2d lc heart cutting on-line of phenolic compounds from three species

Transkrypt

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 4 pp. 885ñ894, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
2D LC HEART CUTTING ON-LINE OF PHENOLIC COMPOUNDS FROM
THREE SPECIES OF THE GENUS SALIX
LORETTA POB£OCKA-OLECH, DANIEL G£”D, BARBARA KR”L-KOGUS
and MIROS£AWA KRAUZE-BARANOWSKA*
Department of Pharmacognosy with Medicinal Plants Garden, Medical University of GdaÒsk,
Al. Gen. J. Hallera 107, 80-416 GdaÒsk, Poland
Abstract: The 2D LC heart-cutting on-line system was elaborated and employed to the analysis of simple phenols and polyphenols occurring in willow barks. Using the test-set of 52 compounds, the conditions of chromatographic separation in each dimension were optimized. The worked-up system was based on RP-separation
in both dimensions and the use of different elution profiles on the first- and second-dimensional columns: gradient and multistep gradient elution, respectively. In all analyses the UV detector was used. Under optimized
separation conditions slightly modified in respect to chemical composition of the each analyzed MeOH extracts
from three willow barks: Salix daphnoides, S. purpurea and S. sachalinensis ëSekkaí the differences in phenolic acid and flavonoid compositions were revealed.
Keywords: two-dimensional HPLC, polyphenols, heart-cutting technique, willow bark, Salix
anthocyanins (21), procyanidins (22, 23) and flavan3-ols (catechins) (27) were presented.
In LC◊LC effluent from the first dimensional
column, divided into small fractions, with proper
modulation time, is analyzed on the second dimensional column (1-5, 8, 9, 19, 28, 40). On the other
hand, in the LC-LC only the selected fractions of
interest are transferred from the first dimension to
the second one (7, 9, 19, 29, 40). Heart-cutting systems are easier to build, as each of the dimensions
may be optimized separately with the use of equipment usually available in every HPLC laboratory.
Moreover, in off-line mode LC-LC enables to detect
analytes appearing in the sample in small concentrations, since the fraction of interest may be evaporated and concentrated after collection (9, 40). Modern
2D LC systems are automated and operated by computer programs which enable the presentation of
two-dimensional separations in the form of 2D
plots.
It is described in literature (41-44), that composition of phenolic compounds, comprising simple
phenols and polyphenols, in bark of willows
evolved dependently on the species of Salix. In our
experiments, 2D LC was employed to compare the
chemical composition of three species from the
genus Salix, namely Salix purpurea L, S. daphnoides
Plant extracts are complex matrices, which
chromatographic analysis is very often difficult due
to the co-elution of analytes and the presence of frequently more than one compound in a single peak.
For their analysis, new chromatographic techniques
are developed. Multidimensional chromatographic
systems, particularly two dimensional liquid chromatography (2D LC) open a new perspective for the
analysis of chemical composition of plant material
(1-11). 2D LC is a powerful tool for the separation
of complex samples, as it offers higher peak capacity, selectivity and resolution power in comparison
with one-dimensional HPLC (1, 4, 7, 12-15). In
recent years, the number of publications concerning
2D LC separation of natural compounds or plant
extracts have significantly increased [1, 3, 8, 11, 1625). Among the two techniques used in various
experimental set-ups of 2D LC: heart-cutting (LCLC) and comprehensive (LC◊LC), the latter is more
popular (1-3, 8, 16, 26-28). However, several applications of heart-cutting technique have also been
described (11, 25, 29-33). In the last decade, special
attention was paid mainly to the polyphenolic plant
secondary metabolites. Researches were performed
on phenolic acids (2, 5, 26, 28, 29, 34-36) and different groups of flavonoids, (2, 5, 15, 16, 24, 25, 30,
31, 33, 35, 37-39). Also the 2D LC separations of
* Corresponding author: e-mail: [email protected]; phone: +48 58 3491960
885
886
LORETTA POB£OCKA-OLECH et al.
Vill. and S. sachalinensis Fr. Schmidt var. ëSekkaí.
The willow bark is herbal remedy used for centuries
as anti-inflammatory, antipyretic and also
antirheumatic (41). As a source of medicinal plant
material some species are used, including S. daphnoides, S. purpurea and S. alba (45). The extracts
from willow bark used in pharmaceutical industry
are standardized only on simple phenol ñ salicin
(45). Salicin and its derivatives decide about pharmacological activity of willow bark. However, also
the other phenols ñ flavonoids, phenolic acids and
catechins are considered as responsible for its
medicinal properties (46, 47). These compounds
possess differentiated antioxidant activity and may
probably participate in an anti-inflammatory activity of willow bark by synergistic effect (47, 48).
In this paper we report the use of elaborated
two-dimensional heart cutting on-line method (LCLC) for the analysis of natural compoundsí complexes present in the MeOH extracts from willow
barks of different origin.
EXPERIMENTAL
General
Standard compounds: kaempferol-3-O-glucoside (2), rutin (6), apigenin-7-O-glucoside (8), luteolin (9), luteolin-7-O-glucoside (10), naringenin-7-
Figure 1. Instrumentation
2D LC heart cutting on-line of phenolic compounds from...
O-glucoside (15), naringin (17), m-hydroxybenzoic
acid (25), isoferulic acid (41) were obtained from
Extrasynthese (Genay, France); kaempferol (1),
quercetin (4), quercetin-3-O-glucoside (5), apigenin
(7), mirycetin (13), catechin (22), salicylic acid (24),
p-hydroxybenzoic acid (26), β-resorcylic acid (28),
gentisinic acid (29), α-resorcylic acid (31), gallic
acid (32), 2,4-dimethoxybenzoic acid (33), veratric
acid (34), m-coumaric acid (38), o-coumaric acid
(39), ferulic acid (40), caffeic acid (42), 4methoxycinnamic acid (44), 3,4-dimethoxycinnamic acid (45), cinnamic acid (46), homovanillic acid
(48), homogentisinic acid (49), chlorogenic acid
(50), rosmarinic acid (51), salicin (52) from Fluka
(Buchs, Switzerland); naringenin (14) and pcoumaric acid (37) from Koch-Light (Colnbrook,
UK), and epicatechin (23), 2,3-dihydroxybenzoic
acid (27), protocatechuic acid (30), vanillic acid
(35), syringic acid (36), sinapinic acid (43), dihydrocaffeic acid (47) from Sigma (Steinheim,
Germany). Kaempferol-3-O-rhamnoside (3),
isorhamnetin (11), isorhamnetin-3-O-glucoside
(12), naringenin-5-O-glucoside (16), amentoflavone
(18), cupressuflavone (19), isosalipurposide (20),
isosalipurposide 6íí-p-coumaroyl ester (21) originated from the collection of standards of the
Department of Pharmacognosy of the Medical
University of GdaÒsk. Some of them ñ compounds
16, 20, 21 were isolated from the willow bark (49)
and their structures were elucidated on the basis of
spectroscopic methods (MS and NMR).
Acetonitrile (ACN) and methanol (MeOH) of
HPLC grade were obtained from Baker (J.T. Baker,
Deventer, The Netherlands). CH3COOH and H3PO4
of analytical grade were obtained from POCH
(Gliwice, Poland), while HCOOH of analytical
grade from Merck (Darmstadt, Germany). H2O was
prepared with a Millipore (Molsheim, France) MilliQ H2O-purification system.
Plant material and samples preparation
Plant material
The bark of Salix purpurea L. and S. daphnoides
Vill. originated from the willow collection of the
University of Warmia and Mazury from Olsztyn
(Poland). The bark of S. sachalinensis Fr. Schmidt var.
ëSekkaí was collected from the Medicinal Plants
Garden of the Medical University of GdaÒsk (Poland).
Sample preparation
Dried and pulverized willow bark (1.0 g) was
exhaustively extracted with MeOH (3 ◊ 30 mL,
60OC). The combined MeOH extracts were evaporated to dryness under reduced pressure. The dried
887
residue was dissolved in MeOH (5 mL) and after filtration through syringe filter (0.22 µm) submitted
for further HPLC analysis.
Chromatographic system
The used 2D LC system (Fig. 1) consisted of
two pumps model L-7100, two UV detectors model
L-7420 (Merck-Hitachi, Germany-Japan), two-position switching valve Model 7000 (Rheodyne, USA),
six-port switching valve Model 7725 (Rheodyne,
USA) and a column oven Jetstream 2 (MerckHitachi, Germany-Japan). The system was operated
under Eurochrom 2000 software (Knauer,
Germany).
2D LC separation of willow bark constituents
The separation in the first dimension (1D) was
performed on a Supelcosil LC-18 column (150 mm
◊ 3 mm, I.D. 3 µm) (Supelco) with linear gradient
elution from 0 to 150 min from 3% to 70% (v/v)
MeOH in H2O with 0.1% H3PO4 (v/v) (program gradient elution I). The flow rate was 0.4 mL/min and
UV detection was perfomed at λ = 280 nm. The volume of an injection loop was 2.5 mL. The flow was
split 1 : 4 by a T connector and 0.125 mm i.d. PEEK
(polyethertherketone) tubing to achieve a flow rate
of 80 µL/min towards the switching valve used as an
interface.
In the second dimension (2D) the Chromolith
Performance RP-18e column (100 mm ◊ 4.6 mm)
(Merck) and the multi-step gradient in mixtures of
ACN in H2O with 0.1% CH3COOH (v/v) (program
elution II), with increasing concentration of ACN:
S1 (5 + 95, v/v), S2 (10 + 90, v/v), S3 (15 : 85, v/v), S4
(20 : 80, v/v), S5 (30 : 70, v/v) at flow rate 1.0
mL/min were used. UV detection was performed at
λ = 210 nm. The separation on both columns was
carried out at ambient temperature. The proper fractions (Fr.) containing unresolved compounds (S.
daphnoides ñ 4 fractions: Fr. I D ñ IV D, S. purpurea
ñ 6 fractions: Fr. I P ñ VI P, S. sachalinensis ëSekkaí
ñ 5 fractions: Fr. I S ñ V S, the mixture of standards
ñ 6 fractions: Fr. I M ñ VI M) were manually transferred via switching valve from the first dimensional column directly on the second dimensional one
(Tab. 1). The volume of transferring fractions was
changeable due to their complexity and elution time
on the first dimensional column (from 128 µL to 744
µL).
RESULTS AND DISCUSSION
However, the use of two different mechanisms
of separation in the first and second dimension is
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Table 1. The characteristic of fractions (Fr.) and compounds transferring from first dimension (1D) and separated in second dimension
(2D) of the elaborated on-line heart-cutting LC-LC method.
Fraction
(Fr.) code
I M / S1
Transferring
time [min]
29.4-32.4
Effluent
volume [µl] *
240
II M / S3
46.5-50.1
288
III M / S2
64.1-67.6
280
IV M / S4
69.4-73.5
328
V M / S5
89.4-95.9
520
VI M / S5
115.0-117.4
192
I D / S1
II D / S2
11.3-14.6
46.0-48.7
264
216
III D / S3
80.7-84.3
288
IV D / S4
90.0-96.0
480
I P / S1
II P / S1
11.2-15.9
16.0-17.6
376
128
III P / S2
47.1-49.4
184
IV P / S2
V P / S3
50.6-54.7
89.4-97.6
328
656
VI P / S4
I S / S1
115.3-117.1
45.0-48.8
144
304
II S / S1
IV S / S3
V S / S4
50.0-53.1
80.1-89.4
89.5-95.6
248
744
488
Separated compounds (no.) (2D)
tR ± SD (2D)
2,3-Dihydroxybenzoic acid (27)
β-Resorcylic acid (28)
Syringic acid (36)
Chlorogenic acid (50)
2,4 -Dimethoxybenzoic acid (33)
m-Coumaric acid (38)
Ferulic acid (40)
o-Coumaric acid (39)
Isoferulic acid (41)
Sinapinic acid (43)
Quercetin-3-O-glucoside (5)
Rutin (6)
Apigenin-7-O-glucoside (8)
Mirycetin (13)
Isosalipurposide (20)
4-Methoxycinnamic acid (44)
Rosmarinic acid (51)
Apigenin (7)
Isorhamnetin (11)
Protocatechic acid (30)
Syringic acid (36)
Naringenin-7-O-glucoside (15)
Cinnamic acid (46)
Mirycetin (13)
Protocatechic acid (30)
α-Resorcylic acid (31)
Salicin (52)
Syringic acid (36)
Salicylic acid (24)
Rutin (6)
Mirycetin (13)
Apigenin (7)
Syringic acid (36)
Salicylic acid (24)
Cinnamic acid (46)
Rutin (6)
Mirycetin (13)
39.31 ± 0.17
38.84 ± 0.18
52.42 ± 0.34
51.71 ± 0.34
76.33 ± 0.61
4.45 ± 0.23
72.55 ± 0.23
90.82 ± 0.56
89.43 ± 0.56
94.71 ± 0.57
122.47 ± 0.53
121.52 ± 0.54
126.51 ± 0.53
129.08 ± 0.54
137.05 ± 0.55
142.71 ± 0.55
131.03 ± 0.54
123.62 ± 0.41
124.76 ± 0.41
18.41 ± 0.021
55.03 ± 0.28
54.04 ± 0.28
88.41 ± 0.15
96.93 ± 0.61
101.07 ± 0.28
106.92 ± 0.27
115.04 ± 0.27
18.73 ± 0.02
22.32 ± 0.02
21.61 ± 0.02
52.13 ± 0.32
51.25 ± 0.32
67.07 ± 0.24
102.02 ± 0.31
105.78 ± 0.31
108.19 ± 0.32
112.62 ± 0.30
123.93 ± 0.39
52.95 ± 0.44
52.17 ± 0.44
64.24 ± 0.33
108.31 ± 0.47
103.83 ± 0.47
101.85 ± 0.46
105.91 ± 0.44
108.47 ± 0.46
Abrreviations: The fractions (Fr.) transferring from first column (1D) to second column (2D): I M ñ VI M ñ standard mixture; I D ñ IV D
ñ Salix daphnoides 1095; I P ñ V P ñ Salix purpurea; I S ñ V S ñ Salix sachalinensis; S1 ñ S5 ñ mobile phases used in the second dimension; *effluent volume calculated as product of flow rate and time of fraction (Fr.) transferring.
generally preferred in 2D LC systems (1, 4, 7), for
our purposes the system consisting of two RP-18
columns was chosen. Such solution, although not
definitely orthogonal, enables significant increase of
2D LC system resolution power (33, 39). The
changes in each dimension selectivity may be performed not only by the choice of the type of RP-18
columns, but also by the use of different organic
modifiers in mobile phases (3, 7, 50). Large choice
of stationary phases in RP-LC gives a possibility to
create many configurations in RP-LC◊RP-LC (50).
Moreover, reverse phases are suitable for many different groups of analytes and therefore they can be
applied to the majority of samples (50).
In the developed 2D LC system three stages of
work can be distinguished, as it is showed on Figure
1. In the first step, the analyzed plant matrix was
separated on the first dimensional column and the
second one was simultaneously reequilibrated with
appropriate mobile phase. A stream of mobile phase
from the first dimension was split in ratio 1 : 4 and
directed in parallel to UV detector and to waste by
switching valve. Next, multi-component effluent
889
fractions were transferred manually by a change of a
position in switching valve. The six-port switching
valve without storage loop was used. A volume of
transferred fraction was changing dependently on its
complexity and elution time on first dimensional
column from 128 µL to 744 µL. However, it is well
known that in 2D LC systems injection of a large
fraction volume on the second-dimensional column
will implicate serious band broadening. This inconvenience can be minimized by employing in second
dimension the mobile phase of higher elution
strength (in comparison to the first dimension) (51).
The third step of elaborated 2D LC system comprised parallel separation processes on first- and second-dimensional columns.
It is stated, that to create a successful 2D LC
system, several parameters should be optimized,
among others: matched flow rates of mobile phases,
dimensions of columns, modulation time, miscibility
and eluent strength of mobile phases in both dimensions (1, 3-7, 20). An optimization process is simpler, when a test-set of compounds covers a wide
range of constituents expected in analyzed sample. In
Figure 2. HPLC chromatogram of heart cutting (LC-LC) separation of the 52-components test-set: I ñ the first dimension; Supelcosil LC18 column (150 ◊ 3 mm, I.D. 3 µm) gradient elution from 0 to 150 min increasing from 3% to 70% (v/v) MeOH in H2O with 0.1% H3PO4
(v/v); II ñ the second dimension; Chromolith Performance RP-18e column (100 ◊ 4.6 mm). Separations performed at differend concentration of ACN in water with 0.1% CH3COOH (v/v): S1 (5 : 95, v/v), S2 (10 : 90, v/v), S3 (15 : 85, v/v), S4 (20 : 80, v/v), S5 (30 : 70, v/v).
Experimental conditions and numbers of compounds, see text
890
Figure 3. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of Salix daphnoides: I ñ the first
dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column (100 ◊
4.6 mm). Experimental conditions and numbers of compounds, see text
Figure 4. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of S. purpurea: I ñ the first dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column (100 ◊ 4.6
mm). Experimental conditions and numbers of compounds, see text
891
Figure 5. HPLC chromatogram of heart cutting (LC-LC) separation of the MeOH extract from the bark of S. sachalinensis ëSekkaí: I ñ the
first dimension Supelcosil LC-18 column (150 ◊ 3 mm, I.D. 3 µm), II ñ the second dimension Chromolith Performance RP-18e column
(100 ◊ 4.6 mm). Experimental conditions and numbers of compounds, see text.
case of some plant extracts, the number of adjacent
and unrecognized peaks, which have to be resolved,
is theoretically unlimited. Regarding this fact, in
response to complication degree of plant matrices,
the use of heart-cutting technique is easier to perform, as both dimensions may be optimized separately and the equipment requirements make it possible to perform in almost every HPLC laboratory.
LC-LC separation of the extracts from willow
bark
To compare the chemical composition of three
willow barks, the 2D RP-LC system consisting of
Supelcosil LC-18 column (150 mm ◊ 3 mm) in the
first dimension (1D) and Chromolith Performance
RP-18e column (100 mm ◊ 4.6 mm) in the second
dimension (2D) was constructed. The separation conditions were optimized for the standard mixture containing 52 constituents, belonging to different groups
of compounds, isolated and identified previously in
willow bark (9-12, 41, 42): flavones (compounds 710), flavonols (1-6, 11-13), flavanones (14-17), chalcones (20, 21), biflavones (18, 19), phenolic acids
(24-51), catechins (22, 23) and salicin (52).
For the separation of standards mixture in the
first dimension on the used conventional porous particle RP-18 column, the program of gradient elution
with increasing concentration of MeOH (from 3 to
70%) in a mixture of MeOH and 0.1% solution
H3PO4 in H2O was adopted. As a result, the resolution of about 45 peaks, attributed to single compounds or co-eluting compounds was obtained at the
time tG = 150 min (Fig. 2).
Selected first-dimensional fractions (Tab. 1)
containing co-eluting compounds were on-line
directly transferred manually by the switching valve
onto the second-dimensional column.
Fractions due to their complexity were
resolved on the second column in different separation time, which varied from 3 min (Fr. VI M / S5)
to 40 min (Fr. V M / S5 (Figs. 3-5).
In two-dimensional LC systems an approach
used extensively for achieving a fast separation in
the second dimension is mainly the employment of
monolithic columns (51). This type of columns
allows very high flow rates without the problem of
back-pressure.
However, the higher flow rate
implies the higher fractions dilutions, what finally
results in worsening the sensitivity. For this reason
in the second dimension, UV detection at λ = 210
nm was used instead of λ = 280 nm as less selective
and specific.
Finally, among 52 analyzed standard compounds, 19 as single peaks were separated in the sec-
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ond dimension (2D) on a monolithic column
employing multi-step gradient in mixtures of ACN
and 0.1% acetic acid solution in water, with increasing concentration of ACN from 5% to 30% as follows: Fr. I M / S1 (5 + 95, v/v), Fr. II M / S2 (10 +
90, v/v), Fr. III M / S3 (15 : 85, v/v), Fr. IV M / S4 (20
: 80, v/v), Fr. V M and VI M / S5 (30 : 70, v/v) (Fig.
2). After each second-dimensional separation, the
column was eluted with the next mixture of solvents, with one exception ñ the second-dimensional
separations of two fractions, Fr. V M and Fr. VI M,
containing compounds 5, 6, 8, 13, 20, 44, 51 (Fr. V
M) and 7, 11 (Fr. VI M), respectively, were performed with the same mobile phase S5 (Fig. 2). An
increase of the ACN concentration was connected
with the decrease of hydrophilic properties of compounds poorly resolved or co-eluting in the first
dimension. However, it was also important to perform fast separation in the second dimension to
make the next first-dimensional fraction transfer
possible.
Among analyzed constituents of the standards
mixture, the following phenolic acids, unresolved on
first-dimensional column, were separated in the second dimension: 2,3-dihydrobenzoic (compound 27)
and β-resorcylic (28) (Fr. I M / S1); syringic (36) and
chlorogenic (50) (Fr. II M / S2); m-coumaric (38),
2,4-dimethoxybenzoic (33) and ferulic (40) (Fr. III
M / S3), sinapinic (43), o-coumaric (39) and isoferulic (41) (Fr. IV M / S4) next to apigenin (7) and
isorhamnetin (11) (Fr. VI M / S5). The most complicated fraction from the first dimension, with volume
ca. 520 µL, contained seven compounds, namely:
rutin (6), quercetin-3-O-glucoside (5), apigenin-7O-glucoside (8), myricetin (13), rosmarinic acid
(51), isosalipurposide (20) and 4-methoxycinnamic
acid (44) (Fr. V M / S5) (Fig. 2). All these compounds were satisfactory resolved in the second
dimension on monolithic column by the use of a
mobile phase of lower or the same elution strength,
as compared to the first-dimension eluent, but possessing different selectivity (52) (Fig. 2). Moreover,
the retention times of some compounds were shorter, in comparison to their separation on the first
dimensional column, as a result of higher flow of
mobile phase and elution volume. The worked-up
system gave repeatable retention times of analyzed
peaks (Tab. 1)
One dimensional HPLC separation of MeOH
extracts of willow barks showed differences in their
chromatographic profiles dependently on the analyzed willow species, comprising the presence of
additional peaks besides peaks of compounds characteristic for this plant material.
As a consequence of this fact the different program
of multi-step gradients for the separation on the second dimension was used (Tab. 1).
Compounds, which were separated under the
same conditions of chromatographic analysis
revealed the repeatable retentions times, for example, protocatechuic acid (30) ñ in Fr. I D / S1 from
Salix daphnoides and Fr. I P / S1 from Salix purpurea
(Figs. 3 and 4). However, for the other compounds
they were different. Depending on matrices complexity some compounds were separated on first column or were carried out to the second column, for
example salicin. This compound was separated in
the Fr. II P / S1 in the extract of Salix purpurea, while
in the case of Salix daphnoides extract salicin was
separated from the other components of matrix on
the first column (Figs. 3 and 4).
The chemical composition of three willow
barks, namely S. purpurea, S. daphnoides and S.
sachalinensis ëSekkaí was compared with the use of
designed set-up of 2D LC system (Figs. 3-5). In all
extracts the presence of chlorogenic (50) and syringic (36) acids was confirmed besides unidentified
compounds, co-eluting with determined phenolic
acids in the first dimension (Figs. 3-5).
Consequently, in the extracts from S. purpurea and
S. sachalinensis ëSekkaí, the resolution on the second-dimensional column revealed the presence of
protocatechuic acid (30) next to the several unidentified peaks (Figs. 3 and 5). Furthermore, the separation of the extract from S. sachalinensis ëSekkaí in
the second dimension did not confirmed the presence of veratric acid (34), eluted as a single peak on
first dimensional chromatogram (Fig. 5). On the
other hand, analyses of the extract from S. purpurea
on the second-dimensional column, allowed to
obtain better separation of salicin (52) and α-resorcylic acid (31) from co-eluting compounds (Fig. 4).
Considering a high concentration of naringenin-7O-glucoside (15) in the extract of S. daphnoides, the
separation in the second dimension provided the resolution of cinnamic acid (46) from this flavanone
glucoside (Fig. 3).
As a result of separation in the second dimension in the extract from S. daphnoides bark
quercetin-3-O-glucoside (5), myricetin (13) and
isosalipurposide (20) as dominating compounds
were identified (Fig. 3). On the other hand, the same
fractions ñ Fr. V P / S3 from the extracts of S. purpurea and Fr. V S / S4 of S. sachalinensis ëSekkaí
which were separated on the second-dimensional
column contained respectively: rutin (6), apigenin7-O-glucoside (8) and myricetin (13) besides isosalipurposide (20), quercetin-3-O-glucoside (5), api-
genin-7-O-glucoside (8) and myricetin (13) (Figs. 4
and 5). Considering the first dimensional-separation
some differences in chromatographic profiles
between the analyzed willow barks were confirmed.
It concerns the presence of salicylic compound:
salicin (52), flavanones: naringenin-5-O-glucoside
(16), naringenin-7-O-glucoside (15) and chalcones:
isosalipurposide and its p-coumaric ester (20 and
21). These compounds were not displayed in the
extract from S. sachalinensis ëSekkaí bark, what
remains in agreement with literature data (11).
Furthermore, isosalipurposide p-coumaric ester (21)
showed to be a distinctive compound only for the
bark of S. daphnoides.
The elaborated system, regarding a wide range
of resolved compounds, may be applied in the quality control of willow bark extracts for medicinal purposes. Moreover, it makes the analysis of phenolic
acids possible without a purification step. Their
analysis in plant material requires mainly a sample
pre-treatment - purification by extraction with solvent or SPE (26, 27).
CONCLUSION
The 2D LC system was worked-up for analysis
of simple phenols and polyphenols occurring in
three willow barks: Salix daphnoides, S. purpurea
and S. sachalinensis ëSekkaí. The two-dimensional
chromatographic separations were performed with
the use of heart-cutting on-line technique. Fifty two
standard compounds were separated and their presence was analyzed in the investigated plant material
by comparison of the tR values.
Compounds belonging to the groups of simple
phenols (salicin derivatives, phenolic acids) and
polyphenols (flavanones, flavones, flavonols, chalcones, flavan-3-ols) were efficiently resolved
(Figs. 3-5). The earlier studies on the chemical
composition of willows with the use of HPLC (42,
43, 53) concerned only two groups of secondary
metabolites ñ salicin derivatives and flavonoids
(chalcones and flavanones). The elaborated 2D LC
heart-cutting on-line system enabled the chromatographic identification of several unrecognized earlier compounds in willow barks, namely apigenin,
luteolin 7-O-glucoside, quercetin, mirycetin,
quercetin 3-O-glucoside, catechin, epicatechin, αresorcylic acid, gallic acid in the Salix daphnoides
bark, quercetin, kaempferol 3-O-glucoside,
mirycetin, kaempferol 3-O-rhamnoside, rutin, apigenin 7-O-glucoside, α-resorcylic acid, gallic acid
in the S. purpurea bark, and naringenin, apigenin 7O-glucoside, luteolin 7-O-glucoside, quercetin 3-
893
O-glucoside, rutin, gallic acid in the S. sachalinensis ëSekkaí bark.
The elaborated 2D LC system can be used to
the routine analysis of the complex of biologically
active compounds present in other willows.
Moreover, the worked-up 2D LC heart-cutting system is simple and easy to construct practically in
each chromatographic laboratory.
Acknowledgments
We would like to give special thank to
Scientific Committee of the 6th Balaton Symposium
on High-Performance Separation Methods held in
Siofok (Hungary) in 2005 for distinction of the
results of this study by Best Poster Award.
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Received: 4. 08. 2015

2d lc heart cutting on-line of phenolic compounds from three species

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