Identification of carbohydrate isomers in flavonoid glycosides after hydrolysis by hydrophilic interaction chromatography

Article abstract of DOI:10.2478/s11696-012-0302-8

A method for carbohydrate isomers (saccharide units) identification in flavonoid glycosides after hydrolysis by an HPLC system with two detectors (diode array UV-VIS detector and evaporative light scattering detector) was set up in this work. Experimental procedure was optimized with two types of model glycosides, namely rutin and hesperidin. The model glycosides were hydrolyzed to saccharide units and aglycone parts; the aglycone was characterized by its UV-VIS spectrum and the saccharide unit was identified by its retention time and elution profile (anomeric signals). Acidic and enzymatic hydrolyses were compared in the first step of the method; in acidic hydrolysis, trifluoracetic acid was used, while glucosidase, galactosidase, and hesperidinase were used in enzymatic hydrolysis. A complete enzymatic hydrolysis was achieved with hesperidin and neohesperidin, but not with 3-O-glycosides. The method was applied for the identification of a glycone from a glycoside isolated from Polygonum lapathifolium (Polygonaceae sp.).

Full text of DOI:10.2478/s11696-012-0302-8

Chemical Papers 67 (4) 357–364 (2013)
DOI: 10.2478/s11696-012-0302-8
ORIGINAL PAPER
Identification of carbohydrate isomers in flavonoid glycosides
after hydrolysis by hydrophilic interaction chromatography
b
aVěra Špačková, Jiří Pazourek*
aDepartment of Natural Drugs, bDepartment of Chemical Drugs, Faculty of Pharmacy,
University of Veterinary and Pharmaceutical Sciences, Palackého 1–3, 612 42 Brno, Czech Republic
Received 28 June 2012; Revised 15 October 2012; Accepted 20 October 2012
A method for carbohydrate isomers (saccharide units) identification in flavonoid glycosides after
hydrolysis by an HPLC system with two detectors (diode array UV-VIS detector and evaporative
light scattering detector) was set up in this work. Experimental procedure was optimized with two
types of model glycosides, namely rutin and hesperidin. The model glycosides were hydrolyzed to
saccharide units and aglycone parts; the aglycone was characterized by its UV-VIS spectrum and
the saccharide unit was identified by its retention time and elution profile (anomeric signals). Acidic
and enzymatic hydrolyses were compared in the first step of the method; in acidic hydrolysis, triflu-
oracetic acid was used, while glucosidase, galactosidase, and hesperidinase were used in enzymatic
hydrolysis. A complete enzymatic hydrolysis was achieved with hesperidin and neohesperidin, but
not with 3-O-glycosides. The method was applied for the identification of a glycone from a glycoside
isolated from Polygonum lapathifolium (Polygonaceae sp.).
c
ꢀ 2012 Institute of Chemistry, Slovak Academy of Sciences
Keywords: glycosides, identification of isomers, acidic hydrolysis, enzymatic hydrolysis, HILIC
Introduction
bond and the position of the glycosidic bond in the
polyhydroxylic aglycone have to be identified for both
basic parts (aglycone and carbohydrate) of the glyco-
side to determine its structure.
Identification of glycosides represents an essen-
tial part of natural compounds analysis. Identifica-
tion of both glycoside parts (aglycone and carbohy-
drate) and the position of the glycosidic bond in the
polyhydroxylic aglycone are essential for the iden-
tification of the structure. Glycosides are molecules
composed of a carbohydrate (glycone) and a non-
carbohydrate part (aglycone). The carbohydrate part
can contain one (monosaccharide) or more (oligosac-
charides) molecules of carbohydrates. The saccharide
part is bond through an anomeric carbon to the rest
of the structure (aglycone) by a glycosidic bond. Not
only oxygen (O-glycosides) but also nitrogen (gly-
cosylamines), sulfur (thioglycosides), and carbon (C-
glycosides) can take part in the glycosidic bond. Gly-
cosides have many functions in living organisms as
storage, defense, and elimination. A lot of plant gly-
cosides are used in therapy. The type of the chemical
The glycosidic bond can be interrupted by chemi-
cal or enzymatic processes. It is known that chemical
hydrolysis proceed non-specifically in contrast to en-
zymatic hydrolysis. Glycosides are sensitive to acidic
hydrolysis (depending on the saccharide part) and in
some cases also to basic hydrolysis (depending on the
aglycone type). Some aglycones exhibit a resistance to
hydrolysis (e.g., triterpens, sterols), where the glyco-
sidic bond is more stable. O-glycosides are the least
resistant to acidic conditions; S- and C-glycosides are
more resistant (Brito-Arias, 2007).
The rate of hydrolytic degradation depends on the
structure of the aglycone and the carbohydrate part of
the glycosides. The hydrolysis resistance of glycosides
depends on their heterocyclic configuration. Carbohy-
drates with furanoside configuration hydrolyze faster
*Corresponding author, e-mail: pazourekj@vfu.cz

358
V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
Identification of saccharides in flavonoids started
already fifty years ago. Chandler and Harper (1961)
developed a systematic approach based on chemical
degradation of flavonoids using several agents (perox-
ide, permanganate, and ozone) which led to absolute
identification of the glycone position. Identification of
the reaction products, however, was done using TLC.
Classical chemical approach (methanol extraction, re-
verse phase liquid chromatographic separation, diode
array and MS detection) was used also for glycosy-
lated flavonoid characterization (Lin & Harnly, 2007).
It can be noted that an MS detector is too expensive
for routine use.
Recently, glycosides have been directly (or after
hydrolysis) analyzed by GC-MS after their conversion
to trifluoroacetylated derivates. Their identification by
comparison with standards for the known glycoside
structures can be done using HPLC-MS (Chassagne
et al., 1998; Chen & Zuo, 2007; Ikegami et al., 2008;
Ma et al., 2011; Thabti et al., 2012; Zhang et al.,
2012). Hyphenation of LC-MS to HRMS (LC-HRMS)
has been recently applied to a “bottom-up” screen-
ing of flavonoids in plant materials (Abrankó et al.,
2012); without using standards, the authors identified
flavonoids only by deduction from their exactly mea-
sured mass. However, identification of glycone parts
is often impossible because monosaccharide isomers
cannot be differentiated even by HRMS. MS anal-
ysis of metal complexes (Al, Mn) with glycosylated
flavonoids can be useful for their identification (Davis
& Brodbelt, 2005; Zhang et al., 2005). Another pos-
sible way (but more demanding) is using hyphenation
HPLC-NMR-MS which provides enough information
for structural identification of glycosides (Lommen et
al., 2000). Also, 2D NMR techniques can be used for
the glycosides identification (Lundborg et al., 2011;
Pieretti et al., 2011; Zahid et al., 2002). Another ap-
proach is the use of pulsed amperometric detector
(HPLC-PAD) (Nehme et al., 2005).
than those with the pyranoside one. The hydrolysis
rate decreases in sequence: L-rha, L-ara, D-glc, D-gal,
D-glucuronic acid. 2-Deoxycarbohydrates hydrolyze
easily. Position of the carbohydrate part is also impor-
tant. C-3 glycosides usually do not hydrolyze partially
because they are quickly fully hydrolyzed by a small
amount of acid. Partial hydrolysis is possible with C-
7 and C-4ꢀ glycosides. An aromatic acyl bond to the
carbohydrate also increases the hydrolysis resistance.
Some O-glycosides can be dissociated by alkaline hy-
drolysis: e.g., phenolic glycoside, 4-hydroxycoumarine,
purin and pyrimidine glycosides, enols, β-substituted
alcohol glycosides, 7-flavonoid glycosides (Brito-Arias,
2007; Garegg, 2004; Harborne, 1965; Quintin & Lewin,
2005). Disaccharides give non-saccharide derivates un-
der basic conditions, however, they provide monomers
under acidic conditions (Garegg, 2004; Litvinenko
& Makarov, 1969; Quintin & Lewin, 2005). Enzy-
matic hydrolysis is carried out using stereo-specific
enzymes, glycosidases, which can split a whole gly-
cone (oligosaccharide) or its parts (oligosaccharide or
monosaccharide). Glycosides with a non-stable agly-
cone hydrolyze enzymatically without any secondary
changes (Kim et al., 2011; Rotrekl, 1998). Configura-
tion of the anomeric carbon can be inverted (inverting
glycosidases) or they can remain unchanged (most of
glycosidases retain glycosidases) (Brito-Arias, 2007).
Generally, β-glucosidases are substrate specific (i.e.,
they have almost absolute requirements on the saccha-
ride part of the substrate = glucose) and they are less
active to other substrates such as fucose or galactose.
β-Galactosidase is able to lyse also glucosides (Chen &
Zuo, 2007). The glycosides of natural compounds are
not always hydrolyzed by glycosidases because they
can act as their inhibitors. For example vanicoside
A and B (from Polygonum sachalinense) inhibit the
β-glucosidase activity (Fan et al., 2010; Kawai et al.,
2006).
The aglycone part of flavonoids can be identified
by its characteristic UV-VIS spectrum (a fit to a UV-
VIS spectra library or a standard). To compare such
a spectrum after the acidic or basic hydrolysis, pH
extremes during the hydrolysis which result in max-
ima shifts have to be considered. Alternatively, the
aglycone part of flavonoids can be identified accord-
ing to its molecular mass by MS. The absence of a
chromophore limits the use of UV-VIS detectors for
glycones identification. A typical alternative, refrac-
tive index detector, can be used only in an isocratic
elution with low sensitivity. On the contrary, an evap-
orative light scattering detector (ELSD) is suitable for
carbohydrates analysis. It is sensitive, provides a sta-
ble baseline and it is applicable under gradient elu-
tion. The ELSD detector is useful in the detection
of compounds without a chromophore or fluorophore
(e.g., phospholipids, fatty acids, and carbohydrates).
A charged aerosol detector (CAD) shows even better
sensitivity than the ELSD one (Eom et al., 2010).
In the last decade HPLC, and recently capil-
lary electrophoresis (CE) methods for high resolu-
tion and reproducible quantitation of carbohydrates
have been developed (Rassi, 1995). A variety of car-
bohydrate isomers considering their polydispersity
and heterogeneity requires separation techniques of
high resolution and high selectivity such as HPLC,
after derivatization on C18-columns, or on grafi-
tized carbon columns; hydrophilic interaction chro-
matography (HILIC); ion-exchange chromatography
(IEC) on cation exchange columns in, typically, K+
,
Ca2+, or Pb2+ cycle; hydrophobic interaction chro-
matography (HIC) and size-exclusion chromatogra-
phy (SEC) or CE. Saccharides (glycones) are typi-
cally separated by HPLC with aminopropyl groups
stationary phases, though novel stationary phases
are reported also for oligomers (Brito-Arias, 2007;
Hernández-Hernández et al., 2012; Ikegami et al.,
2008; Inoue et al., 2012; Kadumi et al., 2011; Qiu et

V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
Table 1. Model flavonoids and glycosides used in this study
359
Aglycone
Radical
Compound name
Quercetin structure
R = H
quercetin
R = glucose
quercetin 3-β-D-glucoside
R = galactose
quercetin 3-β-D-galactoside
R = rhamnose
quercitrin, quercetin 3-α-L-rhamnoside
R = rutinose (rhamnosyl-(1→6)-glucosyl)
rutin, quercetin 3-[6-O-(α-L-rhamnopyranosyl)-
β-D-glucopyranoside]
Hesperitin structure
R = H
hesperetin
R = rutinose
hesperidin, hesperetin 7-[6-O-(α-L-
rhamnopyranosyl)-β-D-glucopyranoside]
R = rhamnosyl-(1→2)-glucosyl
neohesperidin, hesperetin 7-[2-O-(α-L-
rhamnopyranosyl)-β-D-glucopyranoside]
al., 2011; Ricochon et al., 2011). Monosaccharides in
biomass were also quantified by LC-CAD (Godin et
al., 2011).
Acidic hydrolysis
TFA was selected for acidic hydrolysis (Rassi,
1995). Solvents and their mixtures used in this pro-
cedure provided complete dissolution of polar ana-
lytes (glycosides, glycones) and also of the less po-
lar products (aglycones) formed during the hydrolysis
(Molnár-Perl & Fu¨zfai, 2005; Stalikas, 2007; Zahid et
al., 2002). Dissolved samples were mixed with 4 M
TFA in acetonitrile (2 mL, ϕr = 1 : 1). The mixture
in vials was thermostated at 90◦C for 60 min. The vials
were closed in order to prevent losses by evaporation.
The aim of this paper is to present an HPLC
system with two detectors that enables analysis of
both the aglycone and glycone parts of a glyco-
side. The aglycone parts in flavonoids were charac-
terized by their UV-VIS spectrum and glycones in
the form of monosaccharides were identified by a
highly selective LC separation in the HILIC mode
on a DIOL stationary phase (Pazourek, 2010). The
procedure was optimized on model glycosides and ap-
plied to a glycoside isolated from Polygonum lapathi-
folium.
Enzymatic hydrolysis
Experimental
Enzymatic hydrolysis was carried out according to
the manufacturer recommended conditions as follows:
The following enzymes were used in the experi-
ments: β-glucosidase EC 3.2.1.21, β-galactosidase EC
3.2.1.23, hesperidinase 3.2.1.40 (Sigma–Aldrich, Ger-
many). Methanol and hydrochloric acid were pur-
chased from Lachner (Czech Republic). For the LC
mobile phase, acetonitrile of supragradient HPLC
grade and water of gradient HPLC grade were pur-
chased from Scharlau Chemie (Spain).
Hesperidinase (EC 3.2.1.40), also called rhamnosidase
Prior to use, a fresh solution of hesperidinase (0.03
U mL−1) in cold water was prepared. pH of the sample
was adjusted to 3.8 with 1 M TFA. The sample was
mixed with the dissolved enzyme in the ratio sam-
ple/enzyme (ϕr = 4 : 1). The mixture was incubated
for 30 min at 40◦C.
Glycosides, derivates of quercetin (quercetin-3-
β-D-galactoside, quercetin-3-β-D-glucoside, quercitrin,
and rutin), derivatives of hesperetin (hesperidin, neo-
hesperidin), glucose, rhamnose, galactose, and triflu-
oroacetic acid (TFA) were purchased from Sigma–
Aldrich (Germany). Structures of some of the stan-
dards are shown in Table 1. Concentration of glyco-
Beta glucosidase (EC 3.2.1.21)
A fresh solution of glycosidase (2 U mL−1) in cold
water was prepared prior to use. pH of the sample was
adjusted to 5.0 with 1 M TFA. The sample was mixed
with the dissolved enzyme in the ratio sample/enzyme
(ϕr = 4 : 1). The mixture was incubated for 30 min
at 37◦C.
sides used for hydrolysis was 1 mg mL−1
.
Standards
Standards were dissolved in a mixture of methanol
and water (ϕr = 1 : 1); only hesperidin was dissolved
in a 0.67 M NaOH solution in water.
Beta galactosidase (EC 3.2.1.23)
A fresh solution of galactosidase (1 U mL−1) in
cold water was prepared. pH of the sample was ad-

360
V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
justed to 7.3 with 1 M TFA. The sample was mixed
with the dissolved enzyme in the ratio sample/enzyme
(ϕr = 4 : 1.) The mixture was incubated for 30 min
at 37◦C.
tector revealed baseline instability with the col-
umn LiChrospher 100 NH2, Merck (Germany; 5 µm,
4.1 mm × 250 mm); therefore, the s/n ratio was fa-
vorable for a DIOL column (glycidylpropyl stationary
phase). A DIOL column can separate even monosac-
charide anomers (Pazourek, 2010), which explains the
occurrence of more peaks (two peaks for glucose and
galactose, resp., a non-resolved peak for rhamnose) for
a monosaccharide in a chromatogram in the following
experiments.
HPLC analysis
An HPLC method in a HILIC mode allowing sep-
aration of mono- and disaccharides (Pazourek, 2010)
was applied for the separation of the hydrolytic prod-
ucts. The method was modified for a system of HPLC-
diode array detector (DAD)-ELSD on an instrument
YL9100 (YL Instruments, South Korea) with DAD
hyphenated with an ELSD (Agilent, Germany), con-
trolled by the software YL Clarity (DataApex, Czech
Republic).
Column LiChrospher 100 DIOL (Merck, Germany;
5 µm, 4.1 mm × 250 mm) was used. Mobile phase
for the isocratic elution was acetonitrile/water (ϕr =
91 : 9), thermostat temperature was 31◦C and the
flow-rate was 2 mL min−1. Operational parameters
of ELSD were as follows: gain of 11, nitrogen pressure
of 300 kPa, drift tube temperature of 40◦C.
Two types of detectors were used. Glycoside and
its aglycone were detected at an optimal wavelength
with a DAD detector (200–400 nm), while glycones
were detected using ELSD.
The method of saccharide units and aglycone parts
detection after glycoside hydrolysis was developed on
a model system with flavonoid glycosides formed from
two different aglycones with differently bonded gly-
cones. While rutin is a 3-O-glycoside, hesperetin is a
7-O-glycoside (see formulas in Materials and meth-
ods). These standards represent the most typical O-
glycosides of natural origin; other derivatives (e.g., 6-
C-glycosides) are quite rare. There are also several
examples of glycones in positions 3ꢀ,4ꢀ (genkwanin,
luteolin-O-glucoside) that were not tested. However,
speaking of both acidic and enzymatic hydrolysis,
there is no structural reason why acidic (non-specific)
hydrolysis would not occur. It is known that only the
3-O-position is resistant to enzymatic activity due to
presence of a keto-group in the adjacent position 4
(rutin). On the other hand, non-specific hydrolysis
cannot determine the glycosidation position.
For quantitative calculation of the acidic hydrolysis
of hesperidin, calibration of glucose and rhamnose was
performed. Due to the use of ELSD, the calibration
curve was non-linear, so the procedure of one point
calibration was employed (Pazourek, 2010).
Sample preparation (plant material isolation)
for the identification of an unknown glycoside
Plant material of Polygonum lapathifolium (Polyg-
onaceae sp.) was purchased from Mikeš-léčivé rostliny
(Czech Republic) and the identity was verified by Dr.
P. Babula from the Department of Natural Drugs,
Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences, Brno (Czech Republic). The
plant material (1.5 kg) was extracted with methanol
(1 g of the plant material in 2 × 10 mL of methanol,
ultrasonic bath, laboratory temperature, 2 × 30 min)
and evaporated. The amount of 321.18 g of the ex-
tract was obtained. Chlorophyll was removed with
petrol ether (Penta-Švec, Czech Republic). The ex-
tract was suspended in water and extracted by im-
miscible solvents (3 × 350 mL). Chloroform (10 g),
ethyl acetate (80 g), and butanol (25 g) extracts
were obtained. From the ethyl acetate extract, sev-
eral fractions were isolated by preparative HPLC and
identified as quercitrin, isoquercitrin, kaempferol-3-O-
glucoside, and an unknown compound which was ex-
amined by the reported method.
Acidic hydrolysis
First, hydrochloric acid was tested as a hydrolytic
agent. A blank chromatogram of the model glyco-
side treated with HCl exhibited a retained peak co-
eluting with analytes, which can be probably ascribed
to some hydrolytic products from the degradation of
the DIOL stationary phase. Therefore, in the follow-
ing experiments, hydrochloric acid was avoided and
replaced with TFA. TFA passes through the column
with a low retention rate (in the void volume), so
it does not co-elute with the glycone units. Stability
tests showed that under the hydrolysis conditions with
TFA, monosaccharides (glucose, rhamnose, galactose),
and aglycones (quercetin, hesperetin) eluted without
degradation.
According to the experimental results of rutin hy-
drolysis, the method for acidic hydrolysis of flavonoid
glycosides was established as follows: rutin was dis-
solved in methanol/water (ϕr = 1 : 1) and mixed with
TFA (4 M TFA in acetonitrile) (ϕr = 1 : 1). Then, the
mixture was heated to 90◦C for 60 min and analyzed
with the analytical method HPLC-DAD-ELSD.
A typical chromatogram of rutin after acidic hy-
drolysis is shown in Fig. 1 (chromatogram a). The
Results and discussion
Although stationary phases with aminopropyl
groups are recommended for carbohydrates analy-
sis, a higher sensitivity of ELSD used as the de-

V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
361
Fig. 3. Yield of monosaccharide units from the load of hes-
Fig. 1. Identification of products of acidic hydrolysis of rutin;
chromatogram of rutin after hydrolysis with TFA (a),
chromatograms of standards: glucose (b), rhamnose (c),
and rutin (d); 1 – rhamnose, 2 – glucose.
peridin under the conditions of acidic (TFA) hydrol-
ysis;
– rhamnose (slope 0.862), ꢀ – glucose (slope
0.840). Data for the linear regression graph were cal-
culated from chromatograms using the one point cal-
ibration procedure (Pazourek, 2010). Symbols (ꢀ and
) represent average values; corresponding error bars
were plotted as half lines only for clarity and they rep-
resent standard deviations of the experimental values
calculated from three replicates.
about 2 correspond to a standard of glucose (Fig. 1,
chromatogram b). The chromatograms for the glucose
standard and the hydrolyzed glucoside from rutin dif-
fer in the two peaks intensity ratio (ϕr = 1 : 1 instead
of ϕr = 1 : 2), which can be explained by the extremely
low pH during the hydrolysis affecting the monosac-
charide anomeric equilibrium.
Fig. 3 shows the dependence of the number of
monosaccharide moles in the hydrolytic product on
the loaded number of hesperidin moles. Because of
the simple stoichiometry, a unit slope of the graph
represents the 100 % yield, the data show that under
the experimental conditions applied (60 min, 90◦C,
TFA), the acidic hydrolysis proceeds with the yield of
approximately 85 % (86.2 % for rhamnose and 84.0 %
for glucose). A t-test for the equality of the graph
slopes with the theoretical value of 1 was performed.
It showed a significant difference (α = 0.05) confirm-
ing that the hydrolysis was not a quantitative pro-
cess. On the other hand, similar values of both the
slopes (0.862 and 0.840) implies that determination of
a molar ratio of the saccharide units in the hydrolytic
products can provide information on the number of
the bonded saccharide units. It should also be noted
that under certain conditions (lower concentration of
TFA, shorter reaction time), a disaccharide unit (i.e.,
rutinose, rhamnosyl-glucose) was observed among the
reaction products, which suggests that acidic hydro-
lysis can also lead to disaccharide cleavage.
Fig. 2. Comparison of quercetin standard spectra (dashed line)
with the spectrum of hydrolyzed rutin taken from chro-
matogram a in Fig. 1 at 1.59 min; aglycone (full line).
broad peak with the retention time of about 1 min rep-
resents low-absorbing products (TFA) and the agly-
cone part (the least polar compounds) identified by
a characteristic UV-VIS spectrum from a correspond-
ing diode-array signal (Fig. 2). A complete identifica-
tion of aglycones from glycosides by the retention time
was not possible in the presented method because the
column (DIOL), though very selective to glycones, is
very polar resulting in very low aglycones retention.
Also, the UV-VIS spectrum can be affected by other
retained compounds from the hydrolytic products.
Rutin itself is eluted as a single peak with a time
comparable to that of glucose but longer than that
of rhamnose. Monosaccharide units were identified by
their retention times and also by the ELSD-signal
characteristic profile (Fig. 1, chromatogram a): two
peaks in the chromatogram with the resolution of
Enzymatic hydrolysis
Both the type and position of the saccharide unit

362
V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
Table 2. Results of enzymatic hydrolysis of hesperidinase, glucosidase, and galactosidase on model glycosides
Enzymea
Enzyme substrate
Hesperidinase
Glucosidase
Galactosidase
Hesperidin
Hesperidin-glucoside
Rutin
Quercetin-rhamnoside
Quercetin-glucoside
Quercetin-galactoside
Neohesperidin
Hesperetin-glucoside
N/A
Quercetin-glucoside
Not affected
N/A
Not affected
Hesperitin
Not affected
N/A
Not affected
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Not affected
N/A
Neohesperetin-glucoside
Not affected
a) Product obtained after hydrolysis; N/A – not tested.
Fig. 4. Chromatograms of a stepwise hydrolysis; hesperidin
(reaction mixture without an enzyme) (a), products of
the first enzymatic hydrolysis of hesperidin with hes-
peridinase (b), enzymatic hydrolysis of b (hesperetin
glucoside and rhamnose) with glucosidase (c). Reaction
conditions were set up according to the enzymes used;
1 – hesperitin-glucoside, 2 – hesperidin, 3 – rhamnose,
4 – glucose.
Fig. 5. Analysis of an unknown glycoside; chromatogram of the
sample after hydrolysis with TFA (a), chromatogram of
the sample before the hydrolysis (b), chromatograms of
glucose (c) and galactose (d) standards; 1 – galactose.
information not only on the type of the sugar part
but also on its position on the aglycone (terminal po-
sition). Fig. 4 illustrates this approach to the detection
of terminal rhamnose and glucose, respectively, by a
step-wise hydrolysis as recorded in Table 2 (the first
two lines). The chromatogram a in Fig. 4 shows the
substrate of hesperidin without an enzyme addition,
chromatogram b shows the products of enzymatic hy-
drolysis of hesperidin with hesperidinase, namely the
presence of rhamnose and hesperetin-glucoside. Chro-
matogram c shows the products after another addi-
tion of glucosidase to the previous hydrolytic prod-
ucts. Therefore, the presence of glucose cleaved from
hesperetin glucoside can be observed.
play an important role in enzymatic hydrolysis. En-
zymes always cleave the terminal saccharide unit. Hes-
peridinase, galactosidase, and glucosidase cleave ter-
minal rhamnose, galactose, and glucose, respectively.
However, position 3-O- in flavonoid glycoside is quite
resistant to enzymatic hydrolysis and the enzymes
used do not cleave 3-O-galactosides under the tested
conditions (Brito-Arias, 2007). Hesperidinase can be
used in the hydrolysis of hesperidin and rutin, but it
is expected not to affect quercetin-3-O-glucoside. This
was confirmed by experiments with enzymes as sum-
marized in Table 2.
Consequently, no substrate reaction (absence of en-
zymatic cleavage) suggests a bond in the oligosaccha-
rides, e.g. a 3-O-glycoside. As in Fig. 1, acidic hydrol-
ysis enabled separation and detection of all parts of
glycosides (aglycone and monosaccharides) after the
complete hydrolysis. In contrast, enzymatic hydrolysis
is substrate and stereo-specific. Therefore, it provides
Application of the method to an unknown gly-
coside
The method was applied to an unknown glyco-
side isolated from a herb, Polygonum lapathifolium
(Polygonaceae sp.) (Fig. 5). An LC-MS analysis per-
formed previously identified the unknown analyte as

V. Špačková, J. Pazourek/Chemical Papers 67 (4) 357–364 (2013)
363
kaempferol monosaccharide glycoside (according to a
fit of the MS-pattern of a kaempferol standard). After
the hydrolysis with TFA, the UV-VIS spectrum of an
unretained peak (chromatogram a, retention time of
1.69 min) was compared to a standard one and the
aglycone part was confirmed to be kaempferol. The
acid hydrolysis proved the presence of galactose, as it
is obvious from the comparison of the retention times
and the resolution profiles with standards of galactose
and glucose in Fig. 5 (chromatograms c and d). Also
enzymatic hydrolysis was carried out using galactosi-
dase. The compound was not hydrolyzed, so it was
deduced that kaempferol was substituted in position
3 (kaempferol-3-O-galactoside).
Systematic bottom-up approach for flavonoid derivative
screening in plant material using liquid chromatography
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Conclusions
Selectivity achieved in the HILIC mode on a DIOL
column was sufficient to distinguish monosaccharide
isomers cleaved from natural glycosides. A serial con-
nection of DAD and ELSD detectors after an HPLC
column facilitated simultaneous monitoring of agly-
cones and carbohydrate parts of the glycoside. Under
the optimized conditions it was possible to hydrolyze,
separate and identify an unknown glycoside (galactose
and kaempferol were found in the sample after acidic
hydrolysis with TFA). Moreover, because the glyco-
side was not enzymatically hydrolyzed in additional
experiments, it could be deduced that galactose was
bond in position 3 (kaempferol-3-O-galactoside).
This work documents the potential of HPLC hy-
phenation with two detectors (HPLC-DAD-ELSD) re-
sulting in a method for the glycone part identification
and characterization of the aglycone part of glycosides
after hydrolysis. The most important feature of the
method is the identification of carbohydrates which
enables differentiation of saccharide isomers which is
not possible with the most popular methods, e.g. LC-
MS.
Acidic hydrolysis with TFA is fast and cheaper
than the enzymatic one, though enzymatic hydroly-
sis can also provide information on the sequence of
bonded saccharides and partially also on the position
of the glycosidic bond. In other words, enzymatic hy-
drolysis is a very specific method which depends both
on the type of the saccharide and also on its position.
This fact can also be used for glycosides identification;
however, the specificity of the enzymes can be limiting
in the hydrolysis of unknown glycosides.
Acknowledgements. Dr. R. Kubínová is acknowledged for
the generous supply of plant extracts of Polygonum lapathi-
folium. This work was supported by IGA VFU, project No.
10/2010/FaF and by the Grant Agency of the Czech Repub-
lic, project P503/12/0337.
Kadumi, K., Yamazaki, K., Kitahara, K. I., Aikawa, Y., Arai,
S., & Masuda-Hanada, T. (2011). Synthesis of new di-cation
type stationary phases for high performance anion-exchange
chromatographic separation of carbohydrates. Bunseki Ka-
gaku, 60, 959–964. DOI: 10.2116/bunsekikagaku.60.959.
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