Current Organic Chemistry, 2011, 15, 2541-2566 2541

Extraction, Separation, Detection, and Structural Analysis of Flavonoids

Xue Qiao, Wen-zhi Yang, De-an Guo and Min Ye*

The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan
Road, Beijing 100191, China

Abstract: Flavonoids, as an important class of natural products, are the main bioactive constituents of a lot of medicinal or dietary plants.
They have been reported to show extensive benefits to human health, including antioxidant, anti-inflammatory, and anti-cancer activities.
In most cases, flavonoids are present in plants as a series of analogues with similar structures and physico-chemical properties. This
chemical complexity renders the quality control of flavonoid-containing herbal products problematic. Therefore, rapid, accurate and sen-
sitive analytical techniques for flavonoids are of significance. This review summarizes the advances in sample extraction, chroma-
tographic separation, detection, and structural analysis of flavonoids in medicinal and dietary herbs since 2005. Emphasis will be put on
the qualitative and quantitative analysis of flavonoids in botanical extracts by high-performance liquid chromatography (HPLC) and liq-
uid chromatography coupled with mass spectrometry (LC/MS). New techniques like UPLC, HILIC, and 2D-HPLC have significantly
improved the separation capabilities of HPLC. The versatile LC/MS does not only allow fast and accurate structural analysis of flavon-
oids in complicated mixtures, but also provides a rapid and sensitive technique for the quantitative detection of flavonoids in herbal ex-
tracts and even in biological matrices. These techniques will be widely used for the chemical analysis of flavonoids and for the quality
control of related herbal products.

Keywords: Flavonoids; HPLC; LC/MS; separation; detection; structural analysis; medicinal and dietary herb.

1. INTRODUCTION showed protective effects against cardiovascular disease [10,11].

Flavonoids are a big class of secondary metabolites ubiqui-
tously distributed in plants, especially in medicinal and dietary
herbs. Most flavonoids are derived from benzo-
-pyrone, and con-
tain a C6-C3-C6 basic structure skeleton. According to their substitu-
tion pattern, unsaturation degree, and arrangement of the basic
skeleton, flavonoids can be classified into a number of sub-types,
including flavones, flavonols, flavanones, flavanols, isoflavones,
and chalcones. Flavonoids usually bear hydroxyl or methoxyl
groups at C-5, C-7, C-3, and C-4. Some flavonoids occur in plants
in free form, while others occur as conjugated O- or C-glycosides.
For O-glycosides, the sugar residues are usually linked to 3, 7, or
4- hydroxyl groups, while for C-glycosides, the sugar residues are
linked directly to C-6 or C-8. The sugar residues include glucose,
galactose, rhamnose, apiose, etc [1]. Due to the presence of conju-
gated chromophores in the molecules, most flavonoids show yellow
to red color. For example, the color of safflower (Carthamus tincto-
rius L.) is due to the presence of carthamin (a chalcone) and other
phenolic compounds. The basic structures of main flavonoid sub-
classes are depicted in Fig. (1).
Flavonoids exhibit a wide range of biological activities [2], in-
cluding antioxidant [3], anti-inflammatory [4], and anti-cancer [5-8]
activities, which significantly contribute to the biological effects of
medicinal herbs. In recent years, more pharmacological significance
of flavonoids has been discovered, and the mechanism of action has
been elucidated. Flavonoids may exert their anti-inflammatory ef-
fects by modulating pro-inflammatory gene expression, which may
be also related with their effects on cancer and cardiovascular dis-
ease [9]. Dietary polyphenolds (including flavonoids) from cocoa,
grape seeds, tea, soy bean, and flavonoids from medicinal herbs
*Address correspondence to this author at the State Key Laboratory of Natural and
Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan
Road, Beijing 100191, China; Tel: +86 10 82802024; Fax; +86 10 82802024; E-mail:
yemin@bjmu.edu.cn
Pushpavalli et al. found that flavonoids like chrysin exhibited hepa-
toprotective [12]. Flavonoids may act as P-glycoprotein (P-gp)
inhibitors, and improve the effects of anti-cancer drugs like pacli-
taxel and camptothecin. The inhibitory effects could be comparable
to those of well-known P-gp inhibitors verapamil and cyclosporine
[13]. Since a lot of flavonoids show estrogen-like activities, they
are called phytoestrogens. An important medicinal use of phytoes-
trogens is to treat osteoporosis. For instance, isoprenylated flavones
from Epimedium species and flavanols from Drynaria fortunei
could improve the proliferation of osteoblastic cells and inhibit the
function of osteoclastic cells [14-16]. Recently, flavonoids have
been found to possess memory-enhancing [17], antifungal [18],
antiviral [19], anxiolytic [20], and hypolipidemic [21] activities.
Due to the wide distribution of flavonoids in medicinal and die-
tary herbs, and due to their various bioactivities, especially the in-
teractions with chemical drugs, rapid separation and sensitive detec-
tion of flavonoids is of significance for the benefits of human
health. Similar topics have been summarized in several recent re-
views [22-25]. The present review focuses on analytical techniques
for extraction, separation, detection, and structural analysis of fla-
vonoids in medicinal and dietary herbs (including Chinese herbal
medicines). The emphasis will be put on the latest advances in
HPLC and LC/MS techniques since 2005. The application of these
techniques for the analysis of flavonoids in biological matrices will
also be discussed.
2. SAMPLE EXTRACTION
Simple and efficient extraction of target compounds from natu-
ral medicinal sources is definitely an important step for the separa-
tion and analysis of flavonoids. Various extraction methods such as
cool maceration, shaking extraction, heat reflux extraction, Sox-
hlet’s extraction, and ultrasound-assisted extraction have been
commonly used for the extraction of flavonoids from natural medi-
cines. Recently, new extraction technologies have been widely ap-

1385-2728/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

2542 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

Fig. (1). Chemical structures of representative naturally-occurring flavonoids.

plied in this field, of which microwave-assisted extraction (MAE) These new technologies, together with improvements in traditional
and pressurized liquid extraction (PLE) are frequently reported. extraction approaches were described here.
Extraction, Separation, Detection, and Structural Analysis of Flavonoids
Fig. (2). The apparatus of vacuum microwave-assisted extraction (VMAE).
Reprinted from Reference [37].
2.1. Microwave-assisted Extraction (MAE)
The application of microwave energy to improve the extraction
efficiency of organic compounds from solid matrices such as soil,
plants and food can date back to 1986 [26]. Since then, the micro-
wave-assisted extraction technology received more and more atten-
tion in the field of pharmaceutical analysis due to the advantages of
short extraction time, low consumption of extraction solvents, and
high extraction efficiency [25]. MAE could rupture the plant cells,
and thus gave higher recovery than convention extraction methods
[27]. Recently, attempts have been made to couple MAE with ana-
lytical techniques, such as HPLC-UV, LC/MS, HSCCC, or with
biological activity tests.
An optimized MAE method was applied for the extraction of
flavonoids from Fructus Aurantii Immaturus [28]. According to an
L9 (3)4 orthogonal design, the MAE conditions were optimized as
follows: 90% ethanol as extraction solvent, liquid-solid ratio of
15:1, extract for 5 min at 80ºC. This method provided higher recov-
eries for narirutin, naringin, and neohesperidin than those obtained
with heat reflux extraction. A similar efficient MAE method was
established for the extraction of rutin and quercetin from the stalks
of Euonymus alatus [29]. Applying MAE for the extraction of fla-
vonoids from Radix Astragali obtained a similar yield to Soxhlet’s
extraction but higher than ultrasound-assisted extraction and heat
reflux extraction [30]. Other evidences revealed that MAE gave a
higher extraction efficiency with less time consumption than heat
reflux, Soxhlet’s, and ultrasound-assisted extractions [31, 32]. MAE
has also been successfully used for the extraction of flavonoids in
Folium Eriobotryae [33], of scutellarin from Erigeron breviscapus
[27], of isoflavones in soybeans [34], and of flavonoids from tea
[35].
Current Organic Chemistry, 2011, Vol. 15, No. 15 2543
Ionic liquids composed of organic cations and inorganic or or-
ganic anions have been regarded as a green alternative to traditional
organic solvents. An MAE method with ionic liquids was devel-
oped for the extraction of phenolic components from the leaves of
Psidium guajava Linn. and the tubers of Smilax china [36]. A total
of 11 ionic liquids containing different cations and anions were
examined, and key parameters including particle size, liquid/solid
ratio, and extraction temperature and time were fully optimized.
Finally, the optimized one-step extraction yield ranged from 79.5-
93.8%.
A modified MAE method, namely vacuum microwave-assisted
extraction (VMAE), was successfully applied to the extraction of
polyphenolic compounds and pigments from Chinese herbs [37].
Different from conventional MAE, this method was carried out in
vacuum. The apparatus was illustrated in Fig. (2). With optimized
conditions, the VMAE method obtained a higher recovery for
thermo-sensitive compounds like myricetin and safflomin than
conventional MAE. Thus, VMAE may be a suitable method to
extract thermo-sensitive compounds from herbal sources.
Another modified MAE was named dynamic microwave-
assisted extraction (DMAE), with a pump transferring the extract
from the sample matrix with flowed fresh solvent to the outside of
extraction vessel, as shown in Fig. (3). This developed DMAE
method was applied to the extraction of flavonoids from Saussurea
medusa dried cell cultures [38]. The key factors were optimized as
follows: 1200 W of radiation power, 50:1 (v/w) of the liquid/solid
ratio, and 50 ml/s of solvent flow rate. With these conditions, a
4.97% yield of total flavonoids was obtained in 60 minutes. This
DMAE method showed obvious advantages of short duration and
high efficiency to conventional extraction methods. DMAE was
also used for the continuous extraction and on-line derivatization
(with aluminium chloride) and UV detection of total flavonoids in
Platycaldus orientalis [39]. Another cased proved that DMAE gave
higher extraction yields than ultrasonic, heat reflux, and Soxhlet’s
approaches for the extraction of flavonoids from Herba Epimedii
[40] and Saussurea medusa [41].
2.2. Pressurized Liquid Extraction (PLE)
The pressurized liquid extraction technique, originally devel-
oped for sample preparation in environmental analysis, has become
a popular extraction method for the extraction of target compounds
from natural medicines. In PLE, pressure is applied to the extrac-
tion system to achieve a high temperature, compared with the at-
mospheric boiling points of traditional aqueous and organic sol-
vents. Temperature increase leads to enhanced diffusivity of the
solvent, as well as improved speed and efficiency of the extraction
[42].
PLE was used for the HPLC-UV quantification of flavonoids in
onion [43]. Five extraction methods were tested in terms of extrac-
tion efficiency of seven flavonoids from freeze-dried onion. The
lowest extraction efficiency was obtained with the conventional
water bath extraction, while the other methods were similar to each
other. However, PLE showed its unique merits of high automation,
small solvent consumption, and clean extract. In addition, it al-
lowed the safe extraction of light and oxygen-sensitive flavonoids
in a lucifugal and inert atmosphere. PLE was used for the simulta-
neous extraction of flavonoids, saponins, and polyacetylenes in
leaves and roots of ginseng [44]. Wang et al. reported their research
work using PLE extraction for the simultaneous determination of
multiple flavonoids in medicinal herbs by HPLC-UV or capillary
electrochromatography [45-48]. The application of PLE also in-
2544 Current Organic Chemistry, 2011, Vol. 15, No. 15
cluded the analysis of polar antioxidants in rosemary extracts by
capillary electrophoresis coupled with mass spectrometry [49],
antioxidants in apple [50], and flavonoids in spinach [51].
In the above PLE methods, the pressure was mostly set at 1500
psi (equivalent to about 10.3 MPa). A new PLE method with a
pressure of above 200 MPa was reported for the extraction of phe-
nolic compounds from longan fruit pericarp [52]. The obtained
extract showed a high recovery of three phenolic compounds.
2.3. Other New Extraction Methods
Aside from the above new emerging methods, modifications of
traditional extraction methods were also reported. Zhang and
Laursen developed a mild extraction method for the analysis of
natural dyes (flavonoids) in textiles of historical interest [53]. A
mild extraction approach with EDTA and formic acid was devel-
oped, and successfully avoided the use of hydrochloric acid and
then degradation of flavonoid glycosides in the extraction process.
A dynamic ultrasonic extraction (DUE) method coupled with
on-line spectrophotometric detection has been used for the determi-
nation of total flavonoids in Scutellaria barbata and S. baicalensis
[54,55]. The DUE process involves a continuous transport of ana-
lytes out of the extraction vessel, which enhanced the extraction
efficiency and facilitated its coupling with other extraction steps or
on-line detectors. Compared with other extraction methods, DUE
method obtained a high recovery for flavonoids. Furthermore, DUE
possesses obvious advantages of being more gentle, less expensive,
simpler, and less prone to contamination.
A new extraction method, namely negative-pressure cavitation
extraction (NPCE), was used for the extraction and quantification
of five flavonoids in pigeon pea leaves [56]. With optimized pa-
rameters, NPCE showed higher recovery than microwave-assisted
extraction (MAE), ultrasonic extraction (USE), and heat reflux
extraction (HRE). As the samples were extracted with nitrogen at
room temperature, NPCE significantly decreased the degradation
and oxidation of flavonoids. In addition, the NPCE method showed
desirable repeatability and reproducibility.
A solvent-free microwave hydrodiffusion and gravity extraction
(MHG) method was used for the extraction of flavonols in onion
(Allium cepa L.) [57]. The microwaves influence textural properties
of plant material and increase secondary metabolites diffusion by
improving tissue softness and increasing cell permeability. The
MHG procedure was performed on peeled onion bulbs, which was
heated using a fixed power density without addition of solvents or
water [57]. This novel MHG technology definitely meets the de-
mand of consumers for healthier food products, for it was per-
formed under effect of microwaves and earth gravity at atmospheric
pressure, without introducing any solvent.
3. CHROMATOGRAPHIC SEPARATION AND DETECTION
Plants usually contain a series of similar flavonoids, with slight
change in substitution patterns or functional groups. These com-
pounds may show very similar properties, and their separation has
been challenging. Chromatography is the most powerful technology
for the separation of organic compounds. All types of chroma-
tographic technologies have been extensively used for the separa-
tion of flavonoids, including thin-layer chromatography (TLC),
column chromatography (CC), high speed counter-current chroma-
tography (HSCCC), capillary electrophoresis (CE), gas chromatog-
raphy (GC), high-performance liquid chromatography (HPLC), and
liquid chromatography coupled with mass spectrometry (LC/MS).
Qiao et al.
Latest progress in the HSCCC and CE analysis of flavonoids has
been well summarized in recent reviews [22,23]. In this section, we
focus on the advances in the separation and detection of flavonoids
by CC, TLC, GC, and GC/MS. The progress in HPLC and LC/MS
analysis will be summarized in sections 4 and 5.
3.1. Column Chromatography
Flavonoids were present in many plants as an essential class of
bioactive components. During the past decades, various column
chromatography techniques were developed for the isolation of
flavonoids. As stationary phases, silica gel, modified silica gel,
reversed-phase silica gel, polyamide, or Sephadex have been exten-
sively used. However, due to the complicated character of plant
samples and the structural similarity of most flavonoids, the separa-
tion of target flavonoids is sometimes challenging. In recent years,
a series of new packing materials and technologies for column
chromatography were developed for the rapid and efficient separa-
tion of flavonoids.
A novel solid phase material, prepared by copolymerization us-
ing allyl-bromide-modified chitosan as macromonomer and ethyl-
ene glycol dimethacrylate as cross-linker, was utilized in the sepa-
ration of flavonoids [58]. Structurally related flavonoids, isorham-
netin, kaempferol, and morin were successfully separated with this
material. More importantly, the developed SPE column packed with
this material showed three times higher of capacity than other col-
umns. Superose 12 column packed with 12% cross-linked agarose
gel, which have been widely used in the separation and purification
of high molecular weight compounds, was successfully applied to
the isolation of flavonoids and isoflavonoids from Sophora japon-
ica [59].
Medium-pressure liquid chromatography (MPLC) acts as an ef-
fective and useful tool in the preparative scale separation of com-
plicated mixtures. A novel method, using MPLC coupled to elec-
trospray ionization multi-stage mass spectrometry (MPLC/ESI-
MSn), was successfully established for the separation and on-line
characterization of flavonoids from Asparagus officinalis [60]. The
separation procedure was guided by the MS/MS analysis. The de-
veloped approach hyphenating MPLC with ESI-MSn proved effec-
tive in the separation of complicated mixtures with high purity,
desired amounts and simultaneous structural elucidation.
Column chromatography is usually performed on a packed col-
umn. Recently, a novel approach to enrich flavonoids from Ginkgo
biloba L. was developed with expanded bed adsorption (EBA) us-
ing Amberlite XAD7HP as the adsorbent [61]. Different from tradi-
tional packed columns, un-treated crude extracts were directly
loaded onto an expanded adsorption bed from the bottom, and the
elution was carried out in an upward flow. With a two-step elution,
this new method achieved approximately the same quality of final
product with those obtained with packed column and liquid-liquid
extraction, but showed its unique advantages of simplified opera-
tional procedure, shorter time consumption, and lower operational
cost.
3.2. Thin-Layer Chromatography (TLC)
Thin-layer chromatography has been used as a routine approach
for the separation and detection of flavonoids since 1960s and still
plays an indispensable role today. The commonly used TLC sta-
tionary phase includes silica gel G, silica gel GF254, RP-C18, and
polyamide. The separated flavonoids are usually detected under 254
nm as fluorescent quenching spots, or colorful spots under 365 nm
Extraction, Separation, Detection, and Structural Analysis of Flavonoids
Fig. (3). Scheme of the dynamic microwave-assisted extraction (DMAE)
system. Reprinted from Reference [38].
after spraying with visualizing reagents. A densitometer may be
used for quantitative TLC analysis.
Bhandart et al. [62] established a dual-run TLC method for the
determination of flavonoids from eight medicinal plants on a RP-18
F254 TLC plate. Mixtures of 5% formic acid-water/methanol (70:30)
and 5% formic acid-water/methanol (50:50) were used as the mo-
bile phase for the dual-development. The flavonoids were deter-
mined by densitometry at 280 nm in reflectance/absorbance mode.
This method showed good linearity and excellent reproducibility.
Pozharitskaya et al. [63] developed a TLC method for the determi-
nation of icariin in the dry extracts of the aerial parts of Epimedium
koreanum. This method led to very similar results to HPLC method.
TLC was also applied to the determination of rutin from Armaryllis
belladonna flowers with lower accuracy and precision than HPLC
but more convenience and less solvent consumption [64]. HRTLC
(high-resolution TLC) has been used for the simultaneous quantita-
tion of five phenolic flavonoids in Glycyrrhiza glabra [65], the
determination of tetrahydroamentoflavone in Semecarpus anacar-
dium and its formula [66], and the simultaneous determination of
quercetin, rutin, and coumaric acid in the flowers of Rhododendron
arboretum [67].
A TLC method for the rapid identification of different plant
species used as “Bailahuen” and its adulterants was established
coupled with antioxidant activity test [68]. Lederer et al. [69] de-
veloped a simple and rapid method for the differentiation of Chi-
nese star anise and other Illicium species. The separation was car-
ried out on an HRTLC plate, eluted with a mixture of ethyl acetate,
formic acid, acetic acid, and water (100:11:11:26), and detected at
365 nm. Results revealed that Chinese star anise showed a different
TLC fingerprint from all other analyzed Illicium species. This
method could be used for the rapid identification of Chinese star
anise in the quality control of commercial batches. The field thistle
(Cirsium setosum) and Japanese field thistle (C. japonicum) were
recorded in Chinese Pharmacopoeia for the treatment of bleeding
and inflammation. A TLC method was established to distinguish
these two species [70]. Using a mixture of ethyl acetate, formic
acid, acetic acid and water, a satisfying resolution was achieved.
Two clear fluorescent spots of linarin and pectolinarin were ob-
served in the chromatogram of C. japonicum while only the spot
Current Organic Chemistry, 2011, Vol. 15, No. 15 2545
Fig. (4). The separation of flavan-3-ols on HPTLC in oak and tea by differ-
ent solvents. (A), 1-butanol-water-acetic acid; (B), 1-propanol-water-acetic
acid. Reprinted from Reference [71].
due to linarin observed in those of C. setosum, which provided a
simple and feasible approach for the differentiation of the two spe-
cies.
In addition to applications in qualitative and quantitative analy-
sis, researches on the separation mechanism of flavonoids by TLC
were also conducted. Vovk et al. [71] investigated the retention
behaviors of eight flavonoids on cellulose HRTLC. (+)-catechin
(C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-
epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-
epigallocatechin gallate (EGCg), procyanidin B1, and procyanidin
B2. Detection was performed using vanillin-H3PO4 reagent and four
developing solvent systems were tested. Consequently, water en-
abled the separation of epimers C from EC and GC from EGC, as
well as the dimers procianidin B1 and B2. Compounds C, EGC, B1
and B2 were well separated from all the other compounds, as
shown in Fig. (4). Using 1-propanol-water-acetic acid (20:80:1, v/v)
as the mobile phase, the best separation of the five main catechins
(EC, GC, EGC, ECg, EGCg) present in green tea was obtained. The
chromatograms of oak bark extract using solvents with higher water
content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic
acid (20:80:1, v/v)) showed less bands than those developed in
solvents with higher organic modifier content. The presence of
procyanidins besides the main component catechin resulted in such
retention behavior.
3.3. Gas Chromatography and Gas Chromatography Coupled
with Mass Spectrometry (GC and GC/MS)
Gas chromatography is especially suitable for the analysis of
volatile ingredients such as essential oils and small aromatic com-
pounds. Due to their high melting points, most flavonoids cannot be
directly analyzed by GC. A derivatization treatment is usually nec-
essary when flavonoids are analyzed by GC. N,O-bis(trimethylsilyl)
trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS)
[73,75,76,78,80,83,84] or hexamethyldisilazane (HMDS) and
trifluoroacetic acid (TFA) [74,82] are the most commonly used
derivatization reagents. These agents converted flavonoids into
trimethylsilyl derivatives by esterification or etherification on the
hydroxyl groups which enables the analysis of flavonoids by gas
2546 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

Table 1. Separation and Detection of Flavonoids by GC and GC/MS.

Samples Target flavonoids Sample preparation Derivatization Column Detection Ref.

Hop strobilus rutin, quercetin, naringenin, SFE; UE - fused silica capillary column GC/MS [72]
myricetin, kaemprefol, nar- (30m0.32 mm,0.25m) with a
ingin, etc bonded stationary phase: poly(5%
diphenyl 95% dimethyl) siloxane

Greek aro- (+)-catechin, quercetin, UE and HRE with aqueous TMCS+BSTFA a capillary column low-bleed CP-Sil8 GC/EI-MS [73]
matic plants apigenin, naringenin, etc methanol containing BHT CB-MS (30 m0.32 mm, 0.25m),
and HCl

Dracocepha- cyanidin, deophinidin, UE with methanol and ace- HMDS+TFA an SGE column (30 m0.25 mm, 0.25 GC/IT-MS [74]
lum species malvidin, pelargonidin, tone containing HCl, loaded m)
chlorides, hesperidin, nar- on Sephadex LH-20, acid
ingin, rutin tridydrate, etc hydrolysis, and purified on
SPE

Iris ger- glycitein, DAI, genistein, Extracted in 69% aqueous Silyl- a non-polar capillary column (Zebron GC/EI-MS [75]
manica dihrogenistein, 3’-OH-DAI, methanol with HCl, UE and 991(BSTFA ZB-5, 30 m0.25mm, 0.25m)
extract 6-OH-DAI, IRI,8-OH-DAI, HRE, then re-extract with +TMCS)
dihydrodaidzein, 6-OH-GEN, ethyl acetate
etc

Five aromatic (+)-catechin, quercetin, UE with 62.5% aqueous HMDS+TMCS a capillary column Low-bleed CP-Sil 8 GC/EI-MS [76]
plants apigenin, naringenin, luteolin, methanol containing +pyridine CB-MS (30 m 0.32 mm, 0.25m),
rutin, etc BHT (1 g /L) (3:1:9)

Knotwood of naringenin, dihydro- PHWE BSTFA+TMCS an HP-1 column (25 m0.20mm, GC/FID; [77]
aspen (P. kaempfeol, naringin etc 0.11m) GC/MS
tremula)

Medicago 26 isofalvones, 3 flavones, 2 extracted by shaking with MSTFA+TMC a DB-5-MS column (60 m0.25 mm, GC/MS [78]
truncatula flavanones, 2 aurones and a 80% aqueous methanol S+pyridine 0.25 m)
root and cell chalcone identified and
culture quantified

human serum icariin, desmethylicariin, Liquid-liquid extraction with BSTFA in an HP-5MS capillary column (5% GC/EI-MS [79]
icaritin, epimedin A,B,C, etc ethyl acetate pyridine (4:1) phenyl methyl-siloxane)
(30m0.25mm,0.25m)

human diosmetin, hesperetin, dios- Hydrolysed, then extract BSTFA+TMCS an HP-5MS fused-silica capillary GC/EI-MS [80]
plasma and min, naringenin with diethyl ether column (30 m 0.25mm,0.25 m).
urine

Kaempferia 11 flavones for qualitative Extract with dimeth- an HP50+ column (crosslinked 50% GC/FID [81]
parviflora and quantitative analysis ylmethane PHME siloxane, 30m0.32 mm,
0.15m)

Orange, pelargonidin, cyaniding, homogenize HMDS+ TFA an SGE column (30m0.25 mm, GC/MS [82]
grapefruit, malvidin, quercetin, apigenin, 0.25mFT)
and lemon luteolin, naringenin, hes-
peretin

soy milk and biochaninA, daidzein, ge- Liquid-liquid extracted with BSTFA+TMCS a DB-5MS capillary column (5% GC/MS [83]
waste water nistein, prunetin, etc. ethyl acetate phenyl, 95% methylpolysiloxane,
samples 25m0.25mm)

urine, plasma, dietary polyphenols BSTFA+TMCS a VF-5ms (30m0.25mm, 0.25m); a GC/MS [84]
and fecal VF-17ht column
water (30m0.25mm,0.10m); a Phenome-
nex ZB-5ms (30m0.25mm ,0.25m)

Green tea catechin, hypericin Heat extract with water TMS- A ZB-5HT Inferno capillary column GC/MS [85]
leaves diazomathane; (15m0.32mm, 0.10m)
TMAH; TMSH

Note: SFE: solid phase extraction; UE: ultrasonic extraction; HRE: heat reflux extraction; PHWD: pressurized hot water extraction; FID: flame ionization detector; BSTFA: N,O-
bis(trimethylsilyl) trifluoroacetamide; TMCS: trimethylchlorosilane; HMDS: hexamethyldisilazane; TFA: trifluoroacetic acid; TMS: trimethylsilane; TMAH: tetramethylammonium
hydroxide; TMSH: trimethylsulfonium hydroxide.

chromatography. The gas chromatography studies in flavonoids Separation and detection of flavonoids by GC and GC/MS were
analysis before 2006 have been well summarized in a recent review exhibited in Table 1.
[24]. Here we mainly focus on the progress since 2006 [72-85].
Extraction, Separation, Detection, and Structural Analysis of Flavonoids
Fig. (5). Fragmentation patterns of phytoestrogen (isoflavones) trimethylsilyl ethers by GC/MS-MS. Reprinted from Reference [83].
Maul et al. [75] studied the GC-EI-MS/MS fragmentation of
flavonoids after being derivatized into trimethylsily ethers. The
elimination of methyl radicals, tetramethylsilane groups or the
combined loss of two methyl groups were found specific for certain
substitution patterns, which could be used for the characterization
of unknown flavonoids. Fuzfai et al. [82] found that anthocyanidins
and flavanones were readily to form trimethylsilyl derivatives after
being treated with HMDS and TFA, while flavonols and flavones
were difficult. The authors studied the fragmentation behaviors of
the derivatives, and identified 33 flavonoids from grapefruit, lemon,
and orange by one injection without any extraction or isolation
procedures.
Ferrer et al. [83] developed a method by gas chromatography
coupled with ion trap mass spectrometry (GC/MS-MS) for the iden-
tification of eight phytoestrogens (isoflavones) in soybean products
and wastewater samples. The target compounds were derivatized
into trimethylsilyl ethers with trimethylchlorosilane (TMCS) and
N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA). The fragmen-
tation patterns were studied by isolating and fragmenting the pre-
cursor ions. A typical fragmentation involving the loss of a methyl
and a carbonyl group was observed (Fig. 5). These characteristic
fragmentation pathways were used for the identification of phytoes-
trogens in soy milk and waster water.
4. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
4.1. Separation and Detection by HPLC
Recent advances in flavonoids analysis have been made primar-
ily based on HPLC technology. Most HPLC analyses of flavonoids,
as summarized in previous reviews [23], are performed on re-
versed-phase C18 (or C8) columns, with a gradient binary solvent
system. HPLC-UV is widely used for quantification, which gave
good linearity and sufficient dynamic range for flavonoids in plant
extracts. Therefore, HPLC-UV has been employed for quantifica-
tion of multiple flavonoids in herbal extracts, such as Herba Epi-
medii [86], Carthamus tinctorius [87], and Glycin tomentella [88].
As a typical example, a total of 33 compounds, including 21 fla-
Current Organic Chemistry, 2011, Vol. 15, No. 15 2547
vonoids, were determined simultaneously in Glycin tomentella in
120 minutes using HPLC-DAD.
Besides commonly used detectors like DAD (diode-array detec-
tor), FLU (fluorescent detector), and ELSD (evaporative light scat-
tering detector), MS (mass spectrometer) has become a popular
detector for HPLC to obtain abundant structural information. New
detection techniques were also used to obtain biochemical informa-
tion of samples. Typical applications were summarized in Table 2.
In the pursuit of higher speed and resolution, great advances in
HPLC analysis have been achieved in the past five years for fla-
vonoids, in both separation and detection. On the basis of Van
Deemter equation,
H = A + C=a(dp)+b/u+c(dp)2 u,
H=plate height, u=speed, dp=particle size.
The acceleration of HPLC depends mainly on two factors,
namely, particle size and flow rate. Improvements in these aspects
include UPLC with smaller particles, and monolithic column with
higher flow rate, respectively. For the improvement of resolution,
novel stationary phases were developed. The hyphenation of col-
umns with different separation mechanisms produced 2D-HPLC.
4.2. Advance in Speed: UPLC and Monolithic Column
The appearance of ultra-performance liquid chromatography
(UPLC) significantly shortened the analysis time for each sample
comparing to conventional HPLC. The utilization of sub-2.5 m
particles, contributing directly to the plate height of an LC system,
tremendously increased separation efficiency. The negative effect
of particle decrease is the back-pressure increase (about 9 times,
versus 5 m particles), with which the separation was performed
under very high pressure (up to 100MPa). However, generally, it
has no negative influence on the chromatographic system, including
analytical columns. Separation efficiency remains unchanged or is
even improved. Eventually, the UPLC system resulted in enhanced
peak capacity and detection sensitivity.
A selection of UPLC applications in flavonoids were summa-
rized in Table 3. Chen et al. developed an UPLC-UV method to
analyze different Herba Epimedii (Yin-Yang-Huo) samples. A si-
2548 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

Table 2. Selection of Recent Publications on HPLC Detectors for Flavonoids

Strength
Time
Equipment Detector Stationary Phase (mm x Elutent Modifier Analytes Aim Sample Source Ref.
(min)
mm)

Styphnolobium
HPLC-PDA-MS UV, MS Luna C18(2) 150 x 4.6 MeOH-H2O AA, 1% 35 27 Profile [89]
japonicum

LiChrospher 100 RP-

HPLC-DAD-MS UV, MS 150 x 4.0 MeOH-H2O AA, 2% 25 28 Qual Thymus [90]
18

TFA,
HPLC-DAD-MS UV, MS ODS-80Ts QA C18 150 x 4.6 MeCN-H2O 35 15 Qual petals [91]
0.1%

HPLC-DAD-MS UV, MS Waters C18 symmetry 250 x 4.6 MeCN-H2O FA, 0.1% 65 23 Qual Lippia graveolens [92]

Collard Greens,
HPLC-DAD-MS UV, MS Waters C18 symmetry 250 x 4.6 MeCN-H2O FA, 0.1% 45 58 Qual Kale, Chinese [93]
Broccoli

HPLC-DAD-MS UV, MS Waters C18 symmetry 250 x 4.6 MeCN-H2O FA, 0.1% 90 58 Qual propolis [94]
n
HPLC-DAD-FTICR MS MS , MS Dikma C18 150 x 4.6 MeCN-H2O AA, 0.5% 15 4 Qual Plant extract [95]

UV,
HPLC-DAD-SPE-NMR Luna C18(2) 150 x 4.6 MeCN-H2O - 60 15 Qual Kanahia laniflora [96]
NMR

HPLC-PDA-SPE-NMR- UV, MS,

Luna C18(2) 150 x 4.6 MeCN-H2O FA, 0.1% 30 12 Qual St. John’s Wort [97]
MS NMR

HPLC-DPPH-DAD- UV,
Chromolith RP-18e 100 x 4.6 MeCN-H2O AA, 3% 50 9 Qual Olive Leaf [98]
FLU FLU

HPLC-DAD-ELSD ELSD ZORBAX ODS C18 250 x 4.6 MeCN-H2O FA, 0.3% 65 10 Quan Radix Astragali [99]

Note: TFA, trifluoroacetic acid; AA, acetic acid; FA, formic acid; Qual, qualitative analyses; Quan, quantitative analyses; Profile, screening analyses.

Table 3. Selection of Recent Publications on UPLC Analysis of Flavonoids

Strength
Stationary Flow Time Ana- Intention Sample Source:
Equipment (mm x Praticle Modifier Ref.
Phase (ml/min) (min) lytes (LOD, LOQ) Flavonoids
mm)

UPLC-DAD- Acquity 100 x 2.1 1.7 m 0.3 TFA, 2.8 21 Qual Rose species: phenolics [100]
ESI-qTOF-MS BEH C18 0.05%

UPLC-DAD- Acquity 50 x 2.1 1.7 m 0.5 AA, 4.4 13 Qual Aesculus hippocastanum [101]
ESI-IT-MS BEH C18 0.05% Seeds: flavonoids

UPLC-DAD- Acquity 100 x 2.1 1.7 m 0.3 AA, 0.1% 6 15 Qual Garcinia hanburyi: xan- [102]
ESI-qTOF-MS BEH C18 thones

UPLC-DAD Acquity 50 x 2.1 1.7 m 0.35 AA, 0.1% 10 23 Profile Trifolium Species, aerial [103]
BEH C18 parts:
isoflavones, phenolics

UPLC-DAD Acquity 50 x 2.1 1.7 m 0.25 AA, 12 15 Quan Epimedium: flavonoids [46]
BEH C18 50mM (130 pg, 520 pg)

UPLC-DAD- Acquity 100 x 2.1 1.8 m 0.6 AA, 0.5% 12 38 Qual Lupin leaves: malonylated [104]
ESI-qTOF-MS HSS T3 flavonoid glyconjugates

UPLC-DAD- Acquity 150 x 2.1 1.7 m 0.3 AA, 0.1% 25 18+52 Profile licorice root extracts: [105]
ESI-IT-MS BEH C18 flavonoids

UPLC-DAD- ZorBax 50 x 4.6 1.8 m 0.8 FA, 0.3% 30 54 Qual Buyang Huanwu decoc- [106]
ESI-qTOF-MS SB-C18 tion:
quinochalcones, flavon-
oids, isoflavones

UPLC-DAD- Acquity 100 x 2.1 1.7 m 0.25 FA, 0.3% 50 51+18 Qual Epimedium koreanum [107]
ESI-qTOF-MS RP C18 Nakai: flavonoids

UPLC-DAD- Acquity 100 x 2.1 1.7 m 0.25 FA, 0.1% 20 28, 6 Qual, Quan Isatis indigatica Fort., [108]
ESI-QqQ-MS BEH C18 (22.5 pg, 410 pg) leaves: flavonoids
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2549

Table 3. contd….

Strength
Stationary Flow Time Ana- Intention Sample Source:
Equipment (mm x Praticle Modifier Ref.
Phase (ml/min) (min) lytes (LOD, LOQ) Flavonoids
mm)

UPLC-DAD- Acquity 50 x 2.1 1.7 m 0.4 FA, 10% 10 27 Quan Milk-Based Products: [109]
ESI-QqQ-MS BEH C18 anthocyanins and fla-
vonols

UPLC-DAD- Acquity 100 x 2.1 1.8 m 0.4 AA, 0.2% 20 20 Quan Carob Flour: phenolic [110]
ESI-TQ-MS HSS T3 (0.75 ng, 2.5 ng) compounds

UPLC-DAD- Acquity 100 x 2.1 1.7 m 0.3 FA, 0.1% 25 109 Profile strawberry flowers: [111]
ESI-qTOF-MS BEH C18 total flavonoids

UPLC-DAD- Acquity 50 x 2.1 1.7 m 0.25 AA, 17 64 Qual Wen-Pi-Tang: [112]

ESI-qTOF-MS BEH C18 0.05% total flavonoids

Note: Certain common LC conditions of these studies were not listed. Detector, UV and / or MS; Elutent System, MeCN-H2O.
TFA, trifluoroacetic acid; AA, acetic acid; FA, formic acid; Qual, qualitative analyses; Quan, quantitative analyses; Profile, screening analyses.

Fig. (6). UPLC chromatograms of identified constituents (A) and different Herba Epimedii samples (B-F). Reprinted from Reference [46].

multaneous determination of 15 flavonoids was achieved within 12
min as illustrated in Fig. (6). This method was fully validated, with
LOD and LOQ lower than 0.13 and 0.52 ng on column, respec-
tively. The results showed great variations in contents of investi-
gated flavonoids among different samples. The clustering results
were in accordance with their flavonoids contents. Finally, four
flavonoids, epimedin A, B, C and icariin, were selected as markers
for quality control of Yin-Yang-Huo [46].
In compatible with fast speed and low concentration, MS detec-
tors with high sensitivity and scanning rate became optimal for
UPLC. Thus, the whole system is well suitable for trace analysis in
complex matrices, such as herbal decoctions, food products, and
body fluids. A UPLC-MS/MS method was established to analyze
anthocyanins and flavonols in yogurt and ice cream. With the com-
bination of MS, a total of 27 structural-similar analytes were suc-
cessfully separated and determined in 10 minutes, as shown in Fig.
(7). In addition, tandem MS/MS allowed maximal selectivity of ion
pairs, with an LOD of 0.3-30 ng/mL for glycosylated analytes, and
10-300 ng/mL for aglycones [109].
UPLC represents a powerful technology for the chemical analy-
sis of herbal medicines. A number of UPLC methods were estab-
lished for the rapid analysis of flavonoids in herbal extracts, includ-
ing Rose species, Trifolium species, Garcinia hanburyi, Epimedium
koreanum, Isatis indigatica, licorice root, lupin leaves, Aesculus
hippocastanum Seeds, strawberry flowers, carob flour, as well as
Chinese herbal medicine prescriptions, Buyang Huanwu Decoction
and Wen-Pi Decoction. A typical example was that 15 caged xan-
thones in the resin of Garcinia hanburyi were well separated within
6 minutes, using a UPLC-qTOF-MS system [102].
Monolithic columns have only played an important role in
HPLC recently. Completely different from packed columns, mono-
lithic columns consist of one piece of a continuous, porous material
2550 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

Fig. (8). Separation of 11 flavonoid aglycones by a monolithic column.

Reprinted from Reference [113].

Fig. (7). UPLC-MS/MS (A, B, C in different m/z) and UV-VIS (D) chroma-
tograms of anthocyanins and flavonols in a milk product. Reprinted from
Reference [109].

that is hermetically sealed against the wall of a tube, so the stream

of mobile phase cannot bypass any significant length of the bed but
must percolate through it. Besides an increase in the permeability
and theoretical plate, the most significant advantage of monolithic
HPLC columns is a reduction in the viscosity of the mobile phase.
This reduction directly shortens the analysis time while indirectly
increases the limit of maximum pressure, allowing a higher velocity
of mobile phase. This may create unexpected improvements in
resolution, especially for a series of compounds with similar struc-
tures. In Fig. (8), eleven main flavonoid aglycones in a botanical
extract were simultaneously determined in 15 minutes by using a
monolithic column [113]. Fingerprints for herbs like Licorice, Cas-
cara, and Curcuma were established using three hyphenated Chro-
molith Performance RP-18e as the stationary phase. More than 100 Fig. (9). Chromatograms of Lonicera japonica (Rendong) extract separated
on (A) C18 column, (B) HILIC Si column, and (C) C18 - HILIC Si column.
compounds were separated in 50 minutes in each of the three herbal Reprinted from Reference [116].
medicines [114].
Micro-liquid chromatography (LC) in conjunction with multi- mendously improve the resolution of hydrophilic compounds.
stage mass spectrometry (MSn) was introduced to study several HILIC uses similar mobile phase (a mixture of water and organic
major heartsease flavonoid glycosides. High-resolution LC sepa- modifiers) to reversed-phase HPLC, but exerts similar separation
ration was achieved by using a monolithic M column, eluted with mechanism to normal phase HPLC. A variety of packing materials
a flow rate of 2 L per min. With this LC-MSn approach, 16 fla- were available for HILIC columns. Besides classical bare silica and
vonoids were characterized in heartsease methanol extract [115]. aminopropyl-bonded silica, silica gels modified with many polar
functionalities such as amide, diol, cyano, sulfoalkylbetaine, cyclo-
4.3. Advance in Resolution: HILIC and 2D-HPLC dextrin are applicable. Flavonoids, especially hydrophilic ones,
could be well separated by HILIC. When HILIC columns with dif-
Various packing materials of different separation mechanisms
ferent mechanisms are arranged in tandem, the separation resolution
have been used in HPLC, which significantly improves the flexibil-
could be further improved, as shown in Fig. (9) [116]. Selected
ity in choosing a suitable stationary phase. Hydrophilic interaction
recent publications were summarized in Table 4.
chromatography (HILIC) provides a novel stationary phase to tre-
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2551

Table 4. Selection of Recent Publications on HILIC Analysis of Flavonoids

Strength Flow Time

Equipment Stationary Phase Praticle Modifier Analytes Sample Source Ref.
mm x mm (ml/min) (min)

Atlantis HILIC silica col- coeluted flavonoids in RP

HILIC-DAD 250 x 4.6 5.0 m 1 FA, 0.1% 18 12 [117]
umn mode
coeluted flavonoids in RP
HILIC-DAD lab made CD-based column 150 x 4.6 5.0 m 1 FA, 0.1% 25 14 [117]
mode
lab made CD-based analyti- flavonoids from licorice
HILIC-DAD 150 x 4.6 5.0 m 0.1 FA, 0.1% 30 15 [118]
cal column extract
lab made CD-based prepa- flavonoids from licorice
HILIC-DAD 150 x 30 5.0 m 17 FA, 0.1% 30 15 [118]
rative column extract

HILIC-DAD- TSKgel Amide-80 oligomeric proanthocyanid-

250 x 4.6 5.0 m 1 - 50 15 [119]
MS (amide-silica column) ins

HILIC-UV PolyHydroxyethyl A 150 x 0.6 3.0 m plant extract [120]

TSKgel Amide-80
HILIC-UV 250 x 2.0 5.0 m pumpkin phloem [120]
(amide-silica column)
mixed std: daidzein,
HILIC-UV polyols ZIC-pHILIC 50 x 4.6 5.0 m 0.5 AC, 30mM 10 3 [121]
kaempferol, taxifolin

Note: Certain common LC conditions of these studies were not listed. Detector, UV and / or MS; Elutent System, MeCN-H2O.
FA, formic acid; AC, ammonium carbonate.

tems are applied to one sample. 2D-LC can be conducted by trans-

Fig. (10). Contour plot and three-dimensional presentation of a comprehen-
sive 2D separation. Sample: mixed standards of flavones and phenolic ac-
ids; first-dimension column, PEG; second-dimension column, Chromolith
RP-18e. Reprinted from Reference [122].
Two-dimensional liquid chromatography (2D-LC) refers to the
technique in which two independent liquid phase separation sys-
ferring either only the interesting portion of the first dimension
effluent to the second dimension, which is referred to as “heart-
cutting” 2D mode, or by transferring sequentially the entirety of the
first dimension effluent, in many small aliquots, to the second di-
mension, known as “comprehensive” 2D mode. Due to the wide
variety of separation mechanisms that LC offers, 2D-HPLC can be
theoretically carried out in many multi-dimensional combinations,
generating increased peak capacity, selectivity and resolution for
the analysis of flavonoids. To establish a 2D-HPLC system, the
combination of columns should be rationally selected, based on
correlations of the retention and the structure, and suitability and
selectivity. In their analyses of flavones and phenolic acids, Jandera
et al. studied the relationship between structure and retention on all
types of columns [122]. The final optimized comprehensive 2D-
HPLC conditions were employed for separations of complex anti-
oxidants from natural resources, as shown in Fig. (10). Under simi-
lar comprehensive mode, combinations of ILC column - Kromasil
ODS column [123], and BDS cyano CN column - C18 RP column
[124] were also successfully applied to separate flavonoids from
complex herbal extracts.
Compared with the comprehensive mode, heart-cutting mode is
more likely to contribute pure fractions, though more difficult to be
automated. By employing size exclusion chromatography in the
first dimension and reversed-phase chromatography in the second
dimension, a 2D-LC-ESI-MS analysis of the leaves of Maytenus
ilicifolia was carried out, as in Fig. (11). A great number of flavonol
glycosides were identified and quantified within a short time [125].
In the advanced chromatographic techniques presented above,
UPLC is the most favorable and prevalent analytical approach for
flavonoids, as well as for other naturally-occurring compounds.
However, for a routine laboratory technique, some practical con-
cerns, such as sample introduction and reproducibility still need an
improvement due to the ultra-high pressure of LC system. Mono-
lithic column, which gives new possibilities in LC, was also con-
sidered promising. 2D-HPLC exhibits powerful capability for com-
plex extracts, serving different intentions such as comprehensive
2552 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

5. MASS SPECTROMETRY AND LIQUID CHROMATOG-

RAPHY COUPLED WITH MASS SPECTROMETRY (MS
AND LC/MS)
Over the past few years, the coupling of mass spectrometry to
liquid chromatography has been spreading rapidly for the analysis
of flavonoids. Modern mass spectrometric techniques, which nota-
bly improved the determination and structural characterization,
played a key role in flavonoid analyses [22,130]. A number of pa-
pers have summarized advances in LC/MS analysis of flavonoids,
particularly, general MS methods [22,23], structural elucidation
[131-134], MS analysis of flavonoids in plants [135], and biological
samples [136,137]. This review will focus on the advances in (1)
structural analysis, (2) fast screening and profiling, and (3)
quantification.

5.1. Structural Analysis by Mass Spectrometry

Fig. (11). Two-dimensional liquid chromatography plotting for leave ex-
tracts of M. ilicifolia. First-dimension column, size exclusion; second-
dimension column, reversed phase. Reprinted from Reference [125].
separation or pure, single-constituent fractions. Comparatively,
HILIC and other novel stationary phases are more “compound-
dependent”, which limited their universal application.
4.4. Pre- and Post- Column Advances
Pre-column treatment and post-column derivatization could also
improve the features of HPLC (Table 5). Pre-column treatments
mainly include ultrafiltration, and purification with a MIC column
or lab-made click OEG column. Post-column manipulations include
derivatization for UV detection, manganese complexation for MS
ionzation, SPE treatment for NMR determination, and DPPH/ABT
for on-line determination of antioxidant activities of flavonoids.
LC/MS provides abundant structural information, including
pseudomolecular ions and fragment ions, for the identification of
unknown compounds [130]. To deal with various and unpredictable
analytes in low concentrations, higher requirements were set for
both interfaces and analyzers of mass spectrometers. Prasain et al.
compared different ionization techniques for the ionization of fla-
vonoids [136] (Table 6). Among these MS ionization sources, EI
and APCI are mainly employed for flavonoid aglycones, due to
their relative hydrophobility. FAB gives better performance for
flavonoid glycosides. ESI covered a wide range of flavonoids,
which is available for both qualitative and quantitative analyses.
Among various techniques, ESI and APCI were the most widely
used, and were suitable for most types of flavonoids.
Different types of flavonoids could be differentiated according
to their fragment ions in tandem mass spectrometry. Most charac-
teristic ions for flavonoids were due to fragmentations in C ring,
including retro-Diels-Alder (RDA) fragmentation, heterocyclic ring
fragmentation, quinone methide fragmentation, benzofuranforming
fragmentation, etc [138]. Fig. (12) provided a schematic summary

Table 5. Selection of Pre and Post Column Techniques to Analyze Flavonoids

Strength
Stationary Time
Equipment Pre-column mm x Praticle Post-Column Modifier Analytes Intention Ref.
phase (min)
mm

enrich flavonoid
UPLC-DAD-ESI- Acquity RP
click OEG 100 x 2.1 1.7 m - FA, 0.3% 50 51+18 glycosides and [107]
qTOF-MS C18
phenolic acids

enrich flavonoid
SPE-HPLC-UV MIP Target ODS 3 250 x 4.0 5.0 m - [126]
antioxidants

screen and idenitify

LC-DAD-MSn +
ultrafiltration Dikma C18 150 x 4.6 5.0 m - AA, 0.5% 15 4 lucosidase Inhibi- [95]
FTICR MS
tors

LC-PDA-PCD-MS - C18 symmetry 250 x 4.6 5.0 m PCD FA, 0.5% 20 19 identification [127]

distinct isomeric
LC-UV-PMC-MS - C18 symmetry 50 x 2.1 3.5 m PMC, +MnCl2 FA, 0.33% 30 8+7 monoglycosyl [128]
flavonoids

LC-DAD-NMR- screen and identify

- Alltima-C18 150 x 2.1 5.0 m DPPH/ABTS FA, 0.1% 35 15 [129]
DPPH/ABTS of antioxidants

HPLC-DAD-SPE-
- Luna C18(2) 150 x 4.6 3.0 m SPE - 60 4 (15) for NMR analysis [96]
NMR

Note: Certain common LC conditions of these studies were not listed. Elutent System, MeCN and / or methanol-H2O.
Click OEG, click oligo (ethylene glycol); MIP, molecularly imprinted polymer; PCD, post-column derivatisation; PMC, postcolumn manganese complexation; DPPH, diphen-
ylpicrylhydrazyl; ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid).
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2553

Table 6. Comparison of Mass Spectrometry Ionization Techniques for Flavonoid Analysis. Reprinted from Reference [136]

Ionization
Major Application Advantages Disadvantages
Technique

EI Mainly aglycone analysis Easy combination with GC Derivatization needed, labor intensive

(qualitative and quantitative) Highly sensitive Limited mass range

Identification of unknown possible Possible thermal decomposition

High fragmentation

Often results in no observable molecular ion

FAB Flavonoid glycosides Extended mass range up to 7000 Da Low sensitivity

(plant samples) Soft ionization technique Requirement of solubility of sample in matrix

High mass limit High background matrix peaks

MALDI-TOF Flavnoid glycosides, Tolerant of mM concentration of salts Low resolution

proanthocyanidins, High-throughput analysis High matrix background signals little use

and condensed tannins for small molecules

APCI Flavonoid aglycones Practical mass range up to 2000 Da May be not good for laser-sensitive compounds

Highly sensitive (femtomole) Sensitivity may be variable with compound type

HPLC/MS capable Possibility of thermal decomposition

ESI Wide range of flavonoids High mass range Relatively low salt tolerance

(qualitative and quantitative) HPLC/MS capable Multiple charge states can be confused in mixtures

Multiple charge resolution -2000 Analysis may be difficult for non-ionizable compounds

Sensitivity femtomole to picomole No or less tolerance for heterogenous mixtures

Fig. (12). Schematic summary of principal fragment patterns for various types of flavonoids.
2554 Current Organic Chemistry, 2011, Vol. 15, No. 15
Fig. (13). Schemematic fragmentation pathways of two homoisoflavones (compounds 1 and 2) in negative ion mode. Reprinted from Reference [145].
Fig. (14). Tandem mass spectra (A, B, C) and characteristic fragmentation pathways (D) of a proanthocyanidin dimer in positive ion mode. A, MS2 of m/z 563,
B, MS3 of m/z 411, C, MS3 of m/z 437. Reprinted from Reference [138].
of principal fragment patterns for various types of flavonoids, in-
cluding flavones, flavonols, flavanones and isoflavones, xanthones,
anthocyanins, isoflavans, flavan-3-ols, and homoisoflavones
[23,138,140,143-146]. Schematic fragmentation pathways of ho-
moisoflavones [145] and proanthocyanidin dimers [138] were pre-
sented in Fig. (13) and Fig. (14), respectively. In addition, the frag-
mentation behaviors of various flavonoids, including caged
xanthones [102], flavonoid C- and O- glycosides [133,147,148],
isoprenylated [149] and prenylated [105] flavonoids have been
intensively studied.
MALDI MS, combined with CID and SID techniques in tandem
mass spectrometry, were applied to differentiate isomeric com-
pounds according to the abundance of specific fragment ions [132].
Computational methods were employed as well to provide com-
puted structures that indicated significant intramolecular hydrogen
bonding and rotation form a coplanar structure [150,151,131].
March et al. compared the product ion mass spectra of a series
of isomeric deprotonated flavonoid glucosides, including flavones,
flavanones, and isoflavones. These compounds could be explicitly
distinguished by their breakdown curves of major product ions at
different collision energy levels [133]. Geng et al. studied the frag-
mentation behaviors of three isomeric quercetin mono-O-glycosides
(3-, 7-, 3
-) under different CID techniques. These techniques gave
Qiao et al.
different performance on the radical cleavage of the quercetin
mono-O-glycosides, as illustrated in Fig. (15). In addition, the ion
kinetic energies may have a definite effect on the occurrence of
radical product ions [148]. In some cases, when the fragmentation
behaviors of reference compounds were thoroughly studied, it was
possible to unambiguously identify the structures of unknown com-
pounds by LC-UV-MSn [95,105,143,152].
Four flavonoids, with their structures clearly elucidated by a
SORI-CID FTICR MS, were identified from the leaves of haw-
thorn. Moreover, with in vitro
-glucosidase inhibition assay com-
bined to LC-DAD-ESI-MSn, these flavonoids were proposed as
-
glucosidase inhibitors [95]. With the aid of polarity switching in
MS, a total of 14 flavonol glycosides were identified form Mangif-
era indica L. Based on authentic reference compounds, flavonols
and xanthones were analyzed in negative mode, while anthocyanins
and rhamnetin hexosides were analyzed in positive mode. Two of
the 14 compounds were isolated and their structures were authenti-
cated by NMR analysis.
Due to the complicated MS/MS fragmentation behaviors of fla-
vonoids, it should be valuable to propose a structure interpretation
guideline [105] or decision tree [143], as illustrated in Fig. (16).
The decision tree, including UV max absorptions and MS/MS diag-
nostic ions, was successfully applied to the identification of 70
mono- and di-prenylated flavonoids from Glycyrrhiza glabra.
Extraction, Separation, Detection, and Structural Analysis of Flavonoids
Fig. (15). Ion abundance ratio ([Y0-H]- / [Y 0]-) of isomeric flavonol-O-
glycosides under different CID mechanisms. (a), collision energy of 3D ion
trap; (b), collision energy and (c), excitation energy AF2 of the quadrpole
LIT. Reprinted from Reference [148].
Fig. (16). Decision tree for the identification of flavonoids by UV and negative APCI mass spectrometry. Reprinted from Reference [143].
Current Organic Chemistry, 2011, Vol. 15, No. 15 2555
5.2. Fast Screening and Profiling
Fast screening or profiling of flavonoids in complex extracts or
matrix is one of the most demanding fields to which LC/MS can be
applied. The most favorable character of MS is versatile, which
made it a universal detector: choices of the interface, ionization
modes, scan range, and different data acquisition programs. Besides
multiple mass analyzer types, more choices were provided in data
acquisition and data process in qualitative analysis. Options in
source-dependent techniques include polarity switching, collision-
induced dissociation (CID), source-induced dissociation (SID).
Options in analyte-dependent techniques including full scan, selec-
tion ion monitoring (SIM), information-dependent data acquisition
(IDA), neutral loss (NL), product/parent ion scan, data dependent
analysis (DDA), tandem mass spectrometry (MS/MS), multi-stage
mass spectrometry (MSn, n=2-5). Moreover, software in structure
prediction also contributed to data processing [153].
With the aid of the above techniques, LC/MS for flavonoid
screening had been developing rapidly. Selected reports in flavon-
oid profiling were listed in Table 7. Among these studies, analysis
efficiency was improved by shortening separation time and enhanc-
ing mass resolution. As shown in Fig. (17), more than 96 phenolic
compounds were identified in 60 min for the analysis of tea, among
which over 30 phenols were newly reported [157]. The identified
compounds included catechins, proanthocyanidins, C- and O-
glycosylated flavonoids, and purine alkaloids. In another case,
monomeric phenols of cashew apple was screened by HPLC-
DAD/ESI-MS analysis, by which the simultaneous identification of
myricetin and quercetin hexosides, pentosides, rhamnosides, and
anthocyanidin glycosides was achieved [164] in spite of an overlap
in UV detector, as shown in Fig. (18).
2556 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

Table 7. Selection of Recent Applications of LC-MS for Fast Screening of Flavonoids

Scan Program / Scan

Sample Flavonoids Ionization Analyzer LC Eluents Ref.
Mode

71 isoflavones daidzein and genistein

information-dependent MeCN, H2O, FA
yellow soybean aglycones, monoglycosides,multi- ESI(+) LTQ [154]
data acquisition (47 min)
glycosides

MeOH, MeCN,
MS/MS, XIC, neutral loss
Ginkgo biloba 72 flavonoids and 5 terpene lactones ESI(-) IT H2O, FA [153]
data-dependent mode
(140 min)

tandem mass spectrometry

MeCN, H2O, AA
Lotus japonicus 61 flavonoids ESI(+) FTICR predicted conjugate struc- [155]
(48 min)
tures

3 Compositae plants
MeCN, H2O, FA
Chrysanthemum morifolium Ra- 41 flavonoids and 6 caffeoylquinic
APCI(+) IT selection ion monitoring (pH 4) [156]
man, Artemisia annua, and Chry- acids
(90 min)
santhemum coronarium

12 catechins, 6 proanthocyanidins, 6
polarity switching
66 green and fermented teas theaflavins and theaflavates, 19 O-G- MeCN, H2O, FA
ESI(+) IT various fragmentation [157]
Camillia sinensis or its varieties flavonols, 7 C-G-flavones, 28 acylated (105 min)
voltages
glycosylated flavonols, 3 flavonols

7 Epimedium species
E. brevicornum, E. sagittatum, E.
126 compounds tandem mass spectrometry MeCN, H2O, FA
pubescens, E. wushanense, E. ESI(+) IT [158]
flavonoids and quinic acids (MSn, n = 2-5). (70 min)
koreanumNakai, E. myrianthu-
mand E. leptorrhizum

6 rhubarb species 107 phenolic compounds

Rheum officinale, R. palmatum, R. sennosides, anthraquinones, stilbenes, tandem mass spectrometry MeCN, H2O, AA
ESI(-) IT [159]
tanguticum,R. franzenbachii, R. glucose, gallates, naphthalenes, and (MSn, n = 2-4). (40 min)
hotaoense, and R. emodi catechins

2 Cuscuta species 50 phenolic compounds tandem mass spectrometry MeCN, H2O, AA

ESI(-) IT [160]
C. chinensis and C. australis including 23 flavonoids (MSn, n = 2-4). (41 min)

IT tandem mass spectrometry MeCN, H2O, AA

peanut skins 83 A-and B-type proanthocyanidins ESI(-) [161]
LTQ data-dependent mode (60 min)

22 anthocyanins and anthocyanin

derivatives
multi-stage mass spec-
27 anthocyanin-(epi)catechin conden- MeCN, H2O, FA
red wine ESI(+) IT trometry [162]
sation products (80 min)
verified by direct-infusion
6 anthocyanin-ethyl-dimer procyanidin
condensation products

MeCN, H2O
hops 17 A-and B-type proanthocyanidins ESI(+) IT directly loop-inject [163]
(direct infusion)

109 flavonoid metabolites

strawberry flowers ellagitannins, proanthocyanidins, multi-stage mass spec- MeCN, H2O, FA
ESI(+) qTOF [111]
(Fragaria ananassa) flavonols, terpenoids, and spermidine trometry (25 min)
derivatives

58 monomeric phenolsmyricetin and

cashew apples(Anacardium occi- MeCN, H2O, FA
quercetin hexosides, pentosides, rham- ESI(+) IT polarity switching [164]
dentale L.) (80 min)
nosides, anthocyanidin glycosides

Note: XIC, extraction of ion chromatogram; MS/MS, tandem mass spectrometry; AA, acetic acid; FA, formic acid; LTQ, quadrupole-linear ion trap mass spectrometer.

Direct loop-injection without any chromatographic separation,
which was generally used in optimizing ionization and collision
parameters of known compounds, has been employed as an alterna-
tive method for flavonoid profiling. A fraction from a hop extract,
containing oligomeric proanthocyanidins (PAs), was directly loop-
injected into the ESI source. Comprehensive mass spectral frag-
mentation patterns were established for sequencing A-type and B-
type PAs in positive ion mode, as shown in Fig. (19). The A-type
and B-type PAs could be distinguished according to their mass
spectra in three aspects: the 2-Da mass differences in the pseudo-
molecular ions, the propensity to undergo QM fissions, and the
reluctance of A-type linkages to undergo RDA fissions [163].
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2557

Fig. (17). Screening and identification of flavonoids from five tea samples using LC/MS. Reprinted from Reference [157].

Fig. (18). MS profiling of monomeric phenols from apple skin extract under different detection wavelengths. Reprinted from Reference [164].

The authentication of herbal medicines is difficult due to the
presence of adulterants with similar appearance and chemical com-
position. LC/MS represents a powerful tool to distinguish similar
plant species. Tu-Si-Zi is a tonic Chinese herbal medicine to im-
prove sexual function, regulate endocrine and immune system, and
prevent senescence. Two Cuscuta species, Cuscuta chinensis and C.
australis, were equally used as this drug in the market. Ye et al.
fully analyzed the chemical constituents of these two species by
HPLC/DAD/ESI-MSn, and discovered significant difference be-
tween the species [160]. Kaempferol and astragalin were the pre-
dominant constituents of C. australis, while hyperoside was the
major compound in C. chinensis. In another case, seven Epimedium
plants, including both official and unofficial species, were chemi-
cally differentiated, with the identification, profiling and compari-
son of 126 phenolic constituents [158]. Similar techniques could be
used to improve the quality control of Chinese herbal medicines.
5.3. Quantitative Analysis
LC/MS has been proved to be one of the most sensitive tech-
niques for the determination of flavonoids in plants and foodstuffs,
with limits of detection (LOD) at the level of ng/ml [22]. Various
2558 Current Organic Chemistry, 2011, Vol. 15, No. 15
Fig. (19). Mass spectra obtained with direct loop-injection in MS profiling. Ion mode, positive; Interface, ESI; Sample, complex oligomeric proanthocyanidins
from hop extract. Reprinted from Reference [163].
types of mass analyzers were employed for flavonoids analysis,
including ion trap, single quadrupole, triple quadrupole, and time-
of-flight MS. Quantitative analysis of flavonoids, with optimized
MS program of selected ion monitoring (SIM), LOD are even better
than fluorescence or electrochemical detection [22]. Moreover,
highly specific scan modes like selected reaction monitoring (SRM)
and multiple reaction monitoring (MRM) could significantly avoid
the interference of sample matrix [24].
In recent years, studies using LC/MS for flavonoid analysis
have been increasing. In a determination of 14 phenolic com-
pounds, the LODs (0.44-127.78 g/kg) and LOQs (1.11-427.78
g/kg) were lower in LC-MS/MS than LC-fluorescence and LC-
DAD [165]. By using MRM detection mode, LODs and LOQs
ranged from 0.3-30 ng/mL and 1-100 ng/mL for glycosylated ana-
lytes, respectively [109]. By comparing ionization sources, ESI was
found to improve precision, sensitivity, and LOD over APCI for
certain analytes, especially in the case of 7, 4'-isoflavandiol (LODs:
0.3 ng/ml for ESI, 2.7 ng/ml for APCI) [166]. In another study, SIM
scan mode was used for LC/MS quantitation, and showed good
selectivity and sensitivity (0.5-12 ng/mL) for flavonoids than diode-
array detector (2-30 ng/mL) [167]. Generally, as a detector, MS is
more sensitive than UV, DAD, and fluorescence for flavonoids. In
terms of MS scan modes, the sensitivity from higher to lower was:
SRM=MRM > SIM > full scan. A main drawback of MS detector
was its poor repeatability, when compared to DAD detector. As a
common situation for MS detection, the RSD is often up to 10-15%
[167]. Table 8 summarized selected quantitative analysis using
LC/MS, with special emphases on data acquisition programs, cali-
bration processes, and limits of quantification.
By using SRM, a fast separation and determination of isofla-
vonoids genistein, daidzein, formononetin, and biochanin A was
achieved within 1.5 min under isocratic conditions [34]. In a similar
study, 27 similar anthocyanins and flavanols, including isomeric
compounds, were simultaneous determined in 10 min, as shown in
Fig. (7). This method showed a dynamic range of 2-180 000 ng/mL
for glycosylated analytes, and 60-600 000 ng/mL for aglycones
[109].
6. BIOANALYSIS
Due to the popular use of flavonoid-containing medicinal and
dietary herbs, there is an ever-increasing interest in the detection of
Qiao et al.
flavonoids in biological matrices. The most popularly used tech-
nique is HPLC, in combination with UV, fluorescence, and electro-
chemical detection. Furthermore, GC, CE, fluorescence quenching,
and immunoassay were also used [137]. Since most flavonoids have
good UV absorption, less sensitive detectors like ELSD or RI were
not frequently used. When the concentrations of flavonoids are
fairly low in biological matrices, sensitive technologies like LC/MS
are indispensable [136,137]. In addition, LC/MS could provide
abundant structural information of unknown metabolites for their
identification. The bioanalysis of flavonoids had been summarized
[136,137]. This review focuses on the recent advances in sample
treatment and in bioanalysis of flavonoids by LC/MS.
6.1. Pre-treatment of Biological Samples
In most cases, biological samples require a pretreatment to get
rid of endogenous proteins, carbohydrates, salts, and lipids. For
LC/MS analysis, sample pre-treatment could remove matrix com-
ponents that might contaminate the system or cause ion suppression
[137]. Chen et al. made a comprehensive summary of sample
preparation techniques for bioanalysis [174].
6.1.1. Conventional Techniques
Three techniques are the most-frequently used pretreatment
methods in flavonoids bioanalysis: protein precipitation (PPT),
solid phase extraction (SPE) and liquid-liquid extraction (LLE)
[137]. A schematic comparison among these methods was made by
Chang et al. [175], as illustrated in Fig. (20). Protein precipitation
(PPT) is the simplest and most universal approach for sample pre-
treatment [137]. Water miscible organic solvents (acetonitrile or
methanol) are usually used to precipitate protein [176]. However,
the supernatant might still contain a significant amount of un-
precipitated endogenous components. Due to the selectivity of MS
detectors, complicated sample pretreatment is not necessary, and
additional centrifugation is sufficient to separate the resultant pro-
tein precipitates from biofluids before LC/MS analysis [177]. It is
also worth noting that precipitation of proteins with acid may cata-
lyze the hydrolysis of some conjugates such as glucuronides and
sulfates [178]. Recently, PPT method has become fully automated.
Samples could be treated in 96-well plates before LC-MS/MS
analysis [177].
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2559

Table 8. Selection of Recent Applications of LC-MS for Flavonoids Quantitative Analysis

Sample Analytes Ionization Analyzer Scan Calibration LOD / LOQ LC eluents Ref

Hypericum 50 flavonoids ESI(+) TOF SIM polarity switching, continuous LOD: 0.04-0.1 g/mL MeOH, H2O, [168]
perforatum, flavonols, flavanones, exact mass measurement LOQ: 0.2 mg/mL FA
Rhodiola flavones, catechins, IS(+):dextromethorphan Range: 0.2-10 mg/mL (23 min)
rosea, anthocyanins ES(-):quercetin-nonglycosidic (kuromanine)
red grape flavonoids; rutin-diglycosides; 0.2-4 mg/mL
wine, quercitrin-monoglycosides (other standards)
orange juice, ES(+):quercetin-
green tea methoxyflavonoids; kuromanine-
anthocyanin glucosides

tobacco leaves 12 glycosides APCI(+) QTRAP MRM ES: [M + H]+ -> [M + H-162]+ or LOD: 10.9ng/mL MeCN, H2O, [169]
scopolin, rutin, quer- [M + H-146]+ Range: 0.008-1.6 FA
cetin-3-glycoside, and mg/mL (38 min)
kaempferol-3-
rutinoside
soybeans isoflavonoids ESI(-) QqQ MRM m/z 269/133 CE40 eV (genistein) LOD: 0.02-0.08 g/g MeOH, H2O, [34]
genistein, daidzein, m/z 253/224 CE40 eV (dadzein) LOQ: 0.04-0.2 g/g FA
formononetin and m/z 267/252 CE 30 eV (for- Range: 0.1-10 g/L (2 min)
biochanin A mononetin) (daidzein, genistein)
m/z 283/268 CE 30 eV 0.02-2g/L (biochanin
(biochanin A) A, formononetin)
green tea 8 flavonoids ESI(+) IT SIM ES: epigallocatechin gallate, N/A MeCN, H2O, [35]
Camellia total epigallocatechin, epigallocatechin, epicatechin FA
sinensis epicatechin, catechin, gallate, epicatechin, gallocatechin (40 min)
and gallocatechin gallate, gallocatechin, catechin
gallate, catechin
Lycium barba- 52 phenolic acids and ESI(-) QqQ SIM IS: 3-hydroxybenzoic acid, hes- N/A MeCN, H2O, [170]
rum flavonoids peridin FA
Linnaeus ES:caffeic acid, chlorogenic acid, (70 min)
pcoumaric acid, rutin, and
kaempferol-3-O-rutinoside
cocoa sources 4 procyanidins, 10 ESI(+) TQ MRM MRM for all 30 analytes N/A MeCN, H2O, [171]
phenolic acids, 14 FA
flavones, 2 alkaloids (18 min)
almond skin 16 flavonoids and 2 ESI(-) IT SIM IS: daidzein LOD: 0.031-0.27 pmol MeOH, H2O, [172]
polyphenols phenolic acids ES: m/z 137, 153, 289 (0-22.6 LOQ: 0.58 pmol FA
min) (flavonoid glycosides) (33 min)
m/z 137, 287, 433, 447, 463, 477,
593, 609, 623 (22.6-28.2 min)
m/z 253, 271, 285, 287, 301, 315
(28.2-50 min)
grape berries 12 anthocyanins ESI(+) QqQ SIM Qual: MS/MS (-) product ion scan LOD: < 200 ng foran- MeCN, [173]
conjugates, 7 flavan-3- Quan: QqQ (+) full scan thocyanins, MeOH, H2O,
ols, 8 flavonols, 6 < 100 ng for other FA
stilbenes analytes (18 min)
Range: 0.1-200 ng/mL
milk-based 27 anthocyanins and ESI(+) QqQ MRM MRM for all 27 analytes LOD: 0.3-30; 10-300 MeCN, H2O, [109]
food products flavonols ng/mL FA
LOQ: 1-100; 30-1000 (10 min)
ng/mL
Range: 2-180000; 60-
600000 ng/mL
(glycosides; agly-
cones)

Note: AA, acetic acid; FA, formic acid; IS, internal standard; ES, external standard; MRM, multiple reaction monitoring; SRM, selected reaction monitoring; SIM, selection ion
monitoring.

SPE is a popular sample preparation method used in routine
bio-analytical laboratories [176]. In the bioanalysis of natural prod-
ucts, SPE represents high selectivity, effective pre-concentration,
and the potential for automation. Especially, SPE reduces the serum
background [137]. The efficiency of SPE depends on the type of
sorbents, the sample volume and pH, the content of organic modi-
fier, and the volume of elution solvent [179]. SPE can be performed
off-line manually, semi-automated, or on-line. On-line methods
provided lower LOD and higher reproducibility than off-line meth-
ods [180], facilitating high-throughput analyses of biological sam-
2560 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

traction, turbulent chromatography (TFC), salting-out liquid-liquid

Fig. (20). A schematic comparison among various sample preparation
methods. Reprinted from Reference [175].
ples [181,182]. A wide range of cartridges and solvents were avail-
able, which made SPE a versatile technique. SPE cartridges that are
extensively used include: SH, GP, C18, C8, C2, Oasis MAX, Oasis
HLB, and MCX cartridges [183,184].
Liquid-liquid extraction (LLE) is suitable for lipophilic com-
pounds. Flavonoids in biofluids were usually extracted by ethyl
acetate after acidification [137]. Classical LLE operates manually,
which was time-consuming and labor intensive. Improved tech-
niques include membrane-separated liquid extraction (MSLE),
countercurrent chromatography (CCC), and single drop microex-
traction (SDME) [185]. For urine, the expected high concentration
of metabolites allows for a simple dilution of sample prior to analy-
sis. In some cases, the biological sample was subjected to PPT-SPE
or SPE-LLE procedures [186]. Semi- and fully automated LLE was
also reported [181].
extraction (SALLE), stir bar sorptive extraction (SBSE), etc. A
comparison was made in a recent investigation among these tech-
niques on extraction time, selectivity, solvent consumption, etc
[176]. Some of these techniques are not yet employed for scientific
purpose. However, a good prospect could be expected for their
application in sample preparation of blofluids.
Molecularly imprinted polymers (MIPs) as affinity based sepa-
ration media for sample preparation, with specific cavities formed
from a template molecule which possesses specific molecular rec-
ognition mechanism. As a consequence, the MIP selectively ex-
tracts the template molecule, offering an easy, low cost and rapid
preparation procedure, and showing high thermal and chemical
stability. MIP was also proved high robustness and easy to obtain,
which allowed their multiple uses in SPE [187, 174]. MIP was used
for offline extraction of quercetin and rutin from wine, orange juice,
and tea.
Micro-extraction by packed sorbent (MEPS) is a new miniatur-
ized SPE technique which allows on-line connection to GC or LC.
MEPS significantly reduces the volume of solvents and sample
needed. Moreover, MEPS reduces the phospholipids concentration
considerably and gives lower noise in MS compared to PPT and
LLE [188]. The miniaturization and automation using on-line cou-
pling of analytical methods is a very important development as well
[176]. On-line sample preparation was able to integrate sampling,
purifying, desalting and concentrating into one step and was thus
especially useful for treating samples with a limited volume [175].
6.2. Bioanalysis of Flavonoids by LC/MS

6.1.2. Current trends and Advanced Techniques
Newly developed sample preparation techniques include solid
phase micro-extraction (SPME), liquid-liquid micro-extraction
(LLME), pressurized liquid extraction (PLE), extraction using re-
stricted access material (RAM), micro-extraction by packed sorbent
(MEPS), molecularly imprinted polymer (MIP), monolith pin ex-
LC/MS was believed to be the most promising technique for the
measurement of metabolites in biofluids due to the inherent high
selectivity afforded through both of the hyphenated techniques
[189]. LC/MS technique was fully developed for identification and
quantification of metabolites [190]. Selected applications using
LC/MS were listed in Table 9. The hyphenation of UPLC and TOF-

Table 9. Selection of Recent Publications of LC-MS Bioanalyses for Flavonoids

Sample source Biomatrix Extraction Analyzer Scan mode LOD; LOQ Ref.

CTN986 metabolites serum SPE QqQ ESI(+), MRM 2 ng/mL [191]

Cyanidin-3-glucuronide: metabolites plasma SPE qTOF ESI(+), MS/MS - [192]
Scutellarin metabolites plasma SPE QqQ ESI(+), SRM 0.2 ng/mL [180]
Anthocyanin metabolites urine SPE QqQ ESI(+), MS/MS 0.001 M [193]
Koparin metabolites urine LLE IT ESI(-), MSn - [194]
Abelmoschus manihot
urine, plasma PPT qTOF ESI(-), MSn - [195]
Flavonoid metabolites
Xindi soft capsule: metabolites urine PPT qTOF ESI(+), MSn - [196]
Licorice flavonoids:
plasma LLE QqQ ESI(-), SRM - [197]
Isoliquiritigenin,isoliquiritin, liquiritin, and liquiritigenin
Selected chiral flavonoids: hesperetin, Naringenin and
urine, plasma PPT SQ ESI(-), SIM 0.5 g/ml [198]
eriodictyol
0.13; 0.16 ng/ml
Diosmin and diosmetin plasma PPT IT APCI(+), MRM [199]
0.24; 0.25 ng/ml
Ginkgo flavonoids plasma PPT QqQ ESI(-), SRM [200]
Cocoa metabolites:
urine, plasma SPE QqQ ESI(-), SRM 0.030; 0.50 g/L [201]
Epicatechin, procyanidins, and phenolic compounds

Note: SPE, solid phase extraction; LLE, liquid-liquid extraction; PPT, protein precipitation; MRM, multiple reaction monitoring; SRM, selected reaction monitoring; MS/MS, tan-
dem mass spectrometry; SIM, selection ion monitoring; MSn, multi-stage mass spectrometry.
Extraction, Separation, Detection, and Structural Analysis of Flavonoids
Fig. (21). UPLC/MS chromatograms of Abelmoschus manihot extracts (c) and its metabolites in rat urine (a) and plasma (b). Reprinted from Reference [195].
MS was frequently employed, while the ionization technique was
predominantly APCI and ESI for flavonoids. In the case of urinary
metabonomics study of Xindi soft capsule, a TCM preparation by
UPLC-qTOF-MS, the results provided a reasonable explanation to
the mechanism of traditional Chinese medicine preparation [196].
With the aid of automated data analysis software, the post-
acquisition data could be easily processed. For example, UPLC-
qTOF-MS data could be analyzed by a professional software called
MetaboLynx. As illustrated in Fig. (21), a total of 16 and 38 me-
tabolites were identified with the assistant of MetaboLynx from
plasma and urine samples, after administration of flavonoids of
Abelmoschus manihot [195]. This type of investigations demon-
strated the great potential of UPLC-qTOF-MS combined with data
analysis software as an ideal approach for the identification of me-
tabolites from Chinese herbal medicines.
7. CONCLUDING REMARKS
This article summarizes recent progress in the extraction, sepa-
ration, detection, and structural analysis of flavonoids, especially
the chemical analysis of flavonoids in medicinal and dietary herbal
extracts. New technologies like MAE and PLE could extract fla-
vonoids from complicated matrices more efficiently than conven-
Current Organic Chemistry, 2011, Vol. 15, No. 15 2561
tional methods while avoiding degradation. Column chromatogra-
phy and TLC continue to be major approaches for the preparative-
scale separation and routine analysis of flavonoids. New absorption
materials with novel separation mechanisms could significantly
improve the separation efficiency. HPLC is still the most com-
monly used technology for the analysis of flavonoids. New ad-
vances in the past few years, such as UPLC, monolithic column,
HILIC column, and 2D-HPLC significantly enhanced the versatility
and resolution of HPLC, and shortened the analysis time. LC/MS
provides abundant structural information for the rapid identification
of unknown flavonoids, but also provides a highly sensitive and
specific technology for the quantitation of flavonoids in herbal ex-
tracts and biological samples. The above technical advances allow
rapid, accurate, and sensitive analysis of flavonoids, and will be
widely used for the chemical analysis of flavonoids in complicated
matrices like medicinal and dietary herbs, and for the quality con-
trol of related products.
ACKNOWLEDGEMENTS
This work was supported by Peking University Health Science
Center (Grant No. 985-2-119-121).
2562 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.

ABBREVIATIONS [14] Wang, X.L.; Wang, N.L.; Zhang, Y.; Gao, H.; Pang, W.Y.; Wong, M.S.;
Zhang, G.; Qin, L.; Yao, X.S. Effects of eleven flavonoids from the osteo-
DAD = diode-array detector protective fraction of Drynaria fortunei (Kunze) J.Sm. on the osteoblastic
proliferation using an osteoblast-like cell line. Chem. Pharm. Bull., 2008, 56,
ESI = electrospray ionization 46-51.
gas chromatography coupled with mass [15] Zhang, J.F.; Li, G.; Meng, C.L.; Dong, Q.; Chan, C.Y.; He, M.L.; Leung,
GC/MS = P.C.; Zhang, Y.O.; Kung, H.F. Total flavonoids of Herba Epimedii improves
spectrometry osteogenesis and inhibits osteoclastogenesis of human mesenchymal stem
HILIC = hydrophilic interaction chromatography cells. Phytomedicine, 2009, 16, 521-519.
[16] Lee, J.W.; Ahn, J.Y.; Hasegawa, S.; Cha, B.Y.; Yonezawa, T.; Nagai, K.;
HPLC = high-performance liquid chromatography Seo, H.J.; Jeon, W.B.; Woo, J.T. Inhibitory effect of luteolin on osteoclast
liquid chromatography coupled with mass differentiation and function. Cytotechnology, 2009, 61, 125-134.
LC/MS = [17] Spencer, J.P.E. The impact of flavonoids on memory: physiological and
spectrometry molecular considerations. Chem. Soc. Rev., 2009, 38, 1152-1161.
LLE = liquid-liquid extraction [18] Galeotti, F.; Barile, E.; Curir, P.; Dolci, M.; Lanzotti, V. Flavonoids from
carnation (Dianthus caryophyllus) and their antifungal activity. Phytochem.
MALDI = matrix-assisted laser desorption ionization Lett., 2008, 1, 44-48.
[19] Liu, A.L.; Wang, H.D.; Lee, S.M.Y.; Wang, Y.T.; Du, G.H. Structure-
MRM = multiple reaction monitoring
activity relationship of flavonoids as influenza virus neuraminidase inhibitors
n
MS = multistage mass spectrometry and their in vitro anti-viral activities. Bioorg. Med. Chem., 2008, 16, 7141-
7147.
PLE = pressurized liquid extraction [20] Herrera-Ruiz, M.; Roman-Ramos, R.; Zamilpa, A.; Tortoriello, J.; Jimenez-
Ferrer, J.E. Flavonoids from Tilia americana with anxiolytic activity in plus-
RDA = retro-Diels-Alder
maze test. J. Ethnopharmcol., 2008, 118, 312-317.
SIM = selected ion monitoring [21] Blade, C.; Arola, L.; Salvado, M.J. Hypolipidemic effects of proanthocyanid-
ins and their underlying biochemical and molecular mechanisms. Mol. Nut.
SPE = solid phase extraction Food Res., 2010, 54, 37-59.
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TLC = thin-layer chromatography
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TOF = time-of-flight mass spectrometer 2009, 1216, 7143-7172.
[23] De Rijke, E.; Out, P.; Niessen, W.M.A.; Ariese, F.; Gooijer, C.; Brinkman,
UPLC = ultra-performance liquid chromatography U.A.T. Analytical separation and detection methods for flavonoids. J. Chro-
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