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HPLC analysis of flavonoids

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HPLC Analysis of Flavonoids
Marina Stefova
Trajče Stafilov
Svetlana Kulevanova
Sts. Cyril and Methodius University, Skopje, Republic of Macedonia
INTRODUCTION
Flavonoids are widely spread plant secondary metabolites
called C6 – C3 –C6 phenolics, which are classified in three
groups, depending on the nature of the C3 fragment and
the type of the heterocyclic ring, as follows: 1) chromone
derivatives (flavones, flavonols, flavanones, and flavano-
nols); 2) chromane derivatives (catechines and antocya-
nidines); and 3) flavonoids with open propane chain
(chalcones) and with a furane ring (aurones). From all of
these, flavones, flavonols, and flavanones are the most
abundant in the plant kingdom and their skeleton is given
in Fig. 1. Substitution in the positions 3, 5, 6, 7, 8, 2’,
3’, 4’, 5’, and 6’ gives all the compounds from these
groups, with hydroxylation, methoxylation, and glycosy-
lation being the most common substitution. Thousands
of various flavonoids with various substitution patterns
are recognized today as free flavones, flavonols, and
flavanones, i.e., aglycones, and as flavonoid glycosides,
which consist of flavonoid, nonsugar component agly-
cone, connected to the sugar moiety (mostly monosac-
charides and disaccharides). Bonding to sugars makes
flavonoids soluble in water and enables their easy trans-
port within plants.
Flavonoids are a well-defined group of compounds
with established physical and chemical characteristics.
This especially counts for their absorption of ultraviolet
(UV) radiation, which makes their UV spectra very
characteristic and UV spectroscopy a method of choice
for their characterization.[1] Two main absorption bands
are observed: 1) band I (300 –380 nm) due to absorption
of ring B; and 2) band II (240 – 280 nm) due to absorption
of ring A. The position of these bands gives information
about the kind of the flavonoid and its substitution
pattern; thus UV spectroscopy is used as a main method
for the identification and the quantification of flavonoids
for decades.
There are several published information regarding the
isolation and the identification of flavonoids in plant
material using different methods, mainly chromatographic
and spectroscopic. Today, high-performance liquid chro-
matography (HPLC) is established as the most convenient
method which enables separation and identification of
Encyclopedia of Chromatography
DOI: 10.1081/E-ECHR 120016287
Copyright D 2003 by Marcel Dekker, Inc. All rights reserved.
H
flavonoids using various detection systems.[2–4] As for
the quantitative analysis, much data have been published
in the last few years confirming the suitability of this
technique for simultaneous determination of flavonoid
compounds in various samples, which gives an insight
into the distribution of flavonoids in the studied material.
High-performance liquid chromatography methods are
developed for qualitative and quantitative analyses of
flavonoids in fruits and beverages, wine, honey, propolis,
and, especially, in various plant materials[5–14] using dif-
ferent detection systems, from which UV diode array
detectors are settled as the most suitable for these com-
pounds and the most accessible as well.
EXPERIMENTAL
Stationary Phases
High-performance liquid chromatography is the method
of choice for the separation of complex mixtures con-
taining nonvolatile compounds such as various flavonoids
in extracts prepared from different samples. A survey of
literatures revealed that most researchers have used C18-
reversed stationary phases, which proved to be superior to
the normal phase technique. The reversed phases are
suitable for separating flavonoids in a wide range of
polarities, as Vande Casteele et al.[15] have demonstrated
the separation of 141 flavonoids from polar triglycosides
to relatively nonpolar polymetoxylated aglycones belong-
ing to the classes of flavones, flavonols, flavanones,
dihydroflavonols, chalcones, and dihydrochalcones.
The use of normal phase silica columns was also
described but after acetylation of the flavonoids and then
isocratic elution on silica gel.[16] Polystyrene divinylben-
zene as a stationary phase was also found to give satis-
factory separation and good peak shapes without using
acidic mobile phases,[17] which are not so favorable for
use with reversed phases.
The choice of a column involves matching the class
of flavonoids to be separated to the characteristics of the
stationary phase capable of providing satisfactory
1
2 HPLC Analysis of Flavonoids
Fig. 1 Structure of flavone, flavonol, and flavanone.
retention, selectivity, and peak shapes. As for the
dimensions, the most popular are 150 and 250 mm
long, with 5 mm particle size, although phases with 3 mm
particles are becoming more popular because of better
efficiency and less solvent consumption. The use of
guard columns is recommended in order to extend
their lifetime by protecting the analytical column from
impurities in the samples prepared from various natu-
ral products.
Mobile Phases
The preferred solvent system used for the separation of
flavonoids on reversed-phase stationary phases is meth-
anol – water, followed closely by acetonitrile – water.
Usually, acetic or formic acid is added (sometimes
phosphoric acid, potassium dihydrogen phosphate, ammo-
nium dihydrogen phosphate, and perchloric acid), which
enables improved separation and prevention of peak
tailing with respect to the phenolic character of the
flavonoids. Ion pairing has also been used for improving
the separation of neutral glycosides from flavonol
sulfates.[4]
Often, gradient elution with a linear gradient, com-
bined with isocratic steps, usually gives the best sepa-
ration of flavonoids, differing in the degree of saturation,
hydroxylation, methylation, and/or glycosylation. Optim-
ization of the elution program is usually performed by
changing the elution program until a satisfactory resolu-
tion and analysis time is achieved, depending on the
analysis purpose, i.e., qualitative or quantitative analysis.
Detection Systems
Identification of flavonoids separated by HPLC is com-
monly performed by comparing the obtained reten-
tion times with the ones of authentic samples as well
as by analysis of their characteristics collected by
the detector.
Detection of flavonoids separated by HPLC can be
performed using several detection systems. Photodiode
array detectors (DADs) are the most convenient for use
because of their availability and easy maintenance, and
mainly because of the valuable information for the iden-
tification of flavonoids contained in their UV [or
ultraviolet –visible (UV – VIS)] spectra. The sophisticated
software packages enable storage of all the spectra ob-
tained during the elution process, which can later be
analyzed and compared to the spectra from a library
previously prepared from authentic samples of flavonoids.
An experienced analyst working on flavonoids can
recognize the peaks from flavonoids in the chromatogram
from the spectra, which are, as previously mentioned,
very characteristic, although they are not enough for
identification. As for the quantitative analysis, scanning
over a wide wavelength range enables measuring all the
components at wavelengths of their absorption maxima,
which provides maximum sensitivity.
Fluorescence detectors have also been employed for
flavonoid determination, offering higher sensitivity and
selectivity, as suggested by the results in the analysis of
3’,4’,5’-trimethoxyflavone, which has been determined by
excitation at 330 nm and by detection at 440 nm.[18]
HPLC Analysis of Flavonoids
Overcoming the difficulty in introducing the liquid
sample into the mass spectrometer in the last decade
enabled their use for HPLC detection, even in routine
practical applications. The atmospheric pressure chemical
ionization, thermospray, and electrospray ionization sys-
tems have proved to be most convenient for flavonoid
analysis by HPLC –MS.[18,19] Mass spectrometry is a
more specific and extremely selective detection tech-
nique, although it is not enough for structure elucidation
(it reveals the structural fragments that are present, but not
the exact substitution pattern). It offers the molecular
weight of the molecular ion, which helps the tentative
identification of the flavonoid. The use of HPLC –MS –
MS gives additional information about the characteristic
fragmentation pattern—‘‘fingerprint’’ of the substance,
which is very useful for qualitative and quantitative
analyses in complex matrices. An excellent illustration of
the use of HPLC –MS for the identification of flavonoids
is presented in the work of Huck et al.,[19] who have
isolated and characterized polymethoxylated flavones
from Primulae veris flos using HPLC – ESI – MS. The
six detected flavonoid compounds were found to be
mono-, di-, tri-, and pentametoxyflavones, but their exact
substitution pattern could not be revealed, except for
3’,4’,5’-trimetoxyflavone, for which 13C NMR spectral
data were available.
As regards the sensitivity of the MS detection in HPLC
analysis of flavonoids, this technique has proved to be the
most sensitive as compared to UV and fluorescence
detection. A very comprehensive comparison of the four
detection systems—UV, fluorescence, and two MS sys-
tems [atmospheric pressure chemical ionization (APCI)
and electrospray ionization (ESI)]—for the determination
of the previously identified 3’,4’,5’-trimethoxyflavone[18]
is presented in Table 1. Fluorescence detection is 10
times more sensitive than UV detection, whereas MS
detection is 50 times more sensitive than UV detection
and 5 times more sensitive than fluorescence detection.
Similar assay of the detection systems is performed by
Stecher et al.,[9] who analyzed flavonols and stilbenes in
wine and biological products and found that ESI –MS
detection gave two, three, and nine times lower limit of
detection (LOD) for myricetin, quercetin, and kaempferol,
Table 1 Regression equations for the calibration curves (logarithmic), regression coefficients, and detection limits for the
determination of 3’,4’,5’-trimethoxyflavone
Detection method Regression equation
UV at 213 nm y = 0.990x + 13.158
Fluorescence at 330/440 nm y = 0.879x + 12.926
ESI – MS y = 0.692x + 17.349
APCI – MS y = 0.899x + 15.798
(From Ref. [18].)
3
respectively, as compared to UV absorbance detection at
377 nm (absorption maximum of flavonols).
A significant amount of literature regarding the anti-
oxidant properties of flavonoids and other plant poly-
H
phenols is available. As the essence of redox chemistry
involves electron transfer, it seems natural that electro-
chemical detection rivals spectrophotometric detection
techniques for the compounds that are supposed to be
antioxidants. With the improvements in electrochemical
detector geometries and electronics over the last decade,
coupled with a requirement for increased sensitivity,
the use of electrochemical detectors offers significant
additional advantages when combined with the traditio-
nal UV – VIS detection in the analysis of flavonoids and
other plant polyphenols.[20]
APPLICATIONS
Several information regarding HPLC analysis of flavo-
noids, often together with other phenolic constituents, in
various samples, such as fruits, vegetables, juices, wines,
honey, propolis, and, especially, plant material, are pub-
lished. The enormous interest in studying these com-
pounds is a result of their potential importance to health
and antioxidant defense mechanisms, which has imposed
the need for developing methods for their identification
and quantification in various natural products. Reversed-
phase HPLC (RP HPLC) with combined isocratic and
gradient elution with acidic mobile phases and UV diode
array or mass spectrometer detector is the most often used
system for flavonoid analysis. Depending on the nature of
the sample, a variety of sample preparation techniques has
been developed in order to achieve good recovery of the
analyzed compounds in a simple sample preparation pro-
cedure. The procedure includes extraction of the flavo-
noids in a polar solvent; this extract is then used either for
injection onto the column (after filtration) or for further
fractionation and purification of the flavonoids to obtain a
relatively ‘‘clean’’ sample for injection. The latter step
can be carried out in several ways: by liquid – liquid
extraction in suitable solvents; by chromatographic tech-
niques (thin layer or column chromatography) on various
Regression coefficient (R2) Detection limit
0.9986 0.244 ng
0.9946 24.4 pg
0.9956 5.00 pg
0.9997 5.00 pg
4 HPLC Analysis of Flavonoids

stationary phases (silica, reversed phases, Amberlite, and
Sephadex); or by solid-phase extraction techniques using
different adsorbents, which in the last decade has been
found very convenient for the isolation of flavonoids from
complex matrices.
Flavonoids in Fruits, Juices, and Wine
During the past decades, extensive analytical research has
been performed on the separation of phenolic constituents
in various fresh fruits and fruit products. A very thorough
examination was performed by Barberán et al.[11] on
samples of several types of fresh nectarines, peaches, and
plums using RP HPLC –DAD – ESI –MS and combined
isocratic and gradient elution with a mobile phase of
water – methanol with 5% formic acid. The samples were
prepared by extraction of a homogenized frozen fruit
material with water –methanol (2:8), by centrifugation, by
filtration, and by injection into the column. This pro-
cedure recovers 85 –92% of all phenolic compounds in-
cluding hydroxycinnamates, procyanidins, flavonols, and
anthocyanins. In the chromatograms obtained for necta-
rin and peach samples, three peaks from flavonols
(quercetin 3-glucoside, quercetin 3-rutinoside, and, prob-
ably, quercetin 3-galactoside), several peaks from flavan-
3-ols (catechin, epicatechin, and dimer procyanindines),
and two anthocyanin pigments (cyanidine 3-glucoside and
cyanidine 3-rutinoside) have been identified by compar-
ing the retention data, the UV, and the mass spectra of the
detected flavonoids with the ones obtained for authentic
markers. Quantification was done using external stand-
ards: quercetin 3-rutinoside for flavonols at 340 nm,
cyanidine 3-rutinoside for anthocyanins at 510 nm, and
catechin for flavan-3-ols at 280 nm.
An HPLC – DAD method was developed for the se-
paration and the determination of flavonoid and phe-
nolic antioxidants in commercial and freshly prepared
cranberry juice.[6] Two sample preparation procedures
were used: with and without hydrolysis of the glycoside
forms of flavonoids carried out by the addition of HCl
in the step prior to solid-phase extraction (SPE). The
flavonoid and phenolic compounds were then fractio-
nated into neutral and acidic groups via a solid-phase ex-
traction method (Sep-Pak C18), followed by a RP HPLC
separation with gradient elution with water – methanol –
acetic acid and a detection at 280 and 360 nm. A com-
parison of the chromatograms obtained for extracts pre-
pared with and without hydrolysis showed that flavonoids
and phenolic acids exist predominantly in combined forms
such as glycosides and esters. In a freshly squeezed cran-
berry juice, for instance, 400 mg of total flavonoids and
phenolics per liter of sample was found, 56% of which
were flavonoids. Quercetin was the main flavonoid in
the hydrolyzed products, where it accounted for about
75% of the total flavonoids, while it was absent in the
unhydrolyzed products.
The flavonoid constituents of Citrus have attracted
attention in the last decade because of their biological
activities (anticarcinogenic, anti-inflammatory effects,
etc.) together with the chemotaxonomic importance of
the specific flavanone glycosides found in this species.
The presence of 12 flavonoids (9 flavanone glycosides,
2 flavanone aglycones, and 1 flavone glycoside) was de-
tected in samples from leaves and fruits of Citrus au-
rantium.[7] The use of different extraction solvents was
studied (methanol, dioxane – methanol 1:1, 0.1% NaOH
aq., 0.01% KOH methanolic, pyridin, dimethylfor-
mamide, and dimethylsulfoxide), with dimethylsulfoxide

Fig. 2 Separation of 25 flavonoid standards (1—eriocitrin; 2—neoeriocitrin; 3—robinetin; 4—narirutin; 5—naringin; 6—rutin; 7—
hesperidin; 8—neohesperidin; 9—isorhoifolin; 10—rhoifolin; 11—diosmin; 12—neodiosmin; 13—neoponcirin; 14—quercetin; 15—
poncirin; 16—luteolin; 17—kaempferol; 18—apigenin; 19—isorhamnetin; 20—diosmetin; 21—rhamnetin; 22—isosakuranetin; 23—
sinensetin; 24—acacetin; 25—tangeretin) using C18 Lichrospher 100, 250  4.0 mm, 5 mm, Merck; gradient elution with 0.01 M
phosphoric acid – methanol with flow rate of 0.6 mL/min at 40°C and detection at 285 nm. (From Ref. [8].)
HPLC Analysis of Flavonoids
representing the best results. Also, an exhaustive des-
cription of the optimization process by studying the quan-
titative chromatographic parameters—k’, w, a, N, height
equivalent to a theoretical plate (HETP), and R—is given
with the discussion of the effects of the variation of
the methanol (acetonitrile) content in the mobile phase,
the degree of mobile phase acidity, together with the
influence of the structural characteristics of the studied
flavonoids on their behavior in the reversed-phase
chromatographic system.
Another thorough procedure for flavonoid analysis in
Citrus samples is presented in the work of Nogata et al.[8]
They developed a method for separation of 9 flavanones,
10 flavones, and 6 flavonols using a RP HPLC system
with gradient elution with 0.01 M phosphoric acid –
methanol and for detection at 285 nm, which is presented
in Fig. 2. Identification was carried out by comparing the
retention data and the spectra of the sample components
with the ones of the standards. For quantitative analysis,
all flavonoids exhibited good linearity (r = 0.988 – 1.00)
between concentration and peak area in the investigated
concentration range (10 – 200 ppm) with detection limits
from 0.5 to 2.5 ppm.
To investigate the probable health benefits of flavo-
noids and stilbenes in red wine, a RP HPLC method with
enhanced separation efficiency, selectivity, sensitivity,
and speed has been established for the determination of the
flavonols quercetin, myricetin, and kaempferol and the
stilbenes cis- and trans-resveratrol in a single run.[9] The
sample preparation step for wines and grape juices in-
cluded only filtration prior to injection. Identification was
carried out by the comparison of the retention data, the
UV, and the mass spectra of the flavonoids and the stil-
benes with the ones obtained for standards. Quantitative
analysis using external standards showed good linearity
(R2 > 0.999 for UV and R2 > 0.9878 for MS detection),
recoveries between 95% and 105%, and limits of detection
in the nanogram range, with MS being more sensitive.
An interesting improvement in the sample preparation
step has been suggested[10] using only filtering of the
sample prior to injection into the system composed of two
columns: clean-up column (50  2.1 mm packed with C18
stationary phase, 5 mm) and analytical column (250  2.1
mm, C18, 5 mm). A column-switching procedure enables
the sample introduced in the first column to be cleaned
from the disturbing matrix compounds for 2 min (elution
phosphate buffer, pH = 7, with 10% acetonitrile), and
then by a gradient of the mobile phase (phosphate buffer,
pH = 2.5, with acetonitrile); the retained analytes—
flavonoids—are eluted to the analytical column, where
they are separated and detected at 365 nm. The method
was used for the determination of flavonoid profiles of
berry wines containing the flavonols quercetin, myricetin,
kaempferol, rutin, and isoquercitrin.
5
Flavonoids of Honey and Propolis
A very interesting application of flavonoid analysis by
HPLC has been carried out by Barberán et al.[11] on honey
H
samples. They used RP HPLC – DAD for the analysis of
20 flavonoid aglycones, which could be considered as
markers for the floral origin of honey. Different solvent
systems were applied to the analysis of flavonoids from
citrus and rosemary honeys. The sample preparation
included dilution with water and HCl (pH = 2 –3) for
hydrolysis of glycosides, followed by purification using
chromatography on Amberlite XAD-2 and Sephadex LH-
20. The flavonoid markers of the botanical origin,
hesperetin and apigenin, were detected using methanol –
water and acetonitrile – water mixtures with formic acid.
Quercetin and kaempferol were separated using Prizma-
optimized conditions, whereas tectochrysin was eluted
with methanol – water and acetonitrile – water mixtures by
increasing the content of the organic solvent at the end of
the elution programs. The use of diode array detection
was found essential in studies of the floral origin of honey
by flavonoid analysis.
Flavonoid analysis has also been carried out in
propolis, another beehive product rich in flavonoids,
which are partly responsible for its pharmacological
activity.[12] Qualitative and quantitative analyses were
performed by using a reversed-phase column and isocratic
elution with water –methanol – acetic acid (60:75:5) and
by monitoring at 275 and 320 nm. The propolis sample
was cut into small pieces, extracted with boiling me-
thanol, and the methanolic extract was then diluted with
water and, subsequently, extracted with light petroleum
and diethyl ether. The last extract contained the propolis
flavonoids pinocembrin (21.4%), galangin (5%), chrysin
(4.8%), quercetin (2.2%), and tectochrysin (1.1%).
Plant Material
Flavonoids play important roles in plant biochemistry and
physiology; they are responsible for the biological effects
of plants and their extracts as well as preparations on
humans. High-performance liquid chromatography is
found very suitable for the detection and the determina-
tion of flavonoids present in various plants and plant
products; the so-called HPLC fingerprint analysis is
suggested for quality control and standardization because
of the ability of good separation and resolution of complex
mixtures as well as peak purity control. One of the limit-
ations of the assays of flavonoids in plant material is the
fact that many compounds, especially glycosides, are not
commercially available. One of the possible solutions to
this problem is the hydrolysis of flavonoid glycosides
during the extraction procedure, which aids the identifica-
tion on flavonoid aglycones. Such a procedure (the
6 HPLC Analysis of Flavonoids
extraction in acetone with the addition of HCl for hy-
drolysis of glycosides) is proposed for the screening of
flavonols and the determination of quercetin in medici-
nal plants using RP HPLC with gradient elution with
water – acetonitrile – acetic acid and UV diode array detec-
tion.[13] Quercetin was found to be the most abundant,
especially in Hyperici herba and Pruni spinosae flos,
kaempferol in Robiniae pseudoacaciae flos and P. spi-
nosae flos, whereas myricetin was detected only in Betulae
folium, as can be seen in the chromatograms presented
in Fig. 3. The content of quercetin ranged from 0.026%
in Bursae pastoris herba to 0.552% in Hyperici herba.
Another quantitative RP HPLC method, based on the
reduction of the complex flavonoid glycoside pattern by
acid hydrolysis to one major aglycone (quercetin) and one
C-glycoside (vitexin), was developed and employed for
the characterization of Crataegus leaves and flowers.[14]
A qualitative fingerprint method was also developed for
the separation and the identification of all characteris-
tic flavonoids (glycosides and aglycones). Samples for
fingerprint analysis were prepared by extraction with 80%
methanol and then filtered through Bond Elut C18
cartridge prior to injection. Elution was performed by a
mobile phase composed of tetrahydrofuran –acetonitrile –
methanol and 0.5% orthophosphoric acid and was mo-
nitored at 370, 336, and 260 nm. For quantitative analysis,
extraction was carried out with methanol in a Soxhlet
apparatus, HCl was added to the methanolic extracts for
the hydrolysis of glycosides, and, finally, they were
filtered through Bond Elut C18 before injection. Se-
paration and quantification of vitexin and quercetin were
Fig. 3 Chromatograms obtained for extracts of the following:
a) H. herba; b) R. pseudoacaciae flos; c) P. spinosae flos; d)
Sambuci flos; e) B. folium; and f) Primula flos, and for mixture
of authentic samples of myricetin (M), quercetin (Q), and ka-
empferol (K) using: C18 (250  4.6 mm, 5 mm, Varian); gradient
elution with 5% acetic acid – acetonitrile with flow rate of
1.0 mL/min at 30°C and detection at 367 nm. (From Ref. [13].)
performed for characterization and standardization of the
plant material as well as its extracts and preparations.
Structure – RP HPLC Retention
Relationships of Flavonoids
The ability to predict the chromatographic mobility of a
compound under given conditions, based on its structure,
offers many advantages in analysis. The elution sequence
of individual flavonoids can be interpreted by assuming
that the compounds are first adsorbed on the reversed
stationary phase by ‘‘hydrophobic interaction,’’ and then
subsequently eluted with the mobile phase according to
the extent of hydrogen bond formation. Therefore the
hydrogen bond donating and/or accepting ability of a
given substituent as well as its contribution to the
hydrophobic interaction have to be considered. The
retention data of 141 flavonoids[15] imply the balance
between these two effects, resulting in almost identical
retention of tricetin pentamethylether (pentamethoxy
flavone) and unsubstituted flavone.
Hydroxylation in positions other than 3 and 5 decreases
retention owing to increasing polarity (hydrogen bond
formation ability). The presence of an OH group in
positions 3 and 5 is specific because of the formation of
an intramolecular hydrogen bond with the carbonyl group
on C-4, which is the strongest hydrogen bond acceptor in
flavones and isoflavones. This is the reason for the
increase in retention, especially when OH is substituted in
position 5, whereas another OH group in position C-3
only slightly lowers the retention. This produces poor
separation of the so-called critical pairs flavone –flavonol
differing only in the OH group in position 3.
Methylation of the OH groups more or less prevents
their effect, which means that flavonoids and their partial
methyl ethers are easily separated. On the other hand, the
introduction of additional methoxy groups has little or no
effect on retention—introducing another type of critical
pair differing only in one methoxy group.
Glycosylation of an OH group means introducing a
hydrophilic moiety together with shielding (by hydrogen
bonding or just by steric hindrance) some hydrophilic
substituents already present in the vicinity. The shielding
effect plays a role when an OH group located ortho to
another OH group is glycosylated (e.g., glycosylation of
7- or 4’-OH without an adjacent ortho-OH decreases the
tR values by 4.26– 3.83 min, whereas, in the presence of
an ortho-OH, the decrease is only 2.28 –1.55 min[15]). The
fact that the tR value of luteolin-5-b-d-glucopyranoside
is only 0.37 min smaller than that of the corresponding
7-b-d-glucopyranoside can also be explained by the
shielding effect of sugar on the carbonyl group. The
contributions of various types of sugars to the hydro-
philic interaction decrease from hexoses through pen-
toses to methylpentoses.
 HPLC Analysis of Flavonoids
Saturation of the C3 ring, which means transformation
of flavones to flavanones and of flavonols to dihydro-
flavonols, affects the retention in a very complex way.
The saturation itself has a small effect; however, in the
presence of OH groups, the retention is always decreased.
This is explained by the interruption of the conjugation
in the system, affecting the acidity and therefore the
hydrogen bond accepting and donating abilities of the OH
groups, especially the 3-OH groups which are phenolic in
flavones and alcoholic in flavanones.
CONCLUSION
Reversed-phase high-performance liquid chromatography
has proved to be the method of choice for the separation
of a variety of flavonoids in different samples. The
phenolic nature of these compounds requires the use of
acidic mobile phases for satisfactory separation and peak
shapes, whereas the detection is usually carried out with
photodiode array detectors which are also very helpful
for their identification of the characteristic absorption
spectra of the flavonoids. In the last decade, mass spec-
trometers connected to HPLC systems introduced a
greater selectivity and sensitivity in flavonoid analysis.
Improving the characteristics of the stationary phases and
developing more sophisticated instruments as well as
devices for more efficient and faster sample preparation
are the challenges for all modern analysts. Discovering
the beneficial health effects of flavonoids and the ‘‘going-
back-to-nature’’ trend motivates the development of more
efficient and fast procedures for their identification and
quantification, with HPLC remaining the most powerful
technique for their separation from the complex mixtures.
REFERENCES
1. Mabry, T.J.; Markham, K.R.; Thomas, M.B. The System-
atic Identification of Flavonoids; Springer-Verlag: New
York, 1970.
2. Harborne, J.B. Phytochemical Methods (A Guide to
Modern Techniques of Plant Analysis); Chapman and
Hall: London, 1984.
3. Wollenweber, E.; Jay, M. Flavones and Flavonols. In The
Flavonoids; Harborn, J.B., Ed.; Chapman and Hall:
London, 1988.
4. Daigle, D.J.; Conkerton, E.J. Analysis of flavonoids by
HPLC: An update. J. Liq. Chromatogr. 1988, 11 (2), 309 –
325.
5. Barberán, F.A.T.; Gil, M.I.; Cremin, P.; Waterhouse, A.L.;
Hess-Pierce, B.; Kader, A.A. HPLC – DAD – ESIMS ana-
lysis of phenolic compounds in nectarines, peaches, and
plums. J. Agric. Food Chem. 2001, 49, 4748 – 4760.
6. Chen, H.; Zuo, Y.; Deng, Y. Separation and determination
of flavonoids and other phenolic compounds in cranberry
View publication stats
7
juice by high-performance liquid chromatography. J.
Chromatogr. A 2001, 913, 387 – 395.
7. Castillo, J.; Benavente-Garcı́a, O.; Del Rio, J.A. Study and
optimization of Citrus flavanone and flavones elucidation
H
by reverse phase HPLC with several mobile phases:
Influence of the structural characteristics. J. Liq. Chroma-
togr. 1994, 17 (7), 1497 – 1523.
8. Nogata, Y.; Ohta, H.; Yoza, K.I.; Berhow, M.; Hasegawa,
S. High-performance liquid chromatographic determina-
tion of naturally occurring flavonoids in Citrus with a
photodiode-array detector. J. Chromatogr. A 1994, 667,
59 – 66.
9. Stecher, G.; Huch, C.W.; Popp, M.; Bonn, G.K. Deter-
mination of flavonoids and stilbenes in red wine and
related biological product by HPLC and HPLC – ESI –
MS – MS. Fresenius’ J. Anal. Chem. 2001, 371, 73 – 80.
10. Ollanketo, M.; Riekkola, M.L. Column-switching tech-
nique for selective determination of flavonoids in Finnish
berry wines by high-performance liquid chromatography
with diode array detection. J. Liq. Chromatogr. Relat.
Technol. 2000, 23 (9), 1339 – 1351.
11. Barberán, F.A.T.; Ferreres, F.; Blázquez, M.A.; Garcı́a-
Viguera, C.; Tomás-Lorente, F. High-performance liquid
chromatography of honey flavonoids. J. Chromatogr. 1993,
634, 41 – 46.
12. Bankova, V.S.; Popov, S.S.; Marekov, N.L. High-perform-
ance liquid chromatographic analysis of flavonoids from
propolis. J. Chromatogr. 1982, 242, 135 – 143.
13. Stefova, M.; Kulevanova, S.; Stafilov, T. Assay of
flavonols and quantification of quercetin in medicinal
plants by HPLC with UV-diode array detection. J. Liq.
Chromatogr. Relat. Technol. 2001, 24 (15), 2283 – 2292.
14. Rehwald, A.; Meier, B.; Sticher, O. Qualitative and
quantitative reversed-phase high-performance liquid chro-
matography of Crataegus leaves and flowers. J. Chroma-
togr. A 1994, 677, 25 – 33.
15. Vande Casteele, K.; Geiger, H.; Van Sumere, C.F. Sepa-
ration of flavonoids by reversed-phase high-performance
liquid chromatography. J. Chromatogr. 1982, 240, 81 – 94.
16. Galensa, R.; Herrmann, K. Analysis of flavonoids by high-
performance liquid chromatography. J. Chromatogr. 1980,
189, 217 – 224.
17. Jagota, N.K.; Cheathan, S.F. HPLC separation of flavo-
noids and flavonoid glycosides using a polystyrene/
divinylbenzene column. J. Liq. Chromatogr. 1992, 15
(4), 603 – 615.
18. Huck, C.W.; Bonn, G.K. Evaluation of detection methods
for the reversed-phase HPLC determination of 3’,4’,5’-
trimethoxyflavone in different phytopharmaceutical pro-
ducts and in human serum. Phytochem. Anal. 2001, 12,
104 – 109.
19. Huck, C.W.; Huber, C.G.; Ongania, K.H.; Bonn, G.K.
Isolation and characterization of methoxylated flavones
in the flowers of Primula veris by liquid chromatogra-
phy and mass spectrometry. J. Chromatogr. A 2000, 870,
453 – 462.
20. Milbury, P.E. Analysis of complex mixtures of flavonoids
and polyphenols by high-performance liquid chromato-
graphy electrochemical detection methods. Methods Enzy-
mol. 2001, 335, 15 – 26.