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Flavonoids by HPLC 69S. W. Annie Bligh, Olumuyiwa Ogegbo, and Zheng-Tao Wang
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2108
2 Structure and Physicochemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2108
3 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2111
4 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2112
4.1 Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2112
4.2 Mobile Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2115
5 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2118
5.1 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2119
5.2 Combination of Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2123
6 Two-Dimensional (2D, LC � LC) HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2124
7 Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2127
8 Selected Examples of Flavonoids Analysis by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2129
9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2129
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2137
Abstract
Flavonoids are secondary plant metabolites that are synthesized via the
shikimate pathway. HPLC has been an important tool for the separation of
these metabolites in the last 4 decades. The coupling of HPLC with a number
of detection technologies either online, in tandem, or off-line enables the
S.W.A. Bligh (*) • O. Ogegbo
Institute for Health Research and Policy, London Metropolitan University, London, UK
e-mail: [email protected]
Z.-T. Wang
The Ministry of Education (MOE) Key Laboratory for Standardization of Chinese Medicines,
Institute of Traditional Chinese Materia Medica, Shanghai University of Traditional Chinese
Medicine, Shanghai, China
e-mail: [email protected]
K.G. Ramawat, J.M. Merillon (eds.), Natural Products,DOI 10.1007/978-3-642-22144-6_97, # Springer-Verlag Berlin Heidelberg 2013
2107
identification of flavonoids in plant, food, and biological samples. This chapter
provides an overview of flavonoid analysis by HPLC, including extraction
of flavonoids in both aglycones and glycosides, separation by a selection of
stationary and mobile phases, and finally, detection and identification by
UV–VIS, fluorescence, electrochemical, mass spectrometry, and NMR
spectroscopy.
Keywords
Flavonoids • Foods • HPLC • Medicinal plants • Quantitation
1 Introduction
Flavonoids are polyphenols and function as secondary metabolites in plants.
Their biological importance in plants, animals, and microorganisms stems from
their diversity in chemical substitution of the C6–C3–C6 framework (Fig. 69.1),
giving over 10,000 known compounds [1, 2]. The rapid increase of new flavonoids
reported in the last decade is partly due to the intense research on rationalizing of
the molecular contribution of health benefits in traditional herbal medicine and food
and on using flavonoid molecular entities in chemosystematics of plants.
The improved bioassay-guided separation technology and the advance in the
development of the HPLC system, especially the detection modality, have also
contributed to the growth of identification of new flavonoids. A number of reviews
have been published on analytical methods for flavonoids and polyphenols in plant,
food, and biological samples [3–9].
In this chapter, we focus on the analysis of flavonoids strictly based on the
C6–C3–C6 framework (i.e., excluding the isoflavonoids). The chemical diversity,
size, three-dimensional shape, and physical properties of flavonoids are reviewed in
recognition of their importance in determining the extraction and separation strat-
egy. Various detectors coupled to the HPLC with different stationary and mobile
phases are discussed for the feasibility of aiding the full identification of flavonoids.
In addition, the challenges of using two-dimensional HPLC in the screening of
flavonoids for their bioactivity in herbal medicine or food are highlighted.
2 Structure and Physicochemical Properties
Flavonoids are characterized by a C6–C3–C6 framework and typically with
a phenylbenzopyran chemical structure. The heterocyclic benzopyran ring is
known as the C ring. An aromatic ring (A ring) fused with the heterocyclic
benzopyran ring (C ring) and linked to a phenyl moiety (B ring) as shown
in Fig. 69.1. The A and B rings can be hydroxylated, and the hydroxyls can be
O- and C-glycosylated, methylated, acetylated, pyrenylated, or sulphated. Sugar
units can be D-glucose, L-rhamnose, D-galactose, L-arabinose, D-xylose, D-allose,
D-apose, D-mannose, D-glactouronic acids, D-glucuronic acids, di- or trisaccharides.
2108 S.W.A. Bligh et al.
The center heterocycle is either pyran, pyrilium, or g-pyrone. The main subclasses
of flavonoids are flavone, flavonol, flavanonol, flavanone, anthocyanidin, and
chalcone (Fig. 69.1). The latter subclass does not have a heterocycle B ring
structure. Both flavanonol and flavanone have a chiral center at C2 position and
they are stereoisomers.
The solubility of a flavonoid is a crucial factor in controlling its interaction with
the mobile phase in HPLC. Therefore, the properties of these flavonoids such as
hydrophobicity, dipole moment, hydrogen bonding, ionization, and steric effects
are important to take into account when choosing a mobile phase for an effective
separation. Flavonoids without sugar units attached tend to have low solubility in
water and are pH dependent. The solubility of quercetin, isoquercitrin, rutin,
chrysin, naringenin, and hesperetin was quantified in three different organic
solvents (acetonitrile, acetone, and tert-amyl alcohol), and the data did not give
a clear correlation between the solubility of flavonoids and their thermodynamic
properties [10].
The number and the position of hydroxyl groups attached even in the same class
of flavonoids can influence the lipophilicity of the flavonoids. Octanol–water
partition coefficient (log P) values were reported for flavonoids from the flavone,
flavonol, and flavanone [11], showing that aglycones are more lipophilic than
any glycosylated or sulfated conjugates. However, there is not a trend of retention
time of the flavonoid with respect to the log P (Table 69.1). Despite having
the same number and position of hydroxyl substituents in luteolin (flavone)
and eriodictyol (flavanone), they have different log P values but similar retention
times.
The acidity of hydroxyl groups in flavonoids has been studied by theoretical
calculations [12]. Interestingly the 40-OH on the B ring and the 7-OH on the A ring
C6-C3-C6 Flavone Flavonol Chalcone
Flavanone
O
O
O
OH
OHOO
A C
5
8
7
6
2
36′
2′
3′4′
5′B
O O
O
OH
O O+ O
Flavanonol Anthocyanidin Catechin
Fig. 69.1 Skeleton of a C6–C3–C6 defining the flavonoid class and structures of the main
flavonoid subclasses
69 Flavonoids by HPLC 2109
Table 69.1 Log P values and HPLC retention times [11]
Flavonoid Log P � SD Retention time (min)
Luteolin 3.22 � 0.08 20.08
Kaempferol 3.11 � 0.54 23.72
Apigenin 2.92 � 0.06 22.90
Naringenin 2.60 � 0.03 22.30
Eriodictyol 2.27 � 0.02 20.13
Quercetin 1.82 � 0.31 20.50
Quercetin-3-glucoside 0.76 � 0.01 12.21
Quercetin-7-sulfate 0.74 � 0.02 14.72
Quercetin-3-rhamnoglucoside �0.64 � 0.05 10.88
Quercetin-3-sulfate �1.11 � 0.01 11.23
2110 S.W.A. Bligh et al.
are identified as the most suitable deprotonation sites because of the favorable
delocalization of the electron pair. Therefore, the most acidic flavonoids are those
characterized by a high degree of p-electron delocalization, for which
deprotonation gives anionic species that can be readily stabilized by resonance
structure.
3 Extraction
The structural complexity of flavonoids has prohibited a single extraction method
for all classes of flavonoids. Sample handling strategies (pretreatment, extraction,
and clean-up) are important prior to the determination of flavonoids by HPLC.
Sample pretreatment is required in most of the flavonoid-containing matrices, such
as plant materials, food products, and biological samples. Work-up routines before
extraction processes can involve freeze-drying, homogenization, centrifugation,
and/or filtration. The pretreatment for liquid food products and biological samples
is simply centrifugation and for solid plant materials and food products is
homogenization.
Solvent extraction of flavonoids is commonly used after pretreatment.
The factors that contribute to the efficiency of solvent extraction are polarity of
solvent or solvent mixtures, pH, temperature, and particle size. The acidity
of the extraction medium can influence the degree of solubility for soluble
flavonoids and their conjugates, for example, glycones in plants and glucuronide
and sulfate conjugates in biological samples. Solvent such as aqueous,
methanol, ethanol, ethyl acetate, acetone, acetonitrile, or their mixture is
commonly used to isolate flavonoids from powdered plant materials [13].
In anthocyanin extraction, acidified aqueous methanol or ethanol is used to
denature the cell membrane and to solubilize the analyte. The use of weak organic
acids and low concentrations of strong acids was reported to prevent the
hydrolysis of anthocyanins to anthocyanidins [14–17]. Since flavonoids can
exist as various conjugated forms, sample treatment with acid [18–20] or
enzymatic [21, 22] hydrolysis is required to facilitate the identification of the
aglycones.
Flavonoids can be extracted by solvent extraction through Soxhlet extraction,
ultrasound-assisted extraction (USAE), microwave-assisted solvent extraction
(MASE), accelerated solvent extraction (ASE), or supercritical fluid
extraction (SFE) methods [23]. The latter two methods are based on using
compressed fluids as extracting agents [24]. A comparative extraction study of
flavonoids from dry cell cultures of Saussurea medusa Maxim by Soxhlet
extraction, USAE, and MASE showed that MASE is more efficient in
terms of yield and time for extraction. Another comparative study, using the
compressed fluid techniques, accelerated solvent extraction (ASE) using water,
and supercritical fluid extraction (SFE) using CO2 and 10 % EtOH as modifier
versus standard hot water or 70 % ethanol extraction of flavonoids from
Scutellaria lateriflora, was reported [25]. The use of ASE at 85 �C with water
69 Flavonoids by HPLC 2111
as solvent gave the best results for flavonoid glycosides, whereas SFE gave higher
yields of flavonoid aglycones.
Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) are usually
performed on liquid samples, such as beverages or biological fluids. These two
extraction methods are used to concentrate flavonoid analytes based on their
solubility in different solvents and their polarity. Unwanted lipids or lipophilic
materials in crude extract can be eliminated by washing it with nonpolar solvents,
such as hexane or dichloromethane.
Column chromatographic and SPE methods are used in the clean-up step before
injection to the HPLC system. The polar nonphenolic compounds such as organic
acids can be removed by SPE method using a preconditioned C18 cartridge.
Apart from the commonly used C18 cartridge, a variety of adsorbent materials
such as Amberlite, C8, and HLB have also been used successfully for extracting
flavonoid compounds from wine [26]. SPE method has been found useful
in enhancing the extraction of glabridin from licorice (from 0.23 % to 35.2 %
after SPE) [27].
4 Separation
Reversed-phase (RP) liquid chromatography is used for separation of analytes that
dissolve in mixed aqueous–organic solvents. Separation of flavonoids is therefore
commonly carried out in the reversed-phase mode, on C8- or C18-bonded silica
columns with mixed aqueous–organic mobile phase. The aqueous mobile phase is
usually acidified water using a mild organic acid such as formic or acetic acid.
The organic mobile phase is typically either methanol or acetonitrile. Normal-
phased liquid chromatography is seldom used for flavonoid analysis because the
analyte often retains on the column. However, peracetylated flavonoids can be
separated on a cyano-silica column using n-hexane-ethyl acetate mobile phase
under isocratic conditions [28].
4.1 Stationary Phases
A complete separation of naturally occurring mixtures of flavonoids poses
problems due to the wide range of polarities and the tendency for flavonoids
of similar polarity to elute in groups. C18 is normally the stationary phase
of choice. Typically columns have an internal diameter ranging from about
2–5 mm with particle sizes 3–5 mm with a length of 75–250 mm. C8 stationary
phase is also used for the separation of more polar flavonoids. A comparison of
HPLC capacity factors of 27 flavonoids have been reported using Zorbax SB
(250 � 4.6 mm) analytical columns containing C18, C8, and CN stationary
phases [29]. The results showed that the hydrophobic flavonoids (usually
aglycones) had similar capacity factors in C18 and C8 columns and were much
reduced in CN column (Table 69.2). However, for polar flavonoids, the capacity
2112 S.W.A. Bligh et al.
factors were similar in all columns. Optimum separation of flavonoids also
depends on column types of C18; for example, the separation of 51 flavonoids in
a Chinese herbal prescription of Longdan Xiegan Decoction using a Symmetry
column from Waters was better than that of Zorbax (Agilent) and LiChroCART
(Merck) [30].
The anthocyanins are most often separated by HPLC on a C18 column with long
gradients to achieve the best chromatographic resolution [31–33]. A new approach
using a HPLC column that combines both ion-exchange and reversed-phase (RP)
separation mechanisms showed significant improvement in chromatographic
Table 69.2 Capacity factors of flavonoids in chromatography columns with different stationary
phases [29]
Compound
Coefficients of
retention
C18 C8 CN
Apigenin (5,7,40-trihydroxyflavone) 34.24 39.23 22.37
Herbacetin (5,7,8,40-tetrahydroxyflavonol) 11.90 11.07 8.69
Quercetin (5,7,30,40-tetrahydroxyflavonol) 16.24 15.42 11.77
3-Methylquercetin (3-O-methyl-5,7,30,40-tetrahydroxyflavonol) 22.69 22.04 13.63
3-Methylkaempferol (3-O-methyl-5,7,40-trihydroxyflavonol) 51.38 51.14 24.33
Myricetin (5,7,30,40,50-pentahydroxyflavonol) 6.24 6.03 5.81
Patuletin (5,7,30,40-tetrahydroxy-6-methoxyflavonol) 16.78 15.02 10.76
Chrysoeriol (5,7,40-trihydroxy-30-methoxyflavone) 40.97 42.88 25.47
Mono- and diglycosides
Guaiaverin (quercetin-3-arabinoside) 3.17 3.32 3.16
Genistin (5,40-dihydroxyflavone-7-O-b-L-glucoside) 2.79 3.08 2.70
Herbacetin–8-O-glucoside 8.31 7.13 5.30
Hyperoside (quercetin-3-O-galactoside) 3.54 3.49 3.57
Quercetin-30-O-glucoside 4.49 4.37 3.95
Rutin (5,7,30,40-tetrahydroxyflavonol-3-O-b-D-rutinoside) 1.51 1.47 1.69
Linarin (5-hydroxy-40-methoxyflavone-7-O-a-L-rhamno-b-D-glucoside) 15.72 14.52 7.23
Luteolin-3-glucoside (5,7,40-trihydroxyflavonol-3-O-glucoside) 2.21 2.46 2.73
Luteolin-7-O-glucoside 2.25 2.50 2.82
3-Methylkaempferol-7-O-glucoside 7.23 7.03 4.65
Myricetin-3-O-galactoside 1.12 1.17 1.44
Myricetin-3-O-rhamnoside 1.96 2.01 2.12
Patuletin (patuletin-7-O-glucoside) 2.30 2.23 2.23
Pectolinarin (5-hydroxy-6,40-dimethoxyflavone-7-O-(6-O-a-L-rhamnopyranosyl)-b-D-glucopyranoside)
17.97 16.42 7.59
Scoparin (chrysoeriol-8-C-b-D-glucopyranoside) 2.46 2.44 2.52
Scutellarein-7-rhamnoxyloside (5,6,40-trihydroxyflavone-7-O-b-rhamnoxyloside)
2.74 3.12 2.74
Hirsutrin (quercetin-3-O-glucoside) 2.19 2.22 2.31
Eriodictyol (5,30-dihydroxy-40-methoxyflavone-7-O-glucoside) 2.24 2.24 1.85
69 Flavonoids by HPLC 2113
performance, especially for the separation of 3,5-diglucoside anthocyanins from
3-monoglucoside anthocyanins in analyzing grape anthocyanins. A total of 37
anthocyanin peaks were detected in the Concord skin extract using a Primesep
column, i.e., a mixed ion-exchange and reversed-phase mode column [34]. In
Fig. 69.2a, the separation of different anthocyanin subgroups using a Primesep
mixed mode column is achieved avoiding overlaps found with a C18 column
(Fig. 69.2b). A total of 25 compounds were clearly identified. Other column such
as monolithic or rod column has been used to separate 24 anthocyanins in a red
cabbage sample in 18 min [35]. The advantages of using monolithic columns over
312
0
0
4 5 6 7 8 9 10 11 12 13 14 15 16 17
18 19 2021 22 23 24 25 26 27 28 29 30 31 32 34 35 36 3733
10
a
b
20 30 40Retention Time (minutes)
Retention Time (minutes)
50 60 70
5
3
10 15 20 25 30 35
4 5 67 8
18
10
11
14
25
A
19
21,27 28 23,32 24,30 31 34 35 36α β γ
37
B C D E F G HI J K L
O PM
Q
R S T U V W X Y ZN
OH OH
R1 R2 Name
key to compound numbersdelphinidin (DE)OHOMeHOMeOMe
R3R3
R2
R1
HO O+
OHOH
OH
OH
O
OOH
53
HOHHHOMe
cyanidin (CY)petunidin (PT)pelargonidin (PG)peonidin (PN)malvinin (MV)
OHacetatecoumaratecaffeoate
Abbreviation
3, DE-GLC2 (GLC2: 3,5-O-diglucoside);
GLCGLC-ACGLC-COGLC-CA
4, CY-GLC2; 5, PT-GLC2;6, PN-GLC2;7, DE-GLC; 8, CY-GLC; 10, PT-GLC;11, PN-GLC; 14, MV-GLC; 18, DE-AC;19, CY-AC; 21, PT-AC; 23, PN-AC;24, MV-AC; 25, DE-GLC2-CO;27, CY-GLC2-CO; 28, PT-GLC2-CO;30, PN-GLC2-CO; 31, MV-GLC2-CO;32, DE-CO; 34, CY-CO; 35, PT-CO;36, PN-CO; 37, MV-CO
Fig. 69.2 HPLC anthocyanin profiles (520 nm) of Concord (Vitis labrusca) skin extracts
(MeOH–H2O–HCOOH ¼ 70:28:2). Magnified regions focus on acylated anthocyanins. (a)Mixed-mode column (Primesep, SIELC). (b) C18 RP column (Zorbax SB-C18, Agilent) [34]
2114 S.W.A. Bligh et al.
the conventional particulated columns are shorter run times, higher flow rates, and
faster column equilibration [36, 37].
A porous polyamide resin is shown to possess hydrogen bond acceptor properties
suitable for the separation of polyphenolic solutes such as phenolic acids, flavonols,
and flavonoids. The separation is achieved in the presence of solvent mixtures of
acetic acid and ethanol. The extent of hydrogen bond adsorption is reviewed based on
data obtained from the elution behavior of a variety of simple polyphenolic solutes.
Polyamide adsorption chromatography was applied for the purification of resveratrol
and polydatin from Polygonum cuspidatum Sieb. & Zucc [38].
The highly cross-linked 12 % agarose gel, Superose® 12 HR 10/30, possesses
hydrogen bond acceptor properties suitable for the separation of polyphenolic solutes
suchas phenolic acids, flavonols, andflavonoids.The separation is achieved isocratically
in the presence of solvent mixtures of acetic acid and ethanol. The extent of hydrogen
bondadsorption is reviewedbasedondata obtained from the elutionbehavior of a variety
of simple polyphenolic solutes including dihydroxybenzoic acids [39, 40].
Columns of HPLC with monolithic supports generally enable faster separations,
for example, a 4 mL/min elution flow could be utilized achieving an HPLC analysis
[35]. However, the high flow rate makes this type of column not suitable for mass
spectrometry detection. Alternatively, smaller dimension columns packed with
smaller particle sizes than the conventional ones achieve a faster separation while
maintaining resolution. A Zorbax SB C18 column (1.8 mm particle size) has been
used for the determination and identification of flavonoids and isoflavonoids
(genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin,
sissotrin, and biochanin A) in fmol quantities in submicroliter sample volumes by
HPLC/UV–VIS DAD separation method (which takes <1 min) [41].
Immobilized artificial membrane (IAM) stationary phase consists of
a monolayer of phospholipid covalently immobilized on an inert silica
support. The IAM stationary phase mimics the lipid environment found in cell
membranes, and it can be used for elucidating drug-membrane interactions. The
interaction of catechins, flavones, flavonols, anthocyanidins, and anthocyanins with
phosphatidylcholine was investigated by HPLC with an IAM column. The IAM
partition coefficients of the flavonoids correlated well with the amounts flavonoids
incorporation into the liposomes [42].
4.2 Mobile Phases
One of the most important parameters for well separation of flavonoids is the
composition of the eluent. Controlling the solubility of the flavonoids in the eluent
is a crucial factor for determining the combination of solvents used. In RP-HPLC,
analytes are retained on the stationary phase based on their hydrophobicity. Elution
of flavonoids in RP-HPLC is therefore in the order of decreasing polarity. Polarity
increases most by hydroxyls at the fourth position, followed by those at the second
and third positions. Loss of polar hydroxyl groups or additions of methoxy groups
reduce polarity and hence increase retention times.
69 Flavonoids by HPLC 2115
In 1974, the first application of HPLC to flavonoid analysis was
published [43], and 2 years later, 12 flavonoids were separated by RP-HPLC in
a methanol–water–acetic acid (30:65:5) mobile phase system [44]. In 1994, Nogata
et al. reported a separation of 25 naturally occurring Citrus flavonoids (flavones,flavonols, and flavanones) simultaneously with a gradient system of 0.01 M phos-
phoric acid (A) and methanol (B), in three steps: (1) 0–55 min, 70–55 % (v/v) A in
B, (2) 55–95 min, 55-0% A in B, and (3) 95–100 min, isocratic, 100 % B, measured
at 285 nm, Fig. 69.3 [45].
Both isocratic and gradient elution methods have been successful in separating
flavonoids from extracts. However, isocratic elution is less used than that of
gradient because it tends to resolve better of members of the same class of
flavonoids but works well with monolithic columns. An RP-HPLC method using
a monolithic column was developed and validated for the separation and quantifi-
cation of three flavonols, myricetin, quercetin, and kaempferol, in Rhus coriaria L.
The method employed the isocratic mobile phase acetonitrile-10 mM potassium
dihydrogen orthophosphate buffer adjusted to pH 3.0 using orthophosphoric acid at
a flow rate of 4.0 mL/min, a Chromolith Performance RP-18e (100 � 4.6 mm)
monolithic column kept at 40 �C, and UV detection at 370 nm [46]. Successful
attempts in simultaneous determination of different classes of flavonoids (querce-
tin, naringenin, naringin, myricetin, rutin, and kaempferol) using a commercially
available monolithic column and isocratic elution were also achieved [36].
Gradient elution is more often employed in recognizing the complex flavonoid
profiles of plants, food, and drinks. Rutin, quercetin-3-arabinoside, naringin,
myricetin, quercetin, apigenin, and quercetin dimethyl ether in beer samples were
separated by gradient elution using a multichannel electrochemical detection with
a CoulArray detector [47]. A step linear gradient method using amixture of methanol
and 0.1 % formic acid as a mobile phase was validated for the simultaneous determi-
nation of five flavonoids (rutin, quercitrin, quercetin, kaempferol, and isorhamnetin)
in rat plasma [48]. Another example is the analysis of rat urine, bile, and plasma after
the oral dose administration of rhubarb extract using a gradient of 0.1 % formic acid
(A) and methanol (B) starting with 5 % B at 0–10 min, 5–20 % B at 10–30 min,
20–25 % B at 30–40 min, 25–45 % B at 40–160 min, 45–60 % B at 160–180 min,
60–80 % B at 180–200 min, and 80 % at 200–220 min (Fig. 69.4) [49].
Multisolvent gradient elution conditions have been suggested to tackle materials
that are difficult to separate. Three-component solvent system, methanol–
acetonitrile–water, is commonly used in separation of natural products [50, 51].
Recently, a detailed study on the ratio of acetonitrile to methanol in a three-
component solvent system for achieving improved separation capabilities of
11 flavonoids (flavanols, biflavanol, triflavanol, and flavanones) was made [52].
Ionic strength and pH of mobile phase is known to influence the retention of
flavonoids on the column depending on if there is protonation dissociation or
a partial dissociation. An increase of pH enhances the ionization of flavonoids
and could reduce the retention in a reversed-phase separation. Thus, small amounts
of HOAc (2–5 %), H3PO4, or TFA (0.1–1 %) are normally included in the solvent to
suppress ionization of phenolic or carboxylic groups and hence improve resolution
2116 S.W.A. Bligh et al.
0 10
1
20 30 40Time (min)
50 60 70 80 90
2
3
4 5
6
78
9 1011
12
13
14
15
16
17
1819
20
2122
23 24 25
No. Name subclass OH OMe O-glycoside1 Eriocitrin flavanone 5,3�,4� – 7-O-rutinoside
2 Neoeriocitrin flavanone 5,3�,4� – 7-O-neohesperidoside
3 Robinetin flavonol 7,3�,4�,5� – –
4 Narirutin flavanone 5,4� – 7-O-rutinoside
5 Naringin flavanone 5,4� – 7-O-neohesperidoside
6 Rutin flavonol 5,7,3�4� – 3-O-rutinoside
7 Hesperidin flavanone 5,3� 4� 7-O-rutinoside
8 Neohesperidin flavanone 5,3� 4� 7-O-neohesperidoside
9 Isorhoifolin flavone 5,4� – 7-O-rutinoside
10 Rhoifolin flavone 5,4� – 7-O-neohesperidoside
11 Diosmin flavone 5,3� 4� 7-O-rutinoside
12 Neodiosmin flavone 5,3� 4� 7-O-neohesperidoside
13 Neoponcirin flavanone 5 4� 7-O-rutinoside
14 Quercetin flavonol 5,7,3�,4� – –
15 Poncirin flavanone 5 4� 7-O-neohesperidoside
16 Luteolin flavone 5,7,3�,4� – –
17 Kaempferol flavonol 5,7,4� – –
18 Apigenin flavone 5,7,4� – –
19 Isorhamnetin flavonol 5,7,4� 3� –
20 Diosmetin flavone 5,7,3� 4� –
21 Rhamnetin flavonol 5,3�,4� 7 –
22 Isosakuranetin flavanone 5,7 4� –
23 Sinensetin flavone 5,6,7,3�,4� – –
24 Acacetin flavone 5,7 4� –
25 Tangeretin flavone 5,6,7,8,4� – –
Fig. 69.3 Separation of 25 flavonoid standards. The detector monitored the eluent at 285 nm and
measured spectra from 200 to 360 nm. A two-solvent gradient system: (1) 0–55 min, 70–55 %
(v/v) A (0.01 M phosphoric acid) in B (methanol), (2) 55–95 min, 55-0 % A in B, and
(3) 95–100 min, isocratic, 100 % B [45]
69 Flavonoids by HPLC 2117
and reproducibility of each separation [53]. Both acetate and phosphate buffers
have been used as part of the mobile phase for optimizing the analysis time and
enhancing separation [29, 54].
5 Identification
Choosing an appropriate detector in anHPLC analysis of flavonoids is as crucial as the
stationary and mobile phase. The detector reports the chemical composition of the
column effluent via a recorded or digitized signal. The chemical information can be
processed differently depending on the type of detectors used. The selection of a
detector in flavonoid analysis is normally based on the chemical properties and the
sensitivity of the analytes. The two detection techniques widely used in flavonoid
analysis areUV–VIS spectrophotometry andmass spectrometry.Multiple-wavelength
detection, such as diode array detection (DAD), can be used for positive identification
mAU
a
b
c
1000
0 0 100
0
0
0
0
mAU
mAU40
1000
0
20
0
mAU
mAU
4020
0
200100
0
mAU200100
0
mAU200
100
0
mAU200
100
0min
100 min
100 min
100 min
Blank bile sample
Blank urine sample
50 100 150 200 min
50 100 150 200 min
50 100 150 200 min
50
mAU10
100 150 200 min
50 100 150 200 min
0 50 100 150 200 min
Blank plasma sample
Drug-containing plasma sample
Drug-containing urine sample
Drug-containing bile sample
50
mAU1050
Fig. 69.4 HPLC–DAD chromatograms monitored at 280 nm of rat (a) urine, (b) bile, and (c)plasma before and after administration of rhubarb decoction [49]
2118 S.W.A. Bligh et al.
by comparing the retention time and UV spectrumwith authentic standards. However,
if no reference standard is available, detections such as tandemmass spectrometry and
NMR spectroscopy have proved useful in the identification of flavonoids.
5.1 Detection
5.1.1 UV–VIS and Photodiode Array Detection (UV-DAD)UV–VIS spectrophotometry offers a routine detection and quantitation of flavo-
noids in HPLC. The two common solvents used as mobile phase in flavonoid
analysis are acetonitrile and methanol, and their UV cut-off lmax are 190 and
205 nm, respectively. They do not interfere with the two UV–VIS absorption
bands at 240–285 nm and 300–560 nm corresponding to two aromatic rings
(A and B) of the flavonoid aglycones [55]. For flavones, the substitution of OH or
OMe positions in aglycones and the type of glycosides (either C- or O- glycosides)give a slight change of the lmax of both bands [30]. The hydrolyzed anthocyanins,
anthocyanidins, show a characteristic absorbance in the visible region between 515
and 540 nm [3, 56]. On the other hand, there is little or no conjugation between the
A- and B-rings of flavanones and isoflavaones, and hence they only exhibit a low
intensity in band I which often appears as a shoulder to the peak of band II [57].
Multiple-wavelength absorbance detection offers advantages over single-
wavelength absorbance detection in flavonoid analysis of plant and food products
by HPLC. These products normally contain flavonoids of different subclasses and
variable substitutions in the same subclass. Two compounds may elute very close
together within one peak, but they may be identified by the differences in their
spectra. For example, catechins in tea infusions were identified by comparing peak
retention times and online DAD spectra of authentic standards, (�)-epigallocatechin,
(�)-epigallocatechin gallate, (�)-epicatechin, (�)-epicatechin gallate,
(�)-epigallocatechin 3-O-(3-O-methyl) gallate, and (�)-3-O-methyl epicatechin
gallate [58]. The total flavonoid content of leaves of Passiflora incarnata L.,
Passifloraceae harvested from plants cultivated or collected under different condi-
tions was evaluated by high-performance liquid chromatography with ultraviolet
detection (HPLC-UV-DAD) [59]. Figure 69.5 shows an HPLC-UV-DAD
chromatogram measured at l ¼ 337 nm of leaves and the UV-DAD spectra of
flavonoids: orientin, homoorientin, vitexin, and luteolin.
5.1.2 Electrochemical Detection (ECD)Electrochemical detectors measure chemical properties of a compound and rely on
chemical reactions in which electrons are transferred from one compound to
another. There are two types of electrochemical detectors, amperometric or coulo-
metric detectors. The latter one is commonly used because of its high surface of
contact with a structure of porous graphite working electrodes giving 100 % of the
analyte. The magnitude of the current is therefore directly proportional to the
injected compounds, and conveniently the peak areas in an HPLC chromatogram
represent the total current as a function of time.
69 Flavonoids by HPLC 2119
Coulometric detectors are particularly suited to the analysis of flavonoids since
the electroactive hydroxyl group present in rings A and B often has a low potential
of oxidation. The capabilities of electrochemical detection techniques were dem-
onstrated on 11 compounds belonging to three different classes of flavonoids:
flavanone glycosides, flavone and flavonol aglycones. Separation of all compounds
examined has been carried out under reversed-phase conditions on a C18 standard-
bore column and using a porous graphite electrode for electrochemical detection.
Instrumental precision in terms of relative standard deviation was found to be
between 0.6 % and 10 % [60]. Another example of HPLC-ECD using a microbore
column analyzing 15 flavonoids in bottled Japanese green tea samples were
reported. The flavonoids were divided into two groups according to their
hydrophobicity and were resolved by two isocratic systems: methanol–water
(1:1 and 3:7, v/v) containing 0.5 % phosphoric acid. The retention factor (k) of
each flavonoid linearly correlated with the log P values. The detection
limits (S/N ¼ 3) of the flavonoids tested were in the range of 2–25 fmol, that is,
600 times more sensitive than conventional HPLC with UV detection [61].
Multichannel electrochemical coulometric detection or coulometric array detec-
tion has been developed so that different potentials are applied on the electrodes.
A number of chromatograms (8, 12, or 16) can be recorded simultaneously.
Flavonoids can have several oxidation processes across the array of potentials,
giving characteristic profiles for identification. Methods were developed for the
Fig. 69.5 Representative HPLC-UV-DAD (l ¼ 337 nm) chromatogram of leaves of Passifloraincarnata L. and UV-DAD spectra of flavonoids peaks identified as (6) orientin, (8) homoorientin,
(12) vitexin, and (18) luteolin [59]
2120 S.W.A. Bligh et al.
analysis of flavonoids in beverages and plant extracts using gradient HPLC with
multichannel electrochemical coulometric detection. Eight-channel CoulArray
detection offers high selectivity and sensitivity with limits of detection in the low
mg L�1 range, at least an order of magnitude lower than single-channel coulometric
detection using the Coulochem detector [62]. An example is given in Fig. 69.6
showing the chromatogram of a mixture of standard phenolic and flavonoid com-
pounds, at 0.25 mg L�1 each, at the optimized HPLC separation selectivity and
CoulArray sensitivity under gradient conditions on a Purospher Star column.
5.1.3 Fluorescence Detection (FD)Fluorescence detection in conjunction with HPLC post-column treatment is com-
monly used to fulfill the requirements of sensitivity and specificity needed for the
study of flavonoids in body fluid. The number of flavonoids that exhibit native
fluorescence is limited, and derivatization of flavonoids with reagents such as Al3+
[63] and Tb3+ [64] is needed before detection. If a hydroxyl group is replaced by
a methoxy group, fluorescence becomes considerably more intense as demonstrated
by a study using luteolin flavones [65].
Another example of post-column liquid chromatographic reaction system for
the determination of flavonoids in orange juices is based on the use of the long-
wavelength fluorophore cresyl violet and cerium (IV) in a cetyltrimethylammonium
bromide micellar medium [66]. Two flavone aglycones (quercetin and kaempferol),
a flavanone aglycone (naringenin), one flavone-O-glycoside (rutin), and two
flavanone-O-glycosides (hesperidin and naringin) were used as analyte models.
The reaction process involves the interaction between the analyte, cerium(IV), and
cresyl violet giving rise to a decrease in the fluorescence, measured at lex 585,
lem 625 nm, which is proportional to the analyte concentration.
5.1.4 Mass Spectrometry DetectionImprovements in the instrumentation, ionization sources, high-resolution mass
analyzers, and detectors [67–69], in recent years have taken mass spectrometry to
a different level of HPLC-MS for natural product analysis. Mass spectrometry
detection offers excellent sensitivity and selectivity, combined with the ability to
elucidate or confirm chemical structures of flavonoids [70–72]. Both atmospheric
pressure chemical ionization (APCI) and electrospray ionization (ESI) are most
commonly used as ionization sources for flavonoid detection [73–76]. Both nega-
tive and positive ionization sources are applied. These sources do not produce many
fragments, and the subsequent collision-induced dissociation energy can be applied
to detect more fragments. Tandem mass spectrometry (MSn, n � 2) provides
information about the relationship of parent and daughter ions, which enables the
confirmation of proposed reaction pathways for fragment ions and is key to identify
types of flavonoids (e.g., flavones, flavonols, flavanones, or chalcones) [77–80].
Anthocyanins are in glycosylated forms, and their aglycones are known as
anthocyanidins. The positive charge in the tetravalent oxygen makes
anthocyanidins more suitable for MS analysis in positive mode at low voltages
[81]. MS detection of catechins and gallocatechins, which are proanthocyanidins,
69 Flavonoids by HPLC 2121
Fig.69.6
Chromatogram
ofamixture
ofphenolicandflavonoid
antioxidantstandards.Column(Purospher
STAR,RP-18e,150�
2.9
mm,5mm
),gradient
condition,0min:2%
MeC
N;20min:2%
MeC
N;50min:9%
MeC
N;65min:19%
MeC
N;90min:50%
MeC
N,pH
¼3.14,flow
rate
0.4
mLmin
�1.
(Flavonoids:7,(+)-catechin;15,(�
)-epicatechin;20,rutin;21
,quercetin-3-arabinoside;22
,naringin;23,myricetin;24,quercetin;25,apigenin;26
,quercetin
dim
ethylether;30,naringenin;31,hesperetin)[62]
2122 S.W.A. Bligh et al.
can solve the problem suffered from the interferences caused by co-eluting pheno-
lics in UV detection [82]. In the fragmentation of catechins, a loss of 152 mass unit
(168 mass unit for gallocatechins) is produced due to their retro-Diels–Alder
fission. The characteristic signals in mass spectra of catechins and gallocatechins
enable identification of their polymerization [83].
Further details on mass spectrometric analysis of flavonoids are discussed in
▶Chap. 66, “Mass Spectrometric Detection of Phenolic Acids.”
5.2 Combination of Detection
Multiple detections, such as HPLC-UV-MSn, HPLC-UV-NMR,HPLC-DAD-FD-UV,
and HPLC-DAD-FD-MS, have been adopted to identify and characterize the structure
of flavonoids and also used to evaluate the bioactivity of components [72, 84–87].
Inmost cases, single-stageMS is used in combinationwithUVdetection to facilitate the
confirmation of the identity of flavonoids in a sample with the help of standards and
reference data (an example is given in Fig. 69.7, [88]). For the identification of
unknowns, tandem mass spectrometry is used.
A recent example of using the multiple detection system elegantly by incorpo-
rating a simultaneous bioactivity screening into a three-detection system (photodi-
ode array and fluorescence detectors and an electrospray ionization tandem mass
spectrometer, DAD-FD-MS2) [87] demonstrated that 25 flavonoids could be char-
acterized and/or tentatively identified in an aqueous infusion of leaves of Ficusdeltoidea (Moraceae). The main constituents are flavan-3-ol monomers, proantho-
cyanidins, and C-linked flavone glycosides. The proanthocyanidins were dimers
and trimers comprising (epi)catechin and (epi)afzelechin units. The antioxidant
activity of F. deltoidea extract was analyzed using HPLC-DAD-FD-UVantioxidant
detection, showing 85 % of the total antioxidant activity of the aqueous F. deltoideainfusion was attributable to the flavan-3-ol monomers and the proanthocyanidins.
The data obtained from the online HPLC-ABTS antioxidant detection system are
shown in Fig. 69.8 along with absorbance traces at 280 and 365 nm. The chromato-
graphic profiles after 34 min did not exhibit antioxidant activity. The peaks
contributing the main antioxidant activity were the flavanonol monomers
gallocatechin (peak 1), catechin (peak 3), and epicatechin (peak 9), and the flavone
apigenin-6,8-C-diglucoside (peak 11).
HPLC-UV-NMR is a powerful technique for the identification and characteri-
zation of flavonoids. However, there are drawbacks, as NMR remains rather
insensitive because of the need for solvent suppression, which has restricted the
observable NMR range. Recently, two major research developments in HPLC-UV-
NMR are post-column solid-phase extraction (HPLC-UV-SPE-NMR) and combi-
nation of HPLC-UV-SPE with capillary separations and NMR detection [89].
A post-column treatment of analyte focusing and multiple trapping through a SPE
has solved the problem of sensitivity and solvent suppression. The separation and
elucidation of three C-methylated flavanones and five dihydrochalcones
from Myrica gale seeds have been achieved by HPLC-DAD-SPE-NMR and
69 Flavonoids by HPLC 2123
HPLC-DAD-MS [90]. Analysis of flavonoids in Wormwood (Artemisia absinthiumL.) and in the leaves of 12 Litsea and Neolitsea plants has been also achieved by
HPLC-DAD-SPE-NMR and HPLC-DAD-MS [91, 92].
6 Two-Dimensional (2D, LC � LC) HPLC
Online 2D LC � LC separation is achieved by a direct coupling of primary and
secondary columns through switching valves. Two approaches are used. In the first,
eluent containing peaks of interest and monitored during the first dimension of
separation is redirected to the second dimension of separation. In the second,
a comprehensive 2D setup, the whole sample is subjected to both separations.
The advantage of 2D chromatographic techniques over 1D methods is the increase
in peak capacity (resolving power) but the timescale for achieving it is compara-
tively long [93]. Separation of flavonoids in plants and foods requires comprehen-
sive 2D LC � LC approach for full separations. RP-HPLC is normally used in the
1D separation, and hence RP � RP systems, perhaps with different selectivity
stationary phases, is selected for 2D analysis.
A review on the use of different stationary phases in polyphenols,
polycarboxylic acids, and flavonoids has highlighted the differences in selectivity
of these classes of polar or possibly ionized compounds [94, 95]. Figure 69.9 shows
the contour plot and the elution conditions of the comprehensive 2D separation of
phenolic acids and flavones using parallel gradients of acetonitrile in a 5 mM
ammonium acetate buffer on a PEG microcolumn in the first dimension and
Electrochemical detector
Fluorescence detector
UV detector
nA0
−50−100−150−200−250−300
LU
1.81.71.61.51.4
25mAU
20151050
0 10 20 30 40 50 60 70 min
0 10 20 30 40 50 60 70 min
0 10 20
A
A
30 40 50 60 70 min
B CD
E
F
GH
B C
D
E F
G
D
EF G
H
Fig. 69.7 Chromatograms of the flavonoid standards; rutin (A), isoquercitrin (B), luteolin-40-glucoside (C), quercetin-40-glucoside (D), quercetin (E), naringenin (F), luteolin (G), and
apigenin (H) with electrochemical, fluorescence, and UV detectors [88]
2124 S.W.A. Bligh et al.
a short monolithic C18 column in the second [95]. In this study, flavonoids included
for separation are 19 (+)-catechin, 20 (�)-epicatechin, 21 rutin, 22 naringin, 23myricetin, 24 quercetin, 25 apigenin, 27 luteolin, 28 naringenin, 297-hydroxyflavone, 30 hesperidin, 31 morin, 32 hesperetin, and 33 flavone. There
is a clear separation of compounds 19 and 33.In addition, from the same group has developed a comprehensive 2-D LC � LC
system for the separation of phenolic and flavone antioxidants, using a PEG-silica
Fig. 69.8 Reversed-phase HPLC of an aqueous infusion of F. deltoidea leaves with absorbance
detection at 280 and 365 nm and online ABTS+ antioxidant detection at 720 nm [87]
69 Flavonoids by HPLC 2125
column in the first dimension and a C-18 column with porous-shell particles in the
second dimension and the use of electrochemical coulometric detection to com-
pensate the effects of the baseline drift observed in UV during the gradient elution
[96]. Superficially porous columns with fused core particles improve the resolution
and speed of second dimension separation in comparison to a fully porous particle
C18 column. The developed system has been applied to the analysis of flavonoids
and phenolic acids in beer samples.
A comprehensive two-dimensional HPLC system, with an RP column as
a primary column and an immobilized liposome chromatography (ILC) column
as a secondary column, was developed for the screening and analysis of
the membrane-permeable compounds in the traditional Chinese medicine
6050403020100
1.5
18
1.0
0.5
0.0
0 20 40 60
1D [min]
2D [m
in]
% a
ceto
nitr
ile
80 100 120
0 20 40 60 80time [min]
2D1D
100 120 140
8
10
34
12
13
7
9 11
20*
17
14
15
19*
21*
33
16
35
22
26
*
29* 28*25*
24*
27**32
Fig. 69.9 Contour plot and elution conditions (top) showing comprehensive LC� LC separations
of phenolic acids and flavones on a PEG column in the first dimension and on a Chromolith RP-18e
column in the second dimension with parallel gradients of acetonitrile in the two dimensions.
Compounds that are flavonoids in this figure (*) are 19 (+)-catechin, 20 (�)-epicatechin, 21 rutin,
22 naringin, 24 quercetin, 25 apigenin, 27 luteolin, 28 naringenin, 29 7-hydroxyflavone,
32 hesperetin, and 33 flavone [95]
2126 S.W.A. Bligh et al.
prescription Longdan Xiegan Decoction (LXD) [97]. More than 50 components
in LXD were resolved using the developed separation system. Eight flavonoids
and two iridoids were identified interacting with the ILC column, a system
that mimics biomembranes (Fig. 69.10). The results show that the developed
comprehensive two-dimensional chromatography system can be used for
identifying membrane permeable flavonoids in complex matrixes such as
extracts of traditional Chinese medicine prescriptions. A similar system with an
RP column and a silica-bonded human serum albumin (HSA) column was
developed for the biological fingerprinting analysis of bioactive components in
LXD [98].
7 Quantitation
Quantitative analysis of flavonoids in plant, food, and biological samples is
important because these compounds are partially responsible for the biological
activity and medical benefits in these products. Flavonoids are commonly used
as chemical markers for quality control purpose of plant and food products.
300
250
200
150
OD
S c
olum
n (s
ec)
100
50
00 50 100 150 200
ILC column (min)
1
250 300 350
2
3
4
5
6
7
8
9
10
Fig. 69.10 2D chromatogram of Longdan Xiegan Decoction. Chromatographic conditions for the
ILC column: isocratic elution with 10 mM ammonium acetate solution (pH 6.8); flow rate,
0.05 mL/min. Chromatographic conditions for the ODS column: linear gradient elution from
10 %MeCN to 70 %MeCN in 7 min, and then returning to the initial mobile phase and holding for
3 min for re-equilibration; flow rate, 2.0 mL/min; injection volume, 5 mL; detection wavelength,
210 nm. Cycle time for the second dimension is 10 min. Compounds (1–10): geniposide,
gentiopicroside, oroxylin A-7- O-glucuronide, wogonoside, 7-O-b-D-glucuronopyranosylchrysin,baicalin, ononin, liquiritin apioside, 30,40-dihydroxy-5,6-dimethoxy-7-O-glucosideflavone,liquiritin [97]
69 Flavonoids by HPLC 2127
Flavonoids can be determined quantitatively by direct (in glycoside or conjugated
form) or indirect (after hydrolysis) analysis. However, sample preparation
(e.g., particle size) and solvents used in extraction steps can significantly
affect the results [99]. Method development for quantitation is often validated
in terms of selectivity, accuracy, precision, recovery, calibration curve, and
reproducibility. Biological sample methods have to comply with the Food
and Drug Administration (FDA) guidelines for validation of bioanalytical
method [100].
With the coupling of HPLC to different sensitive detection techniques, quan-
titation of flavonoids in all types of samples has been explored whenever the
reference standards are available for calibration [101–103]. Furthermore, the
quantitation potentials of analytes (regardless of the type of compounds) rely
mainly on the sensitivity limits of the coupled detection system. To date, HPLC
coupled to a UV–VIS detector is the most popular quantitation technique used in
flavonoid analysis especially for samples with high flavonoid concentrations.
The wavelengths used to quantify anthocyanins are at the range 510–520 nm,
flavanonols at 280 nm, flavones and flavanols at 270 and 360–370 nm. However,
DAD is the other detection mode for quantitation but only slightly more sensitive
than UV, and it is still not as sensitive as MS. The detection limits of LC-UV/
DAD are usually in the region of mg/mL to the ng/mL levels, while for LC-MS, it
is in the region of ng/mL to the pg/mL levels. The ability for the analysis to attain
the lowest possible limit of detection, characteristic of the detector,
depends on the chromatographic and detection method development, as well as
the sample preparation/clean-up method. These are important factors to consider
in order to prevent interferences, which could cause inaccurate and misleading
measurements. A good review on quantitative analysis of flavonol glycosides,
biflavones, and proanthocyanidins in Ginkgo biloba leaves, extracts, and
phytopharmaceuticals has been published highlighting the different factors from
extraction, separation to detection on the quantitative analysis of one subclass of
flavonoid [104].
Quantitative methods using the HPLC-electrospray ionization-tandem mass
spectrometry method (HPLC–MS2) facilitates the achievement of adequate sensi-
tivity for pharmacokinetic and metabonomic studies with flavonoids. Matrix effects
on signal intensity are important in biological samples, especially during the
preparation of calibration curves, to avoid errors from nonlinear range at high
concentration [50]. Flavonoid kaempferol, for example, is mainly present as glu-
curonides and sulfates, and small amounts of the intact aglycone in rat plasma.
A validated HPLC–MS2 method following FDA guidelines has been reported for
determination of kaempferol and its major metabolite glucuronidated kaempferol in
rat plasma in a study of the pharmacokinetics after oral administration of
kaempferol with different doses [103]. The separation of kaempferol and its
metabolites was carried out on a C18 column (150 � 2.1 mm, 4.5 mm, Waters
Corp.) with isocratic elution at a flow rate of 0.3 mL min�1, and a mobile phase
consisting of 0.5 % formic acid and acetonitrile (50:50, v/v). The quantitative
2128 S.W.A. Bligh et al.
determination was from a Quattro Premier mass spectrometer operating under
a multiple-reaction monitoring mode (MRM), using the electrospray ionization
technique.
8 Selected Examples of Flavonoids Analysis by HPLC
Some examples of more flavonoid analyses by HPLC have been selected and
detailed as presented in Tables 69.3–69.5. These examples are divided into plant
(Table 69.3), food (Table 69.4), and biological (Table 69.5) samples. Notably,
these examples are mainly from work published from 2008 onward, except
for three papers in 2006 and 2007. These examples have been the selected
ones due to the research article details including good representation(s) of
chromatogram(s) to assist other researchers to easily validate their studies.
These examples show the application of this chapter’s aforementioned types of
extractions, separations (in terms of column chemistry, dimensions), and the
detection systems.
9 Conclusion
The number of flavonoids (9,000 in 2004 [105], 9,600 in 2007 [1], 10,380 in 2009
[2]) identified from 2004 to 2009 indicates the high level of interest of this class of
secondary metabolites in plant. The achievements are a result of the advance in
the technology of detection. In 1994, the separation of 25 flavonoids reference
standards was made comfortably relying on the stationary phase technology and
the knowledge of the interaction of mobile phase and analytes. The improvement
of sensitivity and target-specific detection, for example, tandem mass spectrom-
etry detection, has compensated the inability of complete resolution of peaks in
a chromatogram and assisted the identification of sugar units, which partly
contributed to the boom of newly identified flavonoids in recent years. In certain
ambiguous circumstances, for example, the position of substitutions, NMR
spectroscopy can provide a fuller picture of the identity of the flavonoids.
HPLC-MS has been used to screen compounds for drug discovery programs
[106]. In flavonoids, HPLC-UV-MS has been used for screening the antioxidant
activities in teas [87] and using the 2D (LC � LC) system in an herbal decoction
[97, 98].
In many examples, with little regard of the sample type, the analyses of flavo-
noids are usually done in the reversed-phase HPLC mode using nonpolar C18 (in
few cases, C8) columns and polar mobile phase (mixed aqueous–organic solvents)
due to their structural and physicochemical properties. Conversely, with more
regard of the sample type, different extractions and sample pretreatments including
Soxhlet, LLE, SPE, USAE, ASE, and SFE have been used prior to HPLC flavonoid
analysis.
69 Flavonoids by HPLC 2129
Table
69.3
Selectedexam
plesofflavonoid
analysisbyHPLCin
plant(SLEsolidliquid
extraction,aq.aqueous,Q-TOFquadrupole-tim
eofflight,TQtriple
quadrupole)
Plant
Flavonoid
sub-class
Extraction
Stationaryphase
Mobilephase
Detector(s)
References
Glycyrrhiza
L.
(Leguminosaefamily):
licorice
6chalcones
SLE(ultrasonification,
70%
aq.MeO
H)
AgilentZorbax
SB-C
18column
(50�
4.6
mm,
1.8
mm)
Gradient:0.2
%form
icacid
(aq.)andMeC
N
UV-M
S
(+ve,Q-
TOFMS2)
[107]
Bup
leurum
species
1catechin,
2flavones,
8flavonols
SLE(ultrasonification,
MeO
H)
Shim
packODSC18
column
(150�
4.6
mm,
5mm
)
Gradient:0.1
%form
icacid
(aq.)andMeC
N
UV
[108]
Orostachys
japo
nicus
7flavonols,
7catechins
SLE(reflux,70%
aq.
MeO
H;hexane;
EA)
AgilentZorbax
SB-C
18column
(250�
4.6
mm,
5mm
)
Gradient:0.05M
ammonium
form
ate(aq.)andMeO
H
UV-M
S
(+ve,
QTRAP)
[71]
Hypericum
japon
icum
1flavanonol,
4flavonols
SLE(ultrasonification,
70%
aq.MeO
H)
AgilentZorbax
SB-C
18column
(250�
4.6
mm,
5mm
)
Gradient:0.5
%form
icacid
(aq.)andMeC
N
UV-M
S
(+ve,Q-
TOFMS2)
[109]
Iristectorum
Maxim
.
(Iridaceae)
2flavanones,
1flavonol,
1flavanonol
SLE(ultrasonification,
MeO
H)
AgilentEclipse
Plus™
C18column
(150�
3.0
mm,
3.5
mm)
Gradient:0.05%
acetic
acid
(aq.)andMeC
N
DAD-M
S
(�veand
+veMS2)
[110]
Murrayapan
iculata
(L.)Jack
14flavones,
2chalcones
SLE(ultrasonification,
70%
aq.MeO
H)
AgilentZorbax
Eclipse
PlusC18column
(250�
4.6mm,5
mm)
Gradient:0.1
%form
icacid
(aq.)andMeC
N
DAD-M
S
(+veMS2)
[111]
Ziziphu
sjujubaMill.
andZ.jujubavar.
spinosa
(Bunge)
Huex
H.F
3flavonols
SLE(ultrasonification,
80%
aq.MeO
H)
WatersSunfire
C18
column
(250�
4.6
mm,
5mm
)
Gradient:0.2
%acetic
acid
(aq.)andMeC
N
DAD-M
S
(�ve,Q-
TOFMS2)
[112]
2130 S.W.A. Bligh et al.
Scutellaria
baicalensis
Georgi(S.baicalensis)
27flavones,
1flavanol,
2flavanones,
1flavanonol,
1biflavone
SLE(reflux,60%
aq.
MeC
N;CH2Cl 2)
Welch
Materials
Ultim
ateXBC18
column
(250�
4.6
mm,
5mm
)
Gradient:0.06%
acetic
acid
(aq.)andMeC
N
UV-M
S
(�veLCQ
Iontrap
MSn)
[113]
Chrysosplenium
(Turn.)L.(aerialparts)
offloweringC.
alternifolium
4flavonols
SLE(reflux,MeO
H)
AgilentHypersilC18
column(125�
4mm,
5mm
)
Gradient:0.5
%phosphoric
acid
(aq.)andMeC
N
DAD
[114]
Glycintomentella
Hayata(leaves
and
roots)
3flavones,
7flavonols,
6flavanones
SLE(reflux,95%
aq.
EtOH)
ThermoHypersil
GOLD
C18column
(250�
4.6
mm,
5mm
).
Gradient:9%
acetic
acid
(aq.)
andMeO
H
DAD
[115]
Houttuynia
cordata
Thunb
4flavonols
Pressurizedliquid
extractionorhot
soakingwithshaking
(70%
EtOH)
KromasilTurner
YWGC18column
(250�
4.6
mm,
10mm
)
Gradient:water–MeC
N–
phosphoricacid
(400:100:0.2)
andMeC
N–MeO
H–water–
phosphoricacid
(375:75:50:0.1)
UV
[116]
Artem
isia
ann
uaL.
5flavonols,
1flavone
SLE(M
aceration,DCM
orhexane)
Merck
Eurosphers
StarRP-18column
(200�
4.6
mm,
5mm
)
Gradient:form
icacid
aq.
(pH
3.2)andMeC
N
DAD-M
S
(+ve,ion
trap,MS)
[117]
69 Flavonoids by HPLC 2131
Table
69.4
Selectedexam
plesofflavonoid
analysisbyHPLC
infoods(SLEsolidliquid
extraction,LLEliquid–liquid
extraction,aq
.aqueous,Q-TOF
quadrupole-tim
eofflight,TQ
triple
quadrupole)
Food
Flavonoid
subclass
Extraction
Stationaryphase
Mobilephase
Detector(s)
References
Black
currantjuice
3flavones,
2flavonols,
3flavanones
None(directinjection)
AgilentZorbax
Rapid
Resolution
C18column
(50�
2.1
mm,
1.8
mm)
Gradient:0.1
%form
ic
acid
(aq.)and0.1
%
form
icacid
inMeC
N
MS(�
ve,
LTQ-
Orbitrap,
MS2)
[118]
Tomato(Lycop
ersicon
esculentum
Mill.)
8flavonols,
11flavanone,
2dihydrochalcones
SLE(H
omogenization,
sonication,and
centrifugation);SPE
Phenomenex
Luna
C18column
(50�
2.0
mm,
5mm
)
Gradient:0.1
%form
ic
acid
(aq.)and0.1
%
form
icacid
inMeC
N
DAD-M
S
(�ve,LTQ-
Orbitrapand
TQ,MSn)
[119]
Passifloraedulisfruitpulp
2flavones,
1flavonol
LLE(sonication60%
or100%
MeO
Hor
EtOH);SPE
WatersSymmetry
C18column
(250�
4.6
mm,
5mm
)
Gradient:0.2
%form
ic
acid
(aq.)and0.2
%
form
icacid
inMeC
N
DAD-M
S
(�ve,TQ,
MS2)
[120]
Riperedpaprika(C
.an
nuu
m)andyellow
habanero(C
.chinense)
peppers
3flavonols,
2flavones
LLE(homogenization,
EtOH);3M
HCl
Phenomenex
Gem
ini
C18column
(250�
4.6
mm,
5mm
)
Gradient:0.03M
phosphoricacid
(aq.)
andMeO
H.
DAD-M
S
(�veor+ve,
Q-TOF,MS)
[121]
Citrusgrandis,Citrus
paradise(flavedos
“external
layer
ofpeel”
andjuices)
15flavonols,
13flavanones
LLE(sonication,
MeO
H)
AgilentZorbax
SB
C18column
(250�
4.0
mm,
5mm
)
Gradient:1%
acetic
acid
(aq.)and1%
acetic
acid
inMeC
N
DAD-M
S
(�ve,Ion
trap,MS2)
[122]
Rooibosteafrom
Aspalathus
linearis
4flavones,
8flavonols,
2dihydrochalcones
SLE(boiled,water)
Phenomenex
Luna
Phenyl–Hexyl
(250�
4.6
mm,
5mm
)
Gradient:2%
acetic
acid
(aq.)andMeC
N
DAD
[123]
2132 S.W.A. Bligh et al.
Ocimum
gratisimum
L.,
Vernon
iaamygda
linaL.,
Corchorusolitorius
L.,
Man
ihotutilissimaPohl.
6flavonols,
10flavones
SLE(M
eOH;70%
aq.
EtOH,pH2.5)
Phenomenex
Synergi
max
C12column
(150�
4.0
mm,
4mm
)
Gradient:form
icacid
aq.
(pH
3.2)andMeC
N
DAD-M
S
(�ve,Ion
trap,MS)
[124]
Sugarcaneraw
juice
(Saccharumsinense
Roxb.)
1flavone,1
anthocyanin
LLE(n-butanol
(1:1
v/v);MeO
H)
WatersSymmetry
C12column
(150�
4.6
mm,
4.6
mm)
Gradient:0.1
%form
ic
acid
(aq.)andMeO
H
DAD
[125]
Slovenianhoneys:Robinia
pseudoa
cacia,Tilia
spp.,
Castanea
sativa,Abies
alba
Mill.,Picea
abies
(L.)
Karst
4flavonols,
3flavones,
3flavanones,
1flavanonol
LLE(acidified
water
pH2);SPE,MeO
H–
MeC
N(2:1,v:v).
Phenomenex
Luna
C18column
(150�
2.0
mm,
3mm
)
Gradient:1%
form
icacid
(aq.)andMeC
N
DAD-M
S
(�ve,TQ,
MS2)
[126]
Red
grapeskin
(Grapes
from
fourvarieties
of
VitisviniferaL.)
5anthocyanins,
5flavonols,
1catechin
Ultrasonification;SLE
(ultrasonification,
MeO
H/HCl99/1)
WatersXbridgeC18
column
(150�
4.6
mm,
5mm
)
Gradient:2mM
KCl,
water/M
eOH/form
icacid
(83/16/1)orWater/
MeO
H/form
icacid
(68.5/
30/1.5)
ECD
[127]
Buckwheat(Fagop
yrum
esculentum
M€ oench)
4flavones,
5flavonols,
19catechins
SLE(ultrasonification,
80%
EtOH)
AgilentZorbax
Eclipse
plusC18
column
(150�
4.6
mm,
1.8
mm)
Gradient:1%
acetic
acid
(aq.)andamixture
of1%
acetic
acid
(aq.)in
MeC
N
(60:40)
MS(�
ve,Q-
TOFMS2)
[128]
Rosm
arinusofficina
lisL.
(Lam
iaceae)
9flavones,
1flavonol
SLE(stirringand
ultrasonification,EtOH)
Phenomenex
Fusion
C18column
(150�
3.9
mm,
4mm
)
Gradient:0.1
%form
ic
acid
(aq.)andMeC
N
DAD-M
S
(+veand
�ve,Q,MS)
[129]
Sugarcane(Saccha
rum
officina
rumL.,Gramineae)
9flavones
SLE(ultrasonification,
50%
MeO
H)
WatersSymmetry
C18column
(250�
4.6
mm,
5mm
)
Gradient:0.2
%form
ic
acid
(aq.)andMeC
N
UV/DAD
[130]
(con
tinu
ed)
69 Flavonoids by HPLC 2133
Table
69.4
(continued)
Food
Flavonoid
subclass
Extraction
Stationaryphase
Mobilephase
Detector(s)
References
Concord
grapejuice
12procyanidins,
25anthocyanins,
5flavanonols
SLE(vortexing,
ultrasonification,
acetone/water/acetic
acid,70:29.5:0.5,v/v/v)
Phenomenex
Luna
C18column
(250�
4.6
mm,
5mm
)
Gradient:ACN/EtOAc
(7:1,v/v)and0.05%
acetic
acid
(aq.).
FD
[131]
Chocolate
andcocoa-
containingfoodproducts
12procyanidins,
25anthocyanins,
5flavanonols
SLE(vortexing,
ultrasonification,
acetone/water/acetic
acid,70:29.5:0.5,v/v/v)
Phenomenex
Luna
C18column(250�
4.6
mm,5mm
)
Gradient:ACN/EtOAc
(7:1,v/v)and0.05%
acetic
acid
(aq.)
FD
[131]
Vaccinium
macrocarpon
cranberry
concentrate
7anthocyanins,
2proanthocyanidins,
10flavonols
LLE(EtOAc)
WatersAcquityC18
column
(100�
2.1
mm,
1.8
mm)
Gradient:5%
form
icacid
(aq.)andMeO
H.
DAD-M
S
(+veand
�ve,Q,MS2)
[132]
2134 S.W.A. Bligh et al.
Table
69.5
Selectedexam
plesofflavonoid
analysisbyHPLCin
biologicalsamples(SLEsolidliquid
extraction,LLEliquid–liquid
extraction,aq
.aqueous,
Q-TOFquadrupole-tim
eofflight,TQtriple
quadrupole)
Biological
sample
Flavonoid
subclass
Extraction
Stationaryphase
Mobilephase
Detector(s)
References
Ginkgobiloba
inrat
plasm
a
3flavonols
Acidhydrolysis(sam
ple:
10M
HCl:MeO
H2:1:2
v:v:
v);neutralized
with15M
NH3;LL(M
eOH)
WatersC18column
(150�
4.6mm,5
mm)
Isocratic:MeC
N-0.02M
NaH
2PO4(0.2
%
H3PO4),pH
¼2.0,
(35:65)
DAD
[133]
Aspalathuslinearisin
human
urineandblood
plasm
a
8flavones,
4flavonols,
2dihydrochalcones
Urine:
SPE(O
asisWCX
cartridges)
Phenomenex
Luna
Phenyl–Hexyl
(250�
4.6mm,5
mm)
Gradient:2%
aceticacid
(aq.)andMeC
N
UV-M
S
(�ve,ion
trap,MS2)
[123]
Blood:centrifugation
(2,000gfor10min
at4C)–
plasm
a:(LLE,EtOAc)
HerbaEpimediiin
dog
plasm
a
7flavonols
SLE(V
ortex,centrifugation
MeO
H,70%
EtOHaq.)
AgilentZorbax
Eclipse
SB-C
18
column
(50�
2.1
mm,
1.8
mm)
Gradient:0.3
%acetic
acid
(aq.)and0.3
%
acetic
acid
inMeC
N
MS(+ve,
TQ,MS2)
[134]
Haw
thorn
leaves
inrat
plasm
a
2flavones
SLE(V
ortex,centrifugation
MeO
H)
DikmaDiamonsilTM
C18column
(200�
4.6mm,5
mm)
Isocratic:MeO
H–
MeC
N–THF–0.5
%
acetic
acid
(1:1:19.4:78.6)
UV
[135]
Vitislabrusca
vines
(Concord
grapes)in
human
urineandblood
25anthocyanins
Acidifywith50%
form
ic
acid
aq.,SPE(Phenomenex
StrataC18(6
mL/500mg),
1%
form
icacid
containing
10%
MeO
H)
Phenomenex
Synergi
(250�
4.6mm,4
mm)
Gradient:1%
form
ic
acid
(aq.)andMeO
H
DAD-M
S
(+veor�v
e,
iontrap,
MSn)
[131]
(con
tinu
ed)
69 Flavonoids by HPLC 2135
Table
69.5
(continued)
Biological
sample
Flavonoid
subclass
Extraction
Stationaryphase
Mobilephase
Detector(s)
References
Dalbergia
odo
rifera
in
raturine
4neoflavones,
2flavanones,
2chalcones
LLE(V
ortex,centrifugation
EtOAc)
AgilentZorbax
SB
C18column
(250�
4.6mm,5
mm)
Gradient:0.3
%acetic
acid
(aq.)andMeC
N
UV-M
S
(�ve,MS)
[136]
Eriobotryajapon
ica
(Thunb.)Lindl.
9flavonols
SLE(EtOH)
HanbonKromasilC18
column
(200�
4.6mm,5
mm)
Gradient:1%
aceticacid
(aq.)and1%
acetic
acid
inMeO
H
DAD-M
S
(�ve,Ion
Trap,MS)
[137]
Vaccinium
macrocarpon
cranberry
concentratein
ratsurineandblood
2flavonols,
7anthocyanins
Tissue:
LLE
(homogenization,80%
MeO
Hcontaining0.1
%
acetic
acid;vortex,
centrifugation80%
MeO
H
aq.)
Phenomenex
Fusion
(150�
2.0
mm,
1.8
mm)
Gradient:0.1
%form
ic
acid
(aq.)and0.1
%
form
icacid
inMeC
N
MS(�
ve,
TQ,MS2)
[132]
Urine,plasm
a:Hydrolysis,
LLE(diethylether)
2136 S.W.A. Bligh et al.
Indeed, with the development of separation techniques for HPLC to the ultra-
HPLC leading to faster analyses and high throughput, coupled with the advance-
ment of the detection technique, even more flavonoids would both be identified and
quantified quicker in the future.
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