flavonoides x hplc

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

mailto:[email protected]

mailto:[email protected]

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


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


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


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


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]


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.


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


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]


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]


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.


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]


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,


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]


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


http://dx.doi.org/10.1007/978-3-642-22144-6_90

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]


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]


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]


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]


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


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.


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


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


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


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


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


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]


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]


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]


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


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


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)


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]


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


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)


Table

69.5

(continued)

Biological

sample

Flavonoid

subclass

Extraction

Stationaryphase

Mobilephase

Detector(s)

References

Dalbergia

odo

rifera

in

raturine

4neoflavones,

2flavanones,

2chalcones

LLE(V


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)


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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