3)-Quer (isoquercetrin)
Rhap-(1 → 4/5)-Ara-(1 → 3)-Quer
Rhap-(1 → 4/5)-Ara-(1 → 3)-Quer
Rhap-(1 → 2)-Galp/Glcp-(1 → 3)-Kaemp
Rhap(1 → )-Rha-(1 → 3)-Quer
Rhap-(1 → 2)-Galp-(1 → 3)-Quer
Rhap-(1 → 6)-Galp-(1 → 3)-Quer
Rhap-(1 → 2)-Glcp-(1 → 3)-Quer
Rhap-(1 → 6)-Glcp-(1 → 3)-Quer (rutin)
Glcp-(1 → 2/6)-Gal/Glc-(1 → 3)-Quer
Rha-(1 → )-Hex-(1 → 3)-Myr
Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Kaemp
Rhap-(1 → 6)-[Rhap-(1 → 2)]-Glclp-(1 → 3)-Kaemp
Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Quer
Rhap-(1 → 6)-[Rhap-(1 → 2)]-Glcp-(1 → 3)-Quer
Hex-(1 → )-[Rha-(1 → )]-Hex-(1 → )-Quer
Rhap-(1 → )-[Rhap-(1 → )]-Hex-(1 → 3)-Myr
Arap-(1 → 3)-Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Kaemp
Hex-(1 → )-Ara-(1 → )-[Rha-(1 → )]-Rha-(1 → 3)-Kaemp
Arap-(1 → 3)-Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Quer
Hex-(1 → )-Ara-(1 → )-[Rha-(1 → )]-Rha-(1 → 3)-Quer
Glcp-(1 → 3)-Rhap-(1 → 2)-[Rha-(1 → 6)]-Gal-(1 → 3)-Kaemp
–
Arap: arabinopyranose, Araf: arabinofuranose, Rhap: Rhamnopyranose, Hex: hexose (galactose or glucose), Galp: galactopyranose, Glcp: glucopyranose, Quer: quercetin,
Kaemp: kaempferol and Myr: myricetin.
a
Novel structures described in leaves of Maytnus ilicifolia.
b
Expected but not observed fragments.
Thus, the online analysis was also carried out in the negative ESI
detection, which allowed identifying the peaks without generating
in-source fragmentation.
Two gradients were developed in order to obtain the good peak
shape, resolution, and short time running. Considering its complexity, the fraction F3 needed a different gradient from that used in
fractions F4 and F5. The gradient I provided the best resolution for
fraction F3, which contained the majority of flavonol mono-, and
diglycosides. Moreover, scanning the individual pseudo-molecular
ions on the chromatograms, it was observed that of at least two
peaks emerged for each ion scanned (Fig. 7A1–A8), this being
observed due to the presence of isomers. Accordingly, in a previous
study Tiberti et al. [22] observed some flavonol glycoside isomers
in M. ilicifolia. Considering that the glycosylation site is at the 3-OH
position for all flavonol glycosides, the isomerism could only be in
the type of sugar, or in the type of interglycosidic linkages. In fact,
both possibilities were confirmed by methylation/GC–MS analysis
(Table 3). Thus, it was confirmed that the most of flavonols contain
galactose or glucose linked to aglycone. An example was the ion at
m/z 609 [M−H]− , that showed 4 peaks on the chromatogram. The
methylation analysis showed the presence of glucose and galactose,
which were branched by rhamnose in 1 → 6 or 1 → 2 O-glycosidic
linkages. On the other hand, in the case of peaks 1–2 and 7–8,
the isomerism seems to be due to ring configuration of arabinose
residues, that appears to have both, pyranosidic and furanosidic
forms.
Fractions F4 and F5, containing mainly flavonol tri-, and tetraglycosides, were analyzed using the gradient II, and despite of their
simplicity when compared to F3, all exhibit similar behavior when
scanned for individual pseudo-molecular ions, also showing the
presence of two peaks for each ion scanned (Fig. 7B1–B8). The
majority of flavonol triglycosides had isomerism consisting in the
presence of galactose or glucose linked to aglycone, whereas in the
flavonol tetraglycosides the isomerism appeared as result of differences on monosaccharide sequence, as observed in MS2 (Table 4).
Due to the complexity of samples, some peak overlapping
occurred, making the methylation analysis especially laborious.
However the data were well consistent with the monosaccharide sequences obtained in tandem-MS analysis. The HPLC–ESI-MS
results and a structure description are summarized in Table 4.
4. Conclusion
A wide variety of flavonol glycosides has been shown to be produced by M. ilicifolia. The monosaccharide composition showed the
presence of arabinose (Ara), rhamnose (Rha), galactose (Gal) and
glucose (Glc) in all fractions studied. Furthermore, the flavonols
can also contain, mainly, glucose and galactose linked to aglycone,
and the glycosidic chain can be further elongated, with rhamnose, arabinose and glucose. The online analysis proved to be a
powerful technique for the identification of several diastereoisomers, allowing the scan of specific and individual ions throughout
the chromatograms. However, the chromatograms resulted in high
complex mixtures, which would hardly be resolved without the
previous fractionation on silica-gel.
The compound ionization on ESI source was directly affected
by the cone energies, and consequently the H+ and Na+ adducts
obtained interfered with the fragmentation profiles. Thus, following the Domon and Costello nomenclature [33], the protonated
ions gave rise to Yj + fragments, while the sodiated ions gave rise
to Bi + fragments, which allowed the appropriate monosaccharide
sequencing. Moreover, the sodiated ions at high CID energy produced abundant radical aglycone ions, which were used to identify
glycosylation sites as being in 3-OH position, in accordance to pre-
L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109
vious investigations [19–22]. The aglycone moieties were identified
by their CID-MS partners in negative and positive ESI modes, compared with literature [7,38–41]. They were confirmed as kaempferol
and quercetin. However it was also found myricetin-glycosides,
which had not been described in M. ilicifolia to date.
Acknowledgments
The authors wish to thank the Central de Produção e
Comercialização de Plantas Medicinais, Aromáticas e Condimentares do Paraná Ltda. for supplying the plant sample, and the
Brazilian funding agencies Conselho Nacional de Desenvolvimento
Cientifico e Tecnológico (CNPq), Fundação Araucária, and PRONEXCarboidratos.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.chroma.2008.08.032.
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