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HPLC analysis of flavonoids

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HPLC Analysis of Flavonoids
H
Marina Stefova
Trajče Stafilov
Svetlana Kulevanova
Sts. Cyril and Methodius University, Skopje, Republic of Macedonia

INTRODUCTION flavonoids using various detection systems.[2–4] As for


the quantitative analysis, much data have been published
Flavonoids are widely spread plant secondary metabolites in the last few years confirming the suitability of this
called C6 – C3 –C6 phenolics, which are classified in three technique for simultaneous determination of flavonoid
groups, depending on the nature of the C3 fragment and compounds in various samples, which gives an insight
the type of the heterocyclic ring, as follows: 1) chromone into the distribution of flavonoids in the studied material.
derivatives (flavones, flavonols, flavanones, and flavano- High-performance liquid chromatography methods are
nols); 2) chromane derivatives (catechines and antocya- developed for qualitative and quantitative analyses of
nidines); and 3) flavonoids with open propane chain flavonoids in fruits and beverages, wine, honey, propolis,
(chalcones) and with a furane ring (aurones). From all of and, especially, in various plant materials[5–14] using dif-
these, flavones, flavonols, and flavanones are the most ferent detection systems, from which UV diode array
abundant in the plant kingdom and their skeleton is given detectors are settled as the most suitable for these com-
in Fig. 1. Substitution in the positions 3, 5, 6, 7, 8, 2’, pounds and the most accessible as well.
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 EXPERIMENTAL
are recognized today as free flavones, flavonols, and
flavanones, i.e., aglycones, and as flavonoid glycosides, Stationary Phases
which consist of flavonoid, nonsugar component agly-
cone, connected to the sugar moiety (mostly monosac- High-performance liquid chromatography is the method
charides and disaccharides). Bonding to sugars makes of choice for the separation of complex mixtures con-
flavonoids soluble in water and enables their easy trans- taining nonvolatile compounds such as various flavonoids
port within plants. in extracts prepared from different samples. A survey of
Flavonoids are a well-defined group of compounds literatures revealed that most researchers have used C18-
with established physical and chemical characteristics. reversed stationary phases, which proved to be superior to
This especially counts for their absorption of ultraviolet the normal phase technique. The reversed phases are
(UV) radiation, which makes their UV spectra very suitable for separating flavonoids in a wide range of
characteristic and UV spectroscopy a method of choice polarities, as Vande Casteele et al.[15] have demonstrated
for their characterization.[1] Two main absorption bands the separation of 141 flavonoids from polar triglycosides
are observed: 1) band I (300 –380 nm) due to absorption to relatively nonpolar polymetoxylated aglycones belong-
of ring B; and 2) band II (240 – 280 nm) due to absorption ing to the classes of flavones, flavonols, flavanones,
of ring A. The position of these bands gives information dihydroflavonols, chalcones, and dihydrochalcones.
about the kind of the flavonoid and its substitution The use of normal phase silica columns was also
pattern; thus UV spectroscopy is used as a main method described but after acetylation of the flavonoids and then
for the identification and the quantification of flavonoids isocratic elution on silica gel.[16] Polystyrene divinylben-
for decades. zene as a stationary phase was also found to give satis-
There are several published information regarding the factory separation and good peak shapes without using
isolation and the identification of flavonoids in plant acidic mobile phases,[17] which are not so favorable for
material using different methods, mainly chromatographic use with reversed phases.
and spectroscopic. Today, high-performance liquid chro- The choice of a column involves matching the class
matography (HPLC) is established as the most convenient of flavonoids to be separated to the characteristics of the
method which enables separation and identification of stationary phase capable of providing satisfactory

Encyclopedia of Chromatography 1
DOI: 10.1081/E-ECHR 120016287
Copyright D 2003 by Marcel Dekker, Inc. All rights reserved.
2 HPLC Analysis of Flavonoids

Fig. 1 Structure of flavone, flavonol, and flavanone.

retention, selectivity, and peak shapes. As for the Detection Systems


dimensions, the most popular are 150 and 250 mm
long, with 5 mm particle size, although phases with 3 mm Identification of flavonoids separated by HPLC is com-
particles are becoming more popular because of better monly performed by comparing the obtained reten-
efficiency and less solvent consumption. The use of tion times with the ones of authentic samples as well
guard columns is recommended in order to extend as by analysis of their characteristics collected by
their lifetime by protecting the analytical column from the detector.
impurities in the samples prepared from various natu- Detection of flavonoids separated by HPLC can be
ral products. performed using several detection systems. Photodiode
array detectors (DADs) are the most convenient for use
Mobile Phases because of their availability and easy maintenance, and
mainly because of the valuable information for the iden-
The preferred solvent system used for the separation of tification of flavonoids contained in their UV [or
flavonoids on reversed-phase stationary phases is meth- ultraviolet –visible (UV – VIS)] spectra. The sophisticated
anol – water, followed closely by acetonitrile – water. software packages enable storage of all the spectra ob-
Usually, acetic or formic acid is added (sometimes tained during the elution process, which can later be
phosphoric acid, potassium dihydrogen phosphate, ammo- analyzed and compared to the spectra from a library
nium dihydrogen phosphate, and perchloric acid), which previously prepared from authentic samples of flavonoids.
enables improved separation and prevention of peak An experienced analyst working on flavonoids can
tailing with respect to the phenolic character of the recognize the peaks from flavonoids in the chromatogram
flavonoids. Ion pairing has also been used for improving from the spectra, which are, as previously mentioned,
the separation of neutral glycosides from flavonol very characteristic, although they are not enough for
sulfates.[4] identification. As for the quantitative analysis, scanning
Often, gradient elution with a linear gradient, com- over a wide wavelength range enables measuring all the
bined with isocratic steps, usually gives the best sepa- components at wavelengths of their absorption maxima,
ration of flavonoids, differing in the degree of saturation, which provides maximum sensitivity.
hydroxylation, methylation, and/or glycosylation. Optim- Fluorescence detectors have also been employed for
ization of the elution program is usually performed by flavonoid determination, offering higher sensitivity and
changing the elution program until a satisfactory resolu- selectivity, as suggested by the results in the analysis of
tion and analysis time is achieved, depending on the 3’,4’,5’-trimethoxyflavone, which has been determined by
analysis purpose, i.e., qualitative or quantitative analysis. excitation at 330 nm and by detection at 440 nm.[18]
HPLC Analysis of Flavonoids 3

Overcoming the difficulty in introducing the liquid respectively, as compared to UV absorbance detection at
sample into the mass spectrometer in the last decade 377 nm (absorption maximum of flavonols).
enabled their use for HPLC detection, even in routine
practical applications. The atmospheric pressure chemical
A significant amount of literature regarding the anti-
oxidant properties of flavonoids and other plant poly-
H
ionization, thermospray, and electrospray ionization sys- phenols is available. As the essence of redox chemistry
tems have proved to be most convenient for flavonoid involves electron transfer, it seems natural that electro-
analysis by HPLC –MS.[18,19] Mass spectrometry is a chemical detection rivals spectrophotometric detection
more specific and extremely selective detection tech- techniques for the compounds that are supposed to be
nique, although it is not enough for structure elucidation antioxidants. With the improvements in electrochemical
(it reveals the structural fragments that are present, but not detector geometries and electronics over the last decade,
the exact substitution pattern). It offers the molecular coupled with a requirement for increased sensitivity,
weight of the molecular ion, which helps the tentative the use of electrochemical detectors offers significant
identification of the flavonoid. The use of HPLC –MS – additional advantages when combined with the traditio-
MS gives additional information about the characteristic nal UV – VIS detection in the analysis of flavonoids and
fragmentation pattern—‘‘fingerprint’’ of the substance, other plant polyphenols.[20]
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 APPLICATIONS
is presented in the work of Huck et al.,[19] who have
isolated and characterized polymethoxylated flavones Several information regarding HPLC analysis of flavo-
from Primulae veris flos using HPLC – ESI – MS. The noids, often together with other phenolic constituents, in
six detected flavonoid compounds were found to be various samples, such as fruits, vegetables, juices, wines,
mono-, di-, tri-, and pentametoxyflavones, but their exact honey, propolis, and, especially, plant material, are pub-
substitution pattern could not be revealed, except for lished. The enormous interest in studying these com-
3’,4’,5’-trimetoxyflavone, for which 13C NMR spectral pounds is a result of their potential importance to health
data were available. and antioxidant defense mechanisms, which has imposed
As regards the sensitivity of the MS detection in HPLC the need for developing methods for their identification
analysis of flavonoids, this technique has proved to be the and quantification in various natural products. Reversed-
most sensitive as compared to UV and fluorescence phase HPLC (RP HPLC) with combined isocratic and
detection. A very comprehensive comparison of the four gradient elution with acidic mobile phases and UV diode
detection systems—UV, fluorescence, and two MS sys- array or mass spectrometer detector is the most often used
tems [atmospheric pressure chemical ionization (APCI) system for flavonoid analysis. Depending on the nature of
and electrospray ionization (ESI)]—for the determination the sample, a variety of sample preparation techniques has
of the previously identified 3’,4’,5’-trimethoxyflavone[18] been developed in order to achieve good recovery of the
is presented in Table 1. Fluorescence detection is 10 analyzed compounds in a simple sample preparation pro-
times more sensitive than UV detection, whereas MS cedure. The procedure includes extraction of the flavo-
detection is 50 times more sensitive than UV detection noids in a polar solvent; this extract is then used either for
and 5 times more sensitive than fluorescence detection. injection onto the column (after filtration) or for further
Similar assay of the detection systems is performed by fractionation and purification of the flavonoids to obtain a
Stecher et al.,[9] who analyzed flavonols and stilbenes in relatively ‘‘clean’’ sample for injection. The latter step
wine and biological products and found that ESI –MS can be carried out in several ways: by liquid – liquid
detection gave two, three, and nine times lower limit of extraction in suitable solvents; by chromatographic tech-
detection (LOD) for myricetin, quercetin, and kaempferol, niques (thin layer or column chromatography) on various

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 Regression coefficient (R2) Detection limit

UV at 213 nm y = 0.990x + 13.158 0.9986 0.244 ng


Fluorescence at 330/440 nm y = 0.879x + 12.926 0.9946 24.4 pg
ESI – MS y = 0.692x + 17.349 0.9956 5.00 pg
APCI – MS y = 0.899x + 15.798 0.9997 5.00 pg
(From Ref. [18].)
4 HPLC Analysis of Flavonoids

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

representing the best results. Also, an exhaustive des- Flavonoids of Honey and Propolis
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
A very interesting application of flavonoid analysis by
HPLC has been carried out by Barberán et al.[11] on honey
H
with the discussion of the effects of the variation of samples. They used RP HPLC – DAD for the analysis of
the methanol (acetonitrile) content in the mobile phase, 20 flavonoid aglycones, which could be considered as
the degree of mobile phase acidity, together with the markers for the floral origin of honey. Different solvent
influence of the structural characteristics of the studied systems were applied to the analysis of flavonoids from
flavonoids on their behavior in the reversed-phase citrus and rosemary honeys. The sample preparation
chromatographic system. included dilution with water and HCl (pH = 2 –3) for
Another thorough procedure for flavonoid analysis in hydrolysis of glycosides, followed by purification using
Citrus samples is presented in the work of Nogata et al.[8] chromatography on Amberlite XAD-2 and Sephadex LH-
They developed a method for separation of 9 flavanones, 20. The flavonoid markers of the botanical origin,
10 flavones, and 6 flavonols using a RP HPLC system hesperetin and apigenin, were detected using methanol –
with gradient elution with 0.01 M phosphoric acid – water and acetonitrile – water mixtures with formic acid.
methanol and for detection at 285 nm, which is presented Quercetin and kaempferol were separated using Prizma-
in Fig. 2. Identification was carried out by comparing the optimized conditions, whereas tectochrysin was eluted
retention data and the spectra of the sample components with methanol – water and acetonitrile – water mixtures by
with the ones of the standards. For quantitative analysis, increasing the content of the organic solvent at the end of
all flavonoids exhibited good linearity (r = 0.988 – 1.00) the elution programs. The use of diode array detection
between concentration and peak area in the investigated was found essential in studies of the floral origin of honey
concentration range (10 – 200 ppm) with detection limits by flavonoid analysis.
from 0.5 to 2.5 ppm. Flavonoid analysis has also been carried out in
To investigate the probable health benefits of flavo- propolis, another beehive product rich in flavonoids,
noids and stilbenes in red wine, a RP HPLC method with which are partly responsible for its pharmacological
enhanced separation efficiency, selectivity, sensitivity, activity.[12] Qualitative and quantitative analyses were
and speed has been established for the determination of the performed by using a reversed-phase column and isocratic
flavonols quercetin, myricetin, and kaempferol and the elution with water –methanol – acetic acid (60:75:5) and
stilbenes cis- and trans-resveratrol in a single run.[9] The by monitoring at 275 and 320 nm. The propolis sample
sample preparation step for wines and grape juices in- was cut into small pieces, extracted with boiling me-
cluded only filtration prior to injection. Identification was thanol, and the methanolic extract was then diluted with
carried out by the comparison of the retention data, the water and, subsequently, extracted with light petroleum
UV, and the mass spectra of the flavonoids and the stil- and diethyl ether. The last extract contained the propolis
benes with the ones obtained for standards. Quantitative flavonoids pinocembrin (21.4%), galangin (5%), chrysin
analysis using external standards showed good linearity (4.8%), quercetin (2.2%), and tectochrysin (1.1%).
(R2 > 0.999 for UV and R2 > 0.9878 for MS detection),
recoveries between 95% and 105%, and limits of detection Plant Material
in the nanogram range, with MS being more sensitive.
An interesting improvement in the sample preparation Flavonoids play important roles in plant biochemistry and
step has been suggested[10] using only filtering of the physiology; they are responsible for the biological effects
sample prior to injection into the system composed of two of plants and their extracts as well as preparations on
columns: clean-up column (50  2.1 mm packed with C18 humans. High-performance liquid chromatography is
stationary phase, 5 mm) and analytical column (250  2.1 found very suitable for the detection and the determina-
mm, C18, 5 mm). A column-switching procedure enables tion of flavonoids present in various plants and plant
the sample introduced in the first column to be cleaned products; the so-called HPLC fingerprint analysis is
from the disturbing matrix compounds for 2 min (elution suggested for quality control and standardization because
phosphate buffer, pH = 7, with 10% acetonitrile), and of the ability of good separation and resolution of complex
then by a gradient of the mobile phase (phosphate buffer, mixtures as well as peak purity control. One of the limit-
pH = 2.5, with acetonitrile); the retained analytes— ations of the assays of flavonoids in plant material is the
flavonoids—are eluted to the analytical column, where fact that many compounds, especially glycosides, are not
they are separated and detected at 365 nm. The method commercially available. One of the possible solutions to
was used for the determination of flavonoid profiles of this problem is the hydrolysis of flavonoid glycosides
berry wines containing the flavonols quercetin, myricetin, during the extraction procedure, which aids the identifica-
kaempferol, rutin, and isoquercitrin. tion on flavonoid aglycones. Such a procedure (the
6 HPLC Analysis of Flavonoids

extraction in acetone with the addition of HCl for hy- performed for characterization and standardization of the
drolysis of glycosides) is proposed for the screening of plant material as well as its extracts and preparations.
flavonols and the determination of quercetin in medici-
nal plants using RP HPLC with gradient elution with Structure – RP HPLC Retention
water – acetonitrile – acetic acid and UV diode array detec- Relationships of Flavonoids
tion.[13] Quercetin was found to be the most abundant,
especially in Hyperici herba and Pruni spinosae flos, The ability to predict the chromatographic mobility of a
kaempferol in Robiniae pseudoacaciae flos and P. spi- compound under given conditions, based on its structure,
nosae flos, whereas myricetin was detected only in Betulae offers many advantages in analysis. The elution sequence
folium, as can be seen in the chromatograms presented of individual flavonoids can be interpreted by assuming
in Fig. 3. The content of quercetin ranged from 0.026% that the compounds are first adsorbed on the reversed
in Bursae pastoris herba to 0.552% in Hyperici herba. stationary phase by ‘‘hydrophobic interaction,’’ and then
Another quantitative RP HPLC method, based on the subsequently eluted with the mobile phase according to
reduction of the complex flavonoid glycoside pattern by the extent of hydrogen bond formation. Therefore the
acid hydrolysis to one major aglycone (quercetin) and one hydrogen bond donating and/or accepting ability of a
C-glycoside (vitexin), was developed and employed for given substituent as well as its contribution to the
the characterization of Crataegus leaves and flowers.[14] hydrophobic interaction have to be considered. The
A qualitative fingerprint method was also developed for retention data of 141 flavonoids[15] imply the balance
the separation and the identification of all characteris- between these two effects, resulting in almost identical
tic flavonoids (glycosides and aglycones). Samples for retention of tricetin pentamethylether (pentamethoxy
fingerprint analysis were prepared by extraction with 80% flavone) and unsubstituted flavone.
methanol and then filtered through Bond Elut C18 Hydroxylation in positions other than 3 and 5 decreases
cartridge prior to injection. Elution was performed by a retention owing to increasing polarity (hydrogen bond
mobile phase composed of tetrahydrofuran –acetonitrile – formation ability). The presence of an OH group in
methanol and 0.5% orthophosphoric acid and was mo- positions 3 and 5 is specific because of the formation of
nitored at 370, 336, and 260 nm. For quantitative analysis, an intramolecular hydrogen bond with the carbonyl group
extraction was carried out with methanol in a Soxhlet on C-4, which is the strongest hydrogen bond acceptor in
apparatus, HCl was added to the methanolic extracts for flavones and isoflavones. This is the reason for the
the hydrolysis of glycosides, and, finally, they were increase in retention, especially when OH is substituted in
filtered through Bond Elut C18 before injection. Se- position 5, whereas another OH group in position C-3
paration and quantification of vitexin and quercetin were 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
Fig. 3 Chromatograms obtained for extracts of the following:
a) H. herba; b) R. pseudoacaciae flos; c) P. spinosae flos; d)
is only 0.37 min smaller than that of the corresponding
Sambuci flos; e) B. folium; and f) Primula flos, and for mixture 7-b-d-glucopyranoside can also be explained by the
of authentic samples of myricetin (M), quercetin (Q), and ka- shielding effect of sugar on the carbonyl group. The
empferol (K) using: C18 (250  4.6 mm, 5 mm, Varian); gradient contributions of various types of sugars to the hydro-
elution with 5% acetic acid – acetonitrile with flow rate of philic interaction decrease from hexoses through pen-
1.0 mL/min at 30°C and detection at 367 nm. (From Ref. [13].) toses to methylpentoses.
HPLC Analysis of Flavonoids 7

Saturation of the C3 ring, which means transformation juice by high-performance liquid chromatography. J.
of flavones to flavanones and of flavonols to dihydro- Chromatogr. A 2001, 913, 387 – 395.
flavonols, affects the retention in a very complex way.
The saturation itself has a small effect; however, in the
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:
presence of OH groups, the retention is always decreased.
Influence of the structural characteristics. J. Liq. Chroma-
This is explained by the interruption of the conjugation
togr. 1994, 17 (7), 1497 – 1523.
in the system, affecting the acidity and therefore the 8. Nogata, Y.; Ohta, H.; Yoza, K.I.; Berhow, M.; Hasegawa,
hydrogen bond accepting and donating abilities of the OH S. High-performance liquid chromatographic determina-
groups, especially the 3-OH groups which are phenolic in tion of naturally occurring flavonoids in Citrus with a
flavones and alcoholic in flavanones. 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
CONCLUSION
related biological product by HPLC and HPLC – ESI –
MS – MS. Fresenius’ J. Anal. Chem. 2001, 371, 73 – 80.
Reversed-phase high-performance liquid chromatography 10. Ollanketo, M.; Riekkola, M.L. Column-switching tech-
has proved to be the method of choice for the separation nique for selective determination of flavonoids in Finnish
of a variety of flavonoids in different samples. The berry wines by high-performance liquid chromatography
phenolic nature of these compounds requires the use of with diode array detection. J. Liq. Chromatogr. Relat.
acidic mobile phases for satisfactory separation and peak Technol. 2000, 23 (9), 1339 – 1351.
shapes, whereas the detection is usually carried out with 11. Barberán, F.A.T.; Ferreres, F.; Blázquez, M.A.; Garcı́a-
photodiode array detectors which are also very helpful Viguera, C.; Tomás-Lorente, F. High-performance liquid
for their identification of the characteristic absorption chromatography of honey flavonoids. J. Chromatogr. 1993,
spectra of the flavonoids. In the last decade, mass spec- 634, 41 – 46.
12. Bankova, V.S.; Popov, S.S.; Marekov, N.L. High-perform-
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ance liquid chromatographic analysis of flavonoids from
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developing more sophisticated instruments as well as flavonols and quantification of quercetin in medicinal
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16. Galensa, R.; Herrmann, K. Analysis of flavonoids by high-
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