Professional Documents
Culture Documents
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.
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 Current Organic Chemistry, 2011, Vol. 15, No. 15 2543
cluded the analysis of polar antioxidants in rosemary extracts by Latest progress in the HSCCC and CE analysis of flavonoids has
capillary electrophoresis coupled with mass spectrometry [49], been well summarized in recent reviews [22,23]. In this section, we
antioxidants in apple [50], and flavonoids in spinach [51]. focus on the advances in the separation and detection of flavonoids
In the above PLE methods, the pressure was mostly set at 1500 by CC, TLC, GC, and GC/MS. The progress in HPLC and LC/MS
psi (equivalent to about 10.3 MPa). A new PLE method with a analysis will be summarized in sections 4 and 5.
pressure of above 200 MPa was reported for the extraction of phe-
nolic compounds from longan fruit pericarp [52]. The obtained 3.1. Column Chromatography
extract showed a high recovery of three phenolic compounds. Flavonoids were present in many plants as an essential class of
bioactive components. During the past decades, various column
2.3. Other New Extraction Methods chromatography techniques were developed for the isolation of
Aside from the above new emerging methods, modifications of flavonoids. As stationary phases, silica gel, modified silica gel,
traditional extraction methods were also reported. Zhang and reversed-phase silica gel, polyamide, or Sephadex have been exten-
Laursen developed a mild extraction method for the analysis of sively used. However, due to the complicated character of plant
natural dyes (flavonoids) in textiles of historical interest [53]. A samples and the structural similarity of most flavonoids, the separa-
mild extraction approach with EDTA and formic acid was devel- tion of target flavonoids is sometimes challenging. In recent years,
oped, and successfully avoided the use of hydrochloric acid and a series of new packing materials and technologies for column
then degradation of flavonoid glycosides in the extraction process. chromatography were developed for the rapid and efficient separa-
A dynamic ultrasonic extraction (DUE) method coupled with tion of flavonoids.
on-line spectrophotometric detection has been used for the determi- A novel solid phase material, prepared by copolymerization us-
nation of total flavonoids in Scutellaria barbata and S. baicalensis ing allyl-bromide-modified chitosan as macromonomer and ethyl-
[54,55]. The DUE process involves a continuous transport of ana- ene glycol dimethacrylate as cross-linker, was utilized in the sepa-
lytes out of the extraction vessel, which enhanced the extraction ration of flavonoids [58]. Structurally related flavonoids, isorham-
efficiency and facilitated its coupling with other extraction steps or netin, kaempferol, and morin were successfully separated with this
on-line detectors. Compared with other extraction methods, DUE material. More importantly, the developed SPE column packed with
method obtained a high recovery for flavonoids. Furthermore, DUE this material showed three times higher of capacity than other col-
possesses obvious advantages of being more gentle, less expensive, umns. Superose 12 column packed with 12% cross-linked agarose
simpler, and less prone to contamination. gel, which have been widely used in the separation and purification
A new extraction method, namely negative-pressure cavitation of high molecular weight compounds, was successfully applied to
extraction (NPCE), was used for the extraction and quantification the isolation of flavonoids and isoflavonoids from Sophora japon-
of five flavonoids in pigeon pea leaves [56]. With optimized pa- ica [59].
rameters, NPCE showed higher recovery than microwave-assisted Medium-pressure liquid chromatography (MPLC) acts as an ef-
extraction (MAE), ultrasonic extraction (USE), and heat reflux fective and useful tool in the preparative scale separation of com-
extraction (HRE). As the samples were extracted with nitrogen at plicated mixtures. A novel method, using MPLC coupled to elec-
room temperature, NPCE significantly decreased the degradation trospray ionization multi-stage mass spectrometry (MPLC/ESI-
and oxidation of flavonoids. In addition, the NPCE method showed MSn), was successfully established for the separation and on-line
desirable repeatability and reproducibility. characterization of flavonoids from Asparagus officinalis [60]. The
A solvent-free microwave hydrodiffusion and gravity extraction separation procedure was guided by the MS/MS analysis. The de-
(MHG) method was used for the extraction of flavonols in onion veloped approach hyphenating MPLC with ESI-MSn proved effec-
(Allium cepa L.) [57]. The microwaves influence textural properties tive in the separation of complicated mixtures with high purity,
of plant material and increase secondary metabolites diffusion by desired amounts and simultaneous structural elucidation.
improving tissue softness and increasing cell permeability. The Column chromatography is usually performed on a packed col-
MHG procedure was performed on peeled onion bulbs, which was umn. Recently, a novel approach to enrich flavonoids from Ginkgo
heated using a fixed power density without addition of solvents or biloba L. was developed with expanded bed adsorption (EBA) us-
water [57]. This novel MHG technology definitely meets the de- ing Amberlite XAD7HP as the adsorbent [61]. Different from tradi-
mand of consumers for healthier food products, for it was per- tional packed columns, un-treated crude extracts were directly
formed under effect of microwaves and earth gravity at atmospheric loaded onto an expanded adsorption bed from the bottom, and the
pressure, without introducing any solvent. elution was carried out in an upward flow. With a two-step elution,
this new method achieved approximately the same quality of final
3. CHROMATOGRAPHIC SEPARATION AND DETECTION product with those obtained with packed column and liquid-liquid
extraction, but showed its unique advantages of simplified opera-
Plants usually contain a series of similar flavonoids, with slight tional procedure, shorter time consumption, and lower operational
change in substitution patterns or functional groups. These com- cost.
pounds may show very similar properties, and their separation has
been challenging. Chromatography is the most powerful technology 3.2. Thin-Layer Chromatography (TLC)
for the separation of organic compounds. All types of chroma-
tographic technologies have been extensively used for the separa- Thin-layer chromatography has been used as a routine approach
tion of flavonoids, including thin-layer chromatography (TLC), for the separation and detection of flavonoids since 1960s and still
column chromatography (CC), high speed counter-current chroma- plays an indispensable role today. The commonly used TLC sta-
tography (HSCCC), capillary electrophoresis (CE), gas chromatog- tionary phase includes silica gel G, silica gel GF254, RP-C18, and
raphy (GC), high-performance liquid chromatography (HPLC), and polyamide. The separated flavonoids are usually detected under 254
liquid chromatography coupled with mass spectrometry (LC/MS). nm as fluorescent quenching spots, or colorful spots under 365 nm
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2545
Fig. (3). Scheme of the dynamic microwave-assisted extraction (DMAE) Fig. (4). The separation of flavan-3-ols on HPTLC in oak and tea by differ-
system. Reprinted from Reference [38]. ent solvents. (A), 1-butanol-water-acetic acid; (B), 1-propanol-water-acetic
acid. Reprinted from Reference [71].
after spraying with visualizing reagents. A densitometer may be
used for quantitative TLC analysis. due to linarin observed in those of C. setosum, which provided a
Bhandart et al. [62] established a dual-run TLC method for the simple and feasible approach for the differentiation of the two spe-
determination of flavonoids from eight medicinal plants on a RP-18 cies.
F254 TLC plate. Mixtures of 5% formic acid-water/methanol (70:30) In addition to applications in qualitative and quantitative analy-
and 5% formic acid-water/methanol (50:50) were used as the mo- sis, researches on the separation mechanism of flavonoids by TLC
bile phase for the dual-development. The flavonoids were deter- were also conducted. Vovk et al. [71] investigated the retention
mined by densitometry at 280 nm in reflectance/absorbance mode. behaviors of eight flavonoids on cellulose HRTLC. (+)-catechin
This method showed good linearity and excellent reproducibility. (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-
Pozharitskaya et al. [63] developed a TLC method for the determi- epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-
nation of icariin in the dry extracts of the aerial parts of Epimedium epigallocatechin gallate (EGCg), procyanidin B1, and procyanidin
koreanum. This method led to very similar results to HPLC method. B2. Detection was performed using vanillin-H3PO4 reagent and four
TLC was also applied to the determination of rutin from Armaryllis developing solvent systems were tested. Consequently, water en-
belladonna flowers with lower accuracy and precision than HPLC abled the separation of epimers C from EC and GC from EGC, as
but more convenience and less solvent consumption [64]. HRTLC well as the dimers procianidin B1 and B2. Compounds C, EGC, B1
(high-resolution TLC) has been used for the simultaneous quantita- and B2 were well separated from all the other compounds, as
tion of five phenolic flavonoids in Glycyrrhiza glabra [65], the shown in Fig. (4). Using 1-propanol-water-acetic acid (20:80:1, v/v)
determination of tetrahydroamentoflavone in Semecarpus anacar- as the mobile phase, the best separation of the five main catechins
dium and its formula [66], and the simultaneous determination of (EC, GC, EGC, ECg, EGCg) present in green tea was obtained. The
quercetin, rutin, and coumaric acid in the flowers of Rhododendron chromatograms of oak bark extract using solvents with higher water
arboretum [67]. content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic
A TLC method for the rapid identification of different plant acid (20:80:1, v/v)) showed less bands than those developed in
species used as “Bailahuen” and its adulterants was established solvents with higher organic modifier content. The presence of
coupled with antioxidant activity test [68]. Lederer et al. [69] de- procyanidins besides the main component catechin resulted in such
veloped a simple and rapid method for the differentiation of Chi- retention behavior.
nese star anise and other Illicium species. The separation was car-
ried out on an HRTLC plate, eluted with a mixture of ethyl acetate, 3.3. Gas Chromatography and Gas Chromatography Coupled
formic acid, acetic acid, and water (100:11:11:26), and detected at with Mass Spectrometry (GC and GC/MS)
365 nm. Results revealed that Chinese star anise showed a different Gas chromatography is especially suitable for the analysis of
TLC fingerprint from all other analyzed Illicium species. This volatile ingredients such as essential oils and small aromatic com-
method could be used for the rapid identification of Chinese star pounds. Due to their high melting points, most flavonoids cannot be
anise in the quality control of commercial batches. The field thistle directly analyzed by GC. A derivatization treatment is usually nec-
(Cirsium setosum) and Japanese field thistle (C. japonicum) were essary when flavonoids are analyzed by GC. N,O-bis(trimethylsilyl)
recorded in Chinese Pharmacopoeia for the treatment of bleeding trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS)
and inflammation. A TLC method was established to distinguish [73,75,76,78,80,83,84] or hexamethyldisilazane (HMDS) and
these two species [70]. Using a mixture of ethyl acetate, formic trifluoroacetic acid (TFA) [74,82] are the most commonly used
acid, acetic acid and water, a satisfying resolution was achieved. derivatization reagents. These agents converted flavonoids into
Two clear fluorescent spots of linarin and pectolinarin were ob- trimethylsilyl derivatives by esterification or etherification on the
served in the chromatogram of C. japonicum while only the spot hydroxyl groups which enables the analysis of flavonoids by gas
2546 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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 Current Organic Chemistry, 2011, Vol. 15, No. 15 2547
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 vonoids, were determined simultaneously in Glycin tomentella in
flavonoids after being derivatized into trimethylsily ethers. The 120 minutes using HPLC-DAD.
elimination of methyl radicals, tetramethylsilane groups or the Besides commonly used detectors like DAD (diode-array detec-
combined loss of two methyl groups were found specific for certain tor), FLU (fluorescent detector), and ELSD (evaporative light scat-
substitution patterns, which could be used for the characterization tering detector), MS (mass spectrometer) has become a popular
of unknown flavonoids. Fuzfai et al. [82] found that anthocyanidins detector for HPLC to obtain abundant structural information. New
and flavanones were readily to form trimethylsilyl derivatives after detection techniques were also used to obtain biochemical informa-
being treated with HMDS and TFA, while flavonols and flavones tion of samples. Typical applications were summarized in Table 2.
were difficult. The authors studied the fragmentation behaviors of In the pursuit of higher speed and resolution, great advances in
the derivatives, and identified 33 flavonoids from grapefruit, lemon, HPLC analysis have been achieved in the past five years for fla-
and orange by one injection without any extraction or isolation vonoids, in both separation and detection. On the basis of Van
procedures. Deemter equation,
Ferrer et al. [83] developed a method by gas chromatography H = A + C=a(dp)+b/u+c(dp)2 u,
coupled with ion trap mass spectrometry (GC/MS-MS) for the iden-
H=plate height, u=speed, dp=particle size.
tification of eight phytoestrogens (isoflavones) in soybean products
and wastewater samples. The target compounds were derivatized The acceleration of HPLC depends mainly on two factors,
into trimethylsilyl ethers with trimethylchlorosilane (TMCS) and namely, particle size and flow rate. Improvements in these aspects
N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA). The fragmen- include UPLC with smaller particles, and monolithic column with
tation patterns were studied by isolating and fragmenting the pre- higher flow rate, respectively. For the improvement of resolution,
cursor ions. A typical fragmentation involving the loss of a methyl novel stationary phases were developed. The hyphenation of col-
and a carbonyl group was observed (Fig. 5). These characteristic umns with different separation mechanisms produced 2D-HPLC.
fragmentation pathways were used for the identification of phytoes-
trogens in soy milk and waster water. 4.2. Advance in Speed: UPLC and Monolithic Column
The appearance of ultra-performance liquid chromatography
4. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) significantly shortened the analysis time for each sample
(HPLC) comparing to conventional HPLC. The utilization of sub-2.5 m
particles, contributing directly to the plate height of an LC system,
4.1. Separation and Detection by HPLC
tremendously increased separation efficiency. The negative effect
Recent advances in flavonoids analysis have been made primar- of particle decrease is the back-pressure increase (about 9 times,
ily based on HPLC technology. Most HPLC analyses of flavonoids, versus 5 m particles), with which the separation was performed
as summarized in previous reviews [23], are performed on re- under very high pressure (up to 100MPa). However, generally, it
versed-phase C18 (or C8) columns, with a gradient binary solvent has no negative influence on the chromatographic system, including
system. HPLC-UV is widely used for quantification, which gave analytical columns. Separation efficiency remains unchanged or is
good linearity and sufficient dynamic range for flavonoids in plant even improved. Eventually, the UPLC system resulted in enhanced
extracts. Therefore, HPLC-UV has been employed for quantifica- peak capacity and detection sensitivity.
tion of multiple flavonoids in herbal extracts, such as Herba Epi- A selection of UPLC applications in flavonoids were summa-
medii [86], Carthamus tinctorius [87], and Glycin tomentella [88]. rized in Table 3. Chen et al. developed an UPLC-UV method to
As a typical example, a total of 33 compounds, including 21 fla- analyze different Herba Epimedii (Yin-Yang-Huo) samples. A si-
2548 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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
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-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.
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
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 (7). In addition, tandem MS/MS allowed maximal selectivity of ion
min as illustrated in Fig. (6). This method was fully validated, with pairs, with an LOD of 0.3-30 ng/mL for glycosylated analytes, and
LOD and LOQ lower than 0.13 and 0.52 ng on column, respec- 10-300 ng/mL for aglycones [109].
tively. The results showed great variations in contents of investi- UPLC represents a powerful technology for the chemical analy-
gated flavonoids among different samples. The clustering results sis of herbal medicines. A number of UPLC methods were estab-
were in accordance with their flavonoids contents. Finally, four lished for the rapid analysis of flavonoids in herbal extracts, includ-
flavonoids, epimedin A, B, C and icariin, were selected as markers ing Rose species, Trifolium species, Garcinia hanburyi, Epimedium
for quality control of Yin-Yang-Huo [46]. koreanum, Isatis indigatica, licorice root, lupin leaves, Aesculus
In compatible with fast speed and low concentration, MS detec- hippocastanum Seeds, strawberry flowers, carob flour, as well as
tors with high sensitivity and scanning rate became optimal for Chinese herbal medicine prescriptions, Buyang Huanwu Decoction
UPLC. Thus, the whole system is well suitable for trace analysis in and Wen-Pi Decoction. A typical example was that 15 caged xan-
complex matrices, such as herbal decoctions, food products, and thones in the resin of Garcinia hanburyi were well separated within
body fluids. A UPLC-MS/MS method was established to analyze 6 minutes, using a UPLC-qTOF-MS system [102].
anthocyanins and flavonols in yogurt and ice cream. With the com- Monolithic columns have only played an important role in
bination of MS, a total of 27 structural-similar analytes were suc- HPLC recently. Completely different from packed columns, mono-
cessfully separated and determined in 10 minutes, as shown in Fig. lithic columns consist of one piece of a continuous, porous material
2550 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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].
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.
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
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
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
High fragmentation
APCI Flavonoid aglycones Practical mass range up to 2000 Da May be not good for laser-sensitive compounds
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
Fig. (12). Schematic summary of principal fragment patterns for various types of flavonoids.
2554 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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- different performance on the radical cleavage of the quercetin
cluding flavones, flavonols, flavanones and isoflavones, xanthones, mono-O-glycosides, as illustrated in Fig. (15). In addition, the ion
anthocyanins, isoflavans, flavan-3-ols, and homoisoflavones kinetic energies may have a definite effect on the occurrence of
[23,138,140,143-146]. Schematic fragmentation pathways of ho- radical product ions [148]. In some cases, when the fragmentation
moisoflavones [145] and proanthocyanidin dimers [138] were pre- behaviors of reference compounds were thoroughly studied, it was
sented in Fig. (13) and Fig. (14), respectively. In addition, the frag- possible to unambiguously identify the structures of unknown com-
mentation behaviors of various flavonoids, including caged pounds by LC-UV-MSn [95,105,143,152].
xanthones [102], flavonoid C- and O- glycosides [133,147,148], Four flavonoids, with their structures clearly elucidated by a
isoprenylated [149] and prenylated [105] flavonoids have been SORI-CID FTICR MS, were identified from the leaves of haw-
intensively studied. thorn. Moreover, with in vitro -glucosidase inhibition assay com-
MALDI MS, combined with CID and SID techniques in tandem bined to LC-DAD-ESI-MSn, these flavonoids were proposed as -
mass spectrometry, were applied to differentiate isomeric com- glucosidase inhibitors [95]. With the aid of polarity switching in
pounds according to the abundance of specific fragment ions [132]. MS, a total of 14 flavonol glycosides were identified form Mangif-
Computational methods were employed as well to provide com- era indica L. Based on authentic reference compounds, flavonols
puted structures that indicated significant intramolecular hydrogen and xanthones were analyzed in negative mode, while anthocyanins
bonding and rotation form a coplanar structure [150,151,131]. and rhamnetin hexosides were analyzed in positive mode. Two of
the 14 compounds were isolated and their structures were authenti-
March et al. compared the product ion mass spectra of a series
cated by NMR analysis.
of isomeric deprotonated flavonoid glucosides, including flavones,
flavanones, and isoflavones. These compounds could be explicitly Due to the complicated MS/MS fragmentation behaviors of fla-
distinguished by their breakdown curves of major product ions at vonoids, it should be valuable to propose a structure interpretation
different collision energy levels [133]. Geng et al. studied the frag- guideline [105] or decision tree [143], as illustrated in Fig. (16).
mentation behaviors of three isomeric quercetin mono-O-glycosides The decision tree, including UV max absorptions and MS/MS diag-
(3-, 7-, 3-) under different CID techniques. These techniques gave 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 Current Organic Chemistry, 2011, Vol. 15, No. 15 2555
Fig. (16). Decision tree for the identification of flavonoids by UV and negative APCI mass spectrometry. Reprinted from Reference [143].
2556 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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)
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
MeCN, H2O
hops 17 A-and B-type proanthocyanidins ESI(+) IT directly loop-inject [163]
(direct infusion)
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, mentation patterns were established for sequencing A-type and B-
which was generally used in optimizing ionization and collision type PAs in positive ion mode, as shown in Fig. (19). The A-type
parameters of known compounds, has been employed as an alterna- and B-type PAs could be distinguished according to their mass
tive method for flavonoid profiling. A fraction from a hop extract, spectra in three aspects: the 2-Da mass differences in the pseudo-
containing oligomeric proanthocyanidins (PAs), was directly loop- molecular ions, the propensity to undergo QM fissions, and the
injected into the ESI source. Comprehensive mass spectral frag- 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 major compound in C. chinensis. In another case, seven Epimedium
presence of adulterants with similar appearance and chemical com- plants, including both official and unofficial species, were chemi-
position. LC/MS represents a powerful tool to distinguish similar cally differentiated, with the identification, profiling and compari-
plant species. Tu-Si-Zi is a tonic Chinese herbal medicine to im- son of 126 phenolic constituents [158]. Similar techniques could be
prove sexual function, regulate endocrine and immune system, and used to improve the quality control of Chinese herbal medicines.
prevent senescence. Two Cuscuta species, Cuscuta chinensis and C.
australis, were equally used as this drug in the market. Ye et al. 5.3. Quantitative Analysis
fully analyzed the chemical constituents of these two species by
HPLC/DAD/ESI-MSn, and discovered significant difference be- LC/MS has been proved to be one of the most sensitive tech-
tween the species [160]. Kaempferol and astragalin were the pre- niques for the determination of flavonoids in plants and foodstuffs,
dominant constituents of C. australis, while hyperoside was the with limits of detection (LOD) at the level of ng/ml [22]. Various
2558 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
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, flavonoids in biological matrices. The most popularly used tech-
including ion trap, single quadrupole, triple quadrupole, and time- nique is HPLC, in combination with UV, fluorescence, and electro-
of-flight MS. Quantitative analysis of flavonoids, with optimized chemical detection. Furthermore, GC, CE, fluorescence quenching,
MS program of selected ion monitoring (SIM), LOD are even better and immunoassay were also used [137]. Since most flavonoids have
than fluorescence or electrochemical detection [22]. Moreover, good UV absorption, less sensitive detectors like ELSD or RI were
highly specific scan modes like selected reaction monitoring (SRM) not frequently used. When the concentrations of flavonoids are
and multiple reaction monitoring (MRM) could significantly avoid fairly low in biological matrices, sensitive technologies like LC/MS
the interference of sample matrix [24]. are indispensable [136,137]. In addition, LC/MS could provide
In recent years, studies using LC/MS for flavonoid analysis abundant structural information of unknown metabolites for their
have been increasing. In a determination of 14 phenolic com- identification. The bioanalysis of flavonoids had been summarized
pounds, the LODs (0.44-127.78 g/kg) and LOQs (1.11-427.78 [136,137]. This review focuses on the recent advances in sample
g/kg) were lower in LC-MS/MS than LC-fluorescence and LC- treatment and in bioanalysis of flavonoids by LC/MS.
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- 6.1. Pre-treatment of Biological Samples
lytes, respectively [109]. By comparing ionization sources, ESI was
In most cases, biological samples require a pretreatment to get
found to improve precision, sensitivity, and LOD over APCI for
rid of endogenous proteins, carbohydrates, salts, and lipids. For
certain analytes, especially in the case of 7, 4'-isoflavandiol (LODs:
LC/MS analysis, sample pre-treatment could remove matrix com-
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 ponents that might contaminate the system or cause ion suppression
selectivity and sensitivity (0.5-12 ng/mL) for flavonoids than diode- [137]. Chen et al. made a comprehensive summary of sample
array detector (2-30 ng/mL) [167]. Generally, as a detector, MS is preparation techniques for bioanalysis [174].
more sensitive than UV, DAD, and fluorescence for flavonoids. In 6.1.1. Conventional Techniques
terms of MS scan modes, the sensitivity from higher to lower was:
SRM=MRM > SIM > full scan. A main drawback of MS detector Three techniques are the most-frequently used pretreatment
was its poor repeatability, when compared to DAD detector. As a methods in flavonoids bioanalysis: protein precipitation (PPT),
common situation for MS detection, the RSD is often up to 10-15% solid phase extraction (SPE) and liquid-liquid extraction (LLE)
[167]. Table 8 summarized selected quantitative analysis using [137]. A schematic comparison among these methods was made by
LC/MS, with special emphases on data acquisition programs, cali- Chang et al. [175], as illustrated in Fig. (20). Protein precipitation
bration processes, and limits of quantification. (PPT) is the simplest and most universal approach for sample pre-
By using SRM, a fast separation and determination of isofla- treatment [137]. Water miscible organic solvents (acetonitrile or
vonoids genistein, daidzein, formononetin, and biochanin A was methanol) are usually used to precipitate protein [176]. However,
achieved within 1.5 min under isocratic conditions [34]. In a similar the supernatant might still contain a significant amount of un-
study, 27 similar anthocyanins and flavanols, including isomeric precipitated endogenous components. Due to the selectivity of MS
compounds, were simultaneous determined in 10 min, as shown in detectors, complicated sample pretreatment is not necessary, and
Fig. (7). This method showed a dynamic range of 2-180 000 ng/mL additional centrifugation is sufficient to separate the resultant pro-
for glycosylated analytes, and 60-600 000 ng/mL for aglycones
tein precipitates from biofluids before LC/MS analysis [177]. It is
[109].
also worth noting that precipitation of proteins with acid may cata-
lyze the hydrolysis of some conjugates such as glucuronides and
6. BIOANALYSIS
sulfates [178]. Recently, PPT method has become fully automated.
Due to the popular use of flavonoid-containing medicinal and Samples could be treated in 96-well plates before LC-MS/MS
dietary herbs, there is an ever-increasing interest in the detection of analysis [177].
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2559
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 sorbents, the sample volume and pH, the content of organic modi-
bio-analytical laboratories [176]. In the bioanalysis of natural prod- fier, and the volume of elution solvent [179]. SPE can be performed
ucts, SPE represents high selectivity, effective pre-concentration, off-line manually, semi-automated, or on-line. On-line methods
and the potential for automation. Especially, SPE reduces the serum provided lower LOD and higher reproducibility than off-line meth-
background [137]. The efficiency of SPE depends on the type of ods [180], facilitating high-throughput analyses of biological sam-
2560 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
6.1.2. Current trends and Advanced Techniques LC/MS was believed to be the most promising technique for the
measurement of metabolites in biofluids due to the inherent high
Newly developed sample preparation techniques include solid selectivity afforded through both of the hyphenated techniques
phase micro-extraction (SPME), liquid-liquid micro-extraction [189]. LC/MS technique was fully developed for identification and
(LLME), pressurized liquid extraction (PLE), extraction using re- quantification of metabolites [190]. Selected applications using
stricted access material (RAM), micro-extraction by packed sorbent LC/MS were listed in Table 9. The hyphenation of UPLC and TOF-
(MEPS), molecularly imprinted polymer (MIP), monolith pin ex-
Sample source Biomatrix Extraction Analyzer Scan mode LOD; LOQ Ref.
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 Current Organic Chemistry, 2011, Vol. 15, No. 15 2561
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 tional methods while avoiding degradation. Column chromatogra-
predominantly APCI and ESI for flavonoids. In the case of urinary phy and TLC continue to be major approaches for the preparative-
metabonomics study of Xindi soft capsule, a TCM preparation by scale separation and routine analysis of flavonoids. New absorption
UPLC-qTOF-MS, the results provided a reasonable explanation to materials with novel separation mechanisms could significantly
the mechanism of traditional Chinese medicine preparation [196]. improve the separation efficiency. HPLC is still the most com-
With the aid of automated data analysis software, the post- monly used technology for the analysis of flavonoids. New ad-
acquisition data could be easily processed. For example, UPLC- vances in the past few years, such as UPLC, monolithic column,
qTOF-MS data could be analyzed by a professional software called HILIC column, and 2D-HPLC significantly enhanced the versatility
MetaboLynx. As illustrated in Fig. (21), a total of 16 and 38 me- and resolution of HPLC, and shortened the analysis time. LC/MS
tabolites were identified with the assistant of MetaboLynx from provides abundant structural information for the rapid identification
plasma and urine samples, after administration of flavonoids of of unknown flavonoids, but also provides a highly sensitive and
Abelmoschus manihot [195]. This type of investigations demon- specific technology for the quantitation of flavonoids in herbal ex-
strated the great potential of UPLC-qTOF-MS combined with data tracts and biological samples. The above technical advances allow
analysis software as an ideal approach for the identification of me- rapid, accurate, and sensitive analysis of flavonoids, and will be
tabolites from Chinese herbal medicines. widely used for the chemical analysis of flavonoids in complicated
matrices like medicinal and dietary herbs, and for the quality con-
7. CONCLUDING REMARKS trol of related products.
This article summarizes recent progress in the extraction, sepa-
ration, detection, and structural analysis of flavonoids, especially ACKNOWLEDGEMENTS
the chemical analysis of flavonoids in medicinal and dietary herbal
This work was supported by Peking University Health Science
extracts. New technologies like MAE and PLE could extract fla-
Center (Grant No. 985-2-119-121).
vonoids from complicated matrices more efficiently than conven-
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.
[22] Valls, J.; Millan, S.; Marti, M.P.; Borras, E.; Arola, L. Advanced separation
TLC = thin-layer chromatography
methods of food anthocyanins, isoflavones and flavonols. J. Chromatogr. A,
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-
matogr. A, 2006, 1112, 31-63.
[24] Stalikas, C.D. Extraction, separation, and detection methods for phenolic
REFERENCES acids and flavonoids. J. Sep. Sci., 2007, 30, 3268-3295.
[1] Harborne, J.B.; Williams, C.A. Advances in flavonoid research since 1992. [25] Liu, E-H.; Qi, L-W.; Cao, J.; Li, P.; Li, C-Y.; Peng, Y-B. Advances of mod-
Phytochemistry, 2000, 55, 481-504. ern chromatographic and electrophoretic methods in separation and analysis
[2] Havsteen, B.H. The biochemistry and medical significance of the flavonoids. of flavonoids. Molecules, 2008, 13, 2521-2544.
Pharmacol. Ther., 2002, 96, 67-202. [26] Ganzier, K.; Salgo, A.; Valko, K. Microwave extraction: A novel sample
[3] Huang, B.; Ban, X.Q.; He, J.S.; Tong, J.; Tian, J.; Wang, Y.W. Hepatopro- preparation methodfor chromatography. J. Chromatogr. A, 1986, 371, 299-
tective and antioxidant activity of ethanolic extracts of edible lotus (Nelumbo 306.
nucifera Gaertn.) leaves. Food Chem., 2010, 120, 873-878. [27] Gao, M.; Huang, W.; Chowdhury, M.R.; Liu, C.Z. Microwave-assisted
[4] Clavin, M.; Gorzalczany, S.; Macho, A.; Munoz, E.; Ferraro, G.; Acevedo, extraction of scutellarin from Erigeron breviscapus Hand-Mazz and its de-
C.; Martino, V. Anti-inflammatory activity of flavonoids from Eupatorium termination by high-performance liquid chromatography. Anal. Chim. Acta,
arnottianum. J. Ethnopharmcol., 2007, 112, 585-589. 2007, 591, 161-166.
[5] Jagtap, S.; Meganathan, K.; Wagh, V.; Winkler, J.; Hescheler, J.; Sachinidis, [28] Wang, C.; Pan, Y.J.; Fan, G.R.; Chai, Y.F.; Wu, Y.T. Application of an
A. Chemoprotective mechanism of the natural compounds, epigallocatechin- efficient strategy based on MAE, HPLC-DAD-MS/MS and HSCCC for the
3-O-gallate, quercetin and curcumin against cancer and cardiovascular dis- rapid extraction, identification, separation and purification of flavonoids
eases. Curr. Med. Chem., 2009, 16, 1451-1462. from Fructus Aurantii Immaturus. Biomed. Chromatogr., 2010, 24, 235-244.
[6] Ferreres, F.; Taveira, M.; Pereira, D.M.; Valentao, P.; Andrade, P.B. Tomato [29] Zhang, F.; Yang, Y.; Su, P.; Guo, Z.K. Microwave-assisted extraction of
(Lycopersicon esculentum) seeds: new flavonols and cytotoxic effect. J. Ag- rutin and quercetin from the Stalks of Euonymus alatus (Thunb.) Sieb. Phy-
ric. Food Chem., 2010, 58, 2854-2861. tochem. Anal., 2009, 20, 33-37.
[7] Lee, Y.J.; Suh, K.S.; Choi, M.C.; Chon, S.; Oh, S.; Woo, J.T.; Kim, S.W.; [30] Xiao, W.H.; Han, L.J.; Shi, B. Microwave-assisted extraction of flavonoids
Kim, J.W.; Kim, Y.S. Kaempferol protects HIT-T15 pancreatic beta cells from Radix Astragali. Sep. Purif. Technol., 2008, 62, 614-618.
from 2-deoxy-D-ribose-induced oxidative damage. Phytother. Res., 2010, 24, [31] Gao, M., Liu, C.Z. Comparison of techniques for the extraction of flavonoids
419-423. from cultured cells of Saussurea medusa Maxim. World J. Microbiol. Biote-
[8] Yu, C.S.; Lai, K.C.; Yang, J.S.; Chiang, J.H.; Lu, C.C.; Wu, C.L.; Lin, J.P.; chnol., 2005, 21, 1461-1463.
Liao, C.L.; Tang, N.Y.; Wood, W.G.; Chung, J.G. Quercetin inhibited mur- [32] Martino, E.; Colina, S.; Rossi, D.; Bazzoni, D.; Gaggeri, R.; Bracco, F.;
ine leukemia WEHI-3 cells in vivo and promoted immune response. Phy- Azzolina, O. Influence of the extraction mode on the yield of hyperoside,
tother. Res., 2010, 24, 163-168. vitexin and vitexin-2-O-Rhamnoside from Crataegus monogyna Jacq.
[9] Garcia-Lafuente, A.; Guillamon, E.; Villares, A.; Rostagno, M.A.; Martinez, (Hawthorn). Phytochem. Anal., 2008, 19, 534-540.
J.A. Flavonoids as anti-inflammatory agents: implications in cancer and car- [33] Chen, Z.; Zhang, L.Y.; Chen, G. Microwave-assisted extraction followed by
diovascular disease. Inflamm. Res., 2009, 58, 537-552. capillary electrophoresis-amperometric detection for the determination of an-
[10] Ghost, D.; Scheepens, A. Vascular action of polyphenols. Mol. Nutr. Food tioxidant constituents in Folium Eriobotryae. J. Chromatogr. A, 2008, 1193,
Res., 2009, 53, 322-331. 178-181.
[11] Chang, C.L.; Wang, G.J.; Zhang, L.J.; Tsai, W.J.; Chen, R.Y.; Wu, Y.C.; [34] Careri, M.; Crorradini, C.; Eoviri, L.; Mangia, A. Optimization of a rapid
Kuo, Y.H. Cardiovascular protective flavonolignans and flavonoids from Ca- microwave assisted extraction method for the liquid chromatography-
lamus quiquesetinervius. Phytochemistry, 2010, 71, 271-279. electrospray tandem mass spectrometry determination of isoflavonoid agly-
[12] Pushpavalli, G.; Kalaiarasi, P.; Veeramani, C.; Pugalendi, K.V. Effect of cones in soybeans. J. Chromatogr. A, 2007, 1152, 274-279.
chrysin on hepatoprotective and antioxidant status in D-galactosamine- [35] Sultana, T.; Stecher, G.; Mayer, R.; Trojer, L.; Qureshi, M.N.; Abel, G.;
induced hepatitis in rats. Eur. J. Pharmacol., 2010, 631, 36-41. Popp, M.; Bonn, G.K. Quality assessment and quantitative analysis of fla-
[13] Bansal, T.; Jaggi, M.; Khar, R.K.; Talegaonkar, S. Emerging significance of vonoids from tea samples of different origins by HPLC-DAD-ESI-MS. J.
flavonoids as P-glycoprotein inhibitors in cancer chemotherapy. J. Pharm. Agric. Food Chem., 2008, 56, 3444-3453.
Pharmaceut. Sci., 2009, 12, 46-78. [36] Du, F.Y.; Xiao, X.H.; Luo, X.J.; Li, G.K. Application of ionic liquids in the
microwave-assisted extraction of polyphenolic compounds from medicinal
plants. Talanta, 2009, 78, 1177-1184.
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2563
[37] Wang, J.X.; Xiao, X.H.; Li, G.K. Study of vacuum microwave-assisted [58] Xia, Y.Q.; Guo, T.Y.; Zhao, H.L.; Song, M.D.; Zhang, B.H.; Zhang, B.L. A
extraction of polyphenolic compounds and pigment from Chinese herbs. J. novel solid phase for selective separation of flavonoid compounds. J. Sep.
Chromatogr. A, 2008, 1198-1199, 45-53. Sci., 2007, 30, 1300-1306.
[38] Gao, M.; Song, B.Z.; Liu, C.Z. Dynamic microwave-assisted extraction of [59] Qi, Y.Y.; Sun, A.L.; Liu, R.M.; Meng, Z.L.; Xie, H.Y. Isolation and purifica-
flavonoids from Saussurea medusa Maxim cultured cells. Biochem. Engi- tion of flavonoid and isoflavonoid compounds from the pericarp of Sophora
neer. J., 2006, 32, 79-83. japonica L. by adsorption chromatography on 12% cross-linked agarose gel
[39] Chen, L.G.; Ding, L.; Yu, A.M.; Yang, R.L.; Wang, X.P.; Li, J.T.; Jin, H.Y.; media. J. Chromatogr. A, 2007, 1140, 219-224.
Zhang, H.Q. Continuous determination of total flavonoids in Platycladus [60] Hou, Z.G.; Luo, J.G.; Kong, L.Y. Medium-Pressure Liquid chromatography
orientalis (L.) Franco by dynamic microwave-assisted extraction coupled coupled to electrospray ionization mass spectrometry for separation and on-
with on-line derivatization and ultraviolet-visible detection. Anal. Chim. line characterization of flavonoids from Asparagus officinalis. Chroma-
Acta, 2007, 596, 164-170. tographia, 2009, 70, 1447-1450.
[40] Chen, L.G.; Jin, H.Y.; Ding, L.; Zhang, H.R.; Li, J.; Qu, C.L.; Zhang, H.Q. [61] Li, J.; Chase, H.A. Use of expanded bed adsorption to purify flavonoids from
Dynamic microwave-assisted extraction of flavonoids from Herba Epimedii. Ginkgo biloba L. J. Chromatogr. A, 2009, 1216, 8759-8770.
Sep. Purif. Technol., 2008, 59, 50-57. [62] Bhangdari, P.; Kumar, N.; Gupta, A.P.; Singh, B.; Kaul, V.K. A rapid RP-
[41] Yu, R.T.; Yu, R.X.; Zhang, X.W.; Luo, Z.M.; Zhang, H.G.; Shao, Y.; Mei, HPTLC densitometry method for simultaneous determination of major fla-
L.J.; Tao, Y.D. Dynamic microwave-assisted extraction of arctigenin from vonoids in important medicinal plants. J. Sep. Sci., 2007, 30, 2092-2096.
Saussurea medusa Maxim. Chromatographia, 2010, 71, 335-339. [63] Pozharitskaya, O.N.; Kosman, V.M.; Shikov, A.N.; Demchenko, D.V.;
[42] Benthin, B.; Danz, H.; Hamburger, M. Pressurized liquid extraction of me- Eschenko, A.Y.; Makarov, V.G. Comparison between HPLC and HPTLC
dicinal plants. J. Chromatogr. A, 1999, 837, 211-219. densitometry for the determination of icariin from Epimedium koreanum ex-
[43] Søltoft, M.; Christensen, J.H.; Nielsen, J.; Knuthsen, P. Pressurised liquid tracts. J. Sep. Sci., 2007, 30, 708-712.
extraction of flavonoids in onions: method development and validation. Ta- [64] Abou-Donia, A.H.; Toaima, S.M.; Hammoda, H.M.; Shawky, E. Determina-
lanta, 2009, 80, 269-278. tion of rutin in Amaryllis belladonna L. flowers by HPTLC and spectropho-
[44] Qian, Z.M; Lu, J.; Gao, Q.P.; Li, S.P. Rapid method for simultaneous deter- tometry. Chromatographia, 2006, 64, 109-112.
mination of flavonoids, saponins and polyacetylenes in Folium Ginseng and [65] Singh, B.; Mungara, P.; Nivsarkar, M.; Anandjiwala, S. HPTLC densitomet-
Radix Ginseng by pressurized liquid extraction and high-performance liquid ric quantification of glycyrrhizin, glycyrrhetinic acid, apigenin, kaempferol
chromatography coupled with diode array detection and mass spectrometry. and quercetin from Glycyrrhiza glabra. Chromatographia, 2009, 70, 1665-
J. Chromatogr. A, 2009, 1216, 3825-3830. 1672.
[45] Chen, X.J; Guo, B.L.; Li, S.P.; Zhang, Q.W.; Tu, P.F.; Wang, Y.T. Simulta- [66] Aravind, S.G.; Arimboor, R.; Rangan, M.; Madhavan, S.N.; Arumughan, C.
neous determination of 15 flavonoids in Epimedium using pressurized liquid Semi-preparative HPLC preparation and HPTLC quantification of tetrahy-
extraction and high-performance liquid chromatography. J. Chromatogr. A, droamentoflavone as marker in Semecarpus anacardium and its polyherbal
2007, 1163, 96-104. formulations. J. Pharm. Biomed. Anal., 2008, 48, 808-813.
[46] Chen, X.J.; Ji, H.; Zhang, Q.W.; Tu, P.F.; Wang, Y.T.; Guo, B.L.; Li, S.P. A [67] Onkoshi, E.; Nagashima, T.; Sato, H.; Fujii, Y.C.; Nozawa, K.; Nagai, M.
rapid method for simultaneous determination of 15 flavonoids in Epimedium Simple preparation of baicalin from Scutellariae Radix. J. Chromatogr. A,
using pressurized liquid extraction and ultra-performance liquid chromatog- 2009, 1216, 2192-2194.
raphy. J. Pharm. Biomed. Anal., 2008, 46, 226-235. [68] Vogel, H.; González, M.; Faini, F.; Razmilic, I.; Rodrignez, J.; Martin, J.S.;
[47] Jiang, Y.; Li, P.; Li, S.P.; Wang, Y.T.; Tu, P.F. Optimization of pressurized Urbina, F. Antioxidant properties and TLC characterization of four Chilean
liquid extraction of five major flavanoids from Lysimachia clethroide. J. Haplopappus-species known as bailahuen. J. Ethnopharmaol., 2005, 97, 97-
Pharm. Biomed. Anal., 2007, 43, 341-345. 100.
[48] Chen, X.J.; Zhao, J.; Meng, Q.; Li, S.P.; Wang, Y.T. Simultaneous determi- [69] Lederer, I.; Schulzki, G.; Gross, J.; Steffen, J.P. Combination of TLC and
nation of five flavonoids in licorice using pressurized liquid extraction and HPLC-MS/MS methods: approach to a rational quality control of Chinese
capillary electrochromatography coupled with peak suppression diode array star anise. J. Agric. Food Chem., 2006, 54, 1970-1974.
detection. J. Chromatogr. A, 2009, 1216, 7329-7335. [70] Ganzera, M.; Pöcher, A.; Stuppner, H. Differentiation of Cirsium japonicum
[49] Herrero, M.; Arráez-Román, D.; Segura, A.; Kenndler, E.; Gius, Beatrice.; and C. setosum by TLC and HPLC-MS. Phytochem. Anal., 2005, 16, 205-
Raggi, M.A.; Ibáez, E.; Cifuentes, A. Pressurized liquid extraction-capillary 209.
electrophoresis-mass spectrometry for the analysis of polar antioxidants in [71] Vovk, I.; Simonovska, B.; Vuorela, H. Separation of eight selected flavan-3-
rosemary extracts. J. Chromatogr. A, 2005, 1084, 54-62. ols on cellulose thin-layer chromatographic plates. J. Chromatogr. A, 2005,
[50] Wijngaard, H.; Brunton, N. The optimization of extraction of antioxidants 1077, 188-194.
from apple pomace by pressurized liquids. J. Agric. Food Chem., 2009, 57, [72] Helmja, K.; Vaher, M.; Püssa, T.; Kamsol, K.; Orav, A.; Kaljurand, M.
10625-10631. Bioactive components of the hop strobilus: comparison of different extrac-
[51] Howard, L.; Pandjaitan, N. Pressurized liquid extraction of flavonoids from tion methods by capillary electrophoretic and chromatographic methods. J.
spinach. J. Food Sci., 2008, 73, 151-157. Chromatogr. A, 2007, 1155, 222-229.
[52] Prasada, K.N.; Yang, B.; Shi, J.; Yu, C.Y.; Zhao, M.M.; Xue, P.; Jiang, Y.M. [73] Proestos, C.; Boziaris, I.S.; Nychas, G.-J.E.; Komaitis, M. Analysis of fla-
Enhanced antioxidant and antityrosinase activities of longan fruit pericarp by vonoids and phenolic acids in Greek aromatic plants: investigation of their
ultra-high-pressure-assisted extraction. J. Pharm. Biomed. Anal., 2010, 51, antioxidant capacity and antimicrobial activity. Food Chem., 2006, 95, 664-
471-477. 971.
[53] Zhang, X.; Laursen, R.A. Development of mild extraction methods for the [74] Kakasy, A.; Füzfai, Z.; Kursinszki, L.; Molnar-Perl, I.; Lemberkovics, É.
analysis of natural dyes in textiles of historical interest using LC-diode array Analysis of non-volatile constituents in Dracocephalum species by HPLC
detector-MS. Anal. Chem., 2005, 77, 2022-2025. and GC-MS. Chromatographia, 2006, 63, S17-S22.
[54] Hu, X.L.; You, J.Y.; Bao, C.L.; Zhang, H.R.; Meng, X.Z.; Xiao, T.T.; Zhang, [75] Maul, R.; Schebb, N.H.; Kulling, S.E. Application of LC and GC hyphenated
K.; Wang, Y.T.; Wang, H.M.; Zhang, H.Q.; Yu, A.M. Determination of total with mass spectrometry as tool for characterization of unknown derivatives
flavonoids in Scutellaria barbata D. Don by dynamic ultrasonic extraction of isoflavonoids. Anal. Bioanal. Chem., 2008, 391, 239-250.
coupled with on-line spectrophotometry. Anal. Chim. Acta, 2008, 610, 217- [76] Proestos, C.; Sereli, D.; Komaitis, M. Determination of phenolic compounds
223. in aromatic plants by RP-HPLC and GC-MS. Food Chem., 2006, 95, 44-52.
[55] You, J.Y.; Gao, S.Q.; Jin, H.Y.; Li, W.J.; Zhang, H.Q.; Yu, A.M. On-line [77] Hartonen, K.; Parshintsev, J.; Sandberg, K.; Bergelin, E.; Nisula, L.;
continuous flow ultrasonic extraction coupled with high performance liquid Riekkola, M.L. Isolation of flavonoids from aspen knotwood by pressurized
chromatographic separation for determination of the flavonoids from root of hot water extraction and comparison with other extraction techniques. Ta-
Scutellaria baicalensis Georgi. J. Chromatogr. A, 2010, 1217, 1875-1881. lanta, 2007, 74, 32-38.
[56] Liu, W.; Fu, Y.J.; Zu, Y.G.; Kong, Y.; Zhang, L.; Zu, B.S.; Efferth, T. Nega- [78] Farag, M.A.; Huhman, D.V.; Lei, Z.T.; Sumner, L.W. Metabolic profiling
tive-pressure cavitation extraction for the determination of flavonoids in pi- and systematic identification of flavonoids and isoflavonoids in roots and cell
geon pea leaves by liquid chromatography-tandem mass spectrometry. J. suspension cultures of Medicago truncatula using HPLC-UV-ESI-MS and
Chromatogr. A.. 2009, 1216, 3841-3850. GC-MS. Phytochemistry, 2007, 68, 342-354.
[57] Zill-e-Huma; Abert-Vian, M.; Maingonnat, J.F.; Chemat, F. Clean recovery [79] Shen, P.; Wong, S.P.; Tong, E.L. Sensitive and rapid method to quantify
of antioxidant flavonoids from onions: optimising solvent free microwave icaritin and desmethylicaritin in human serum using gas chromatography-
extraction method. J. Chromatogr. A, 2009, 1216, 7700-7707. mass spectrometry. J. Chromatogr. B, 2007, 857, 47-52.
[80] Spanakis, M.; Kasmas, S.; Niopas, L. Simultaneous determination of the
flavonoid aglycones diosmetin and hesperetin in human plasma and urine by
2564 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
a validated GC/MS method: in vivo metabolic reduction of diosmetin to hes- [100] Kumar, N.; Bhandari, P.; Singh, B.; Bari, S.S. Antioxidant activity and ultra-
peretin. Biomed. Chromatogr., 2009, 23, 124-131. performance LC-electrospray ionization-quadrupole time-of-flight mass
[81] Sutthanut, K.; Sripanidkulchai, B.; Yenjai, C.; Jay, M. Simultaneous identifi- spectrometry for phenolics-based fingerprinting of Rose species: Rosa dam-
cation and quantitation of 11 flavonoid constituents in Kaempferia parviflora ascena, Rosa bourboniana and Rosa brunonii. Food Chem. Toxicol., 2009,
by gas chromatography. J. Chromatogr. A, 2007, 1143, 227-233. 47, 361-367.
[82] Füzfai, Z.; Molnár-Perl, I. Gas chromatographic-mass spectrometric frag- [101] Kapusta, I.; Janda, B.; Szajwaj, B.; Stochmal, A.; Piacente, S.; Pizza, C.;
mentation study of flavonoids as their trimethylsilyl derivatives: analysis of Franceschi, F.; Franz, C.; Oleszek, W. Flavonoids in horse chestnut (Aescu-
flavonoids, sugars, carboxylic and amino acids in model systems and in cit- lus hippocastanum) seeds and powdered waste water byproducts. J. Agric.
rus fruits. J. Chromatogr. A, 2007, 1149, 88-101. Food Chem., 2007, 55, 8485-8490.
[83] Ferrer, I.; Barber, L.B.; Thurman, E.M. Gas chromatographic-mass spectro- [102] Zhou, Y.; Liu, X.; Yang, J.; Han, Q.B.; Song, J.Z.; Li, S.L; Qiao, C.F.; Ding,
metric fragmentation study of phytoestrogens as their trimethylsilyl deriva- L.S.; Xu, H.X. Analysis of caged xanthones from the resin of Garcinia han-
tives: identification in soy milk and wastewater samples. J. Chromatogr. A, buryi using ultra-performance liquid chromatography/electrospray ionization
2009, 1216, 6024-6032. quadrupole time-of-flight tandem mass spectrometry. Anal. Chim. Acta.,
[84] Crün, C.H.; van Dorsten, F.A.; Jacobs, D.M.; Belleguic, M.L.; van Velzen, 2008, 629,104-118.
E.J.J.; Bingham, M.O.; Janssen, H.G.; van Duynhoven, J.P.M. GC-MS [103] Wieslaw O.; Anna S.; Bogdan J. Concentration of isoflavones and other
methods for metabolic profiling of microbial fermentation products of die- phenolics in the aerial parts of Trifolium species. J. Agric. Food Chem.,
tary polyphenols in human and in vitro intervention studies. J. Chromatogr. 2007, 55, 8095-8100.
B, 2008, 871, 212-219. [104] Muth, D.; Marsden-Edwards, E.; Kachlicki, P.; Stobiecki, M. Differentiation
[85] Shadkami, F.; Helleur, R. Use of an injection port for thermochemolysis-gas of isomeric malonylated flavonoid glyconjugates in plant extracts with
chromatography/mass spectrometry: rapid profiling of biomaterials. J. UPLC-ESI/MS/MS. Phytochem. Anal.. 2008, 19, 444-452.
Chromatogr. A, 2009, 1216, 5903-5910. [105] Simons, R.; Vincken, J.P.; Bakx, E.J.; Verbruggen M.A.; Gruppen, H. A
[86] Huang, H; Liang, M.J.; Zhang, X.M.; Zhang, C.; Shen, Z.Y.; Zhang, W. D. rapid screening method for prenylated flavonoids with ultra-high-
Simultaneous determination of nine flavonoids and qualitative evaluation of performance liquid chromatography/ electrospray ionisation mass spectrome-
Herba Epimedii by high performance liquid chromatography with ultraviolet try in licorice root extracts. Rapid Commun. Mass Spectrom., 2009, 23,
detection. J. Sep. Sci., 2007, 30, 3207-3213. 3083-3093.
[87] Fan, L.; Zhao, H.Y.; Xu, M.; Zhou, L.; Guo, H.; Han, J.; Wang, B.R.; Guo, [106] Liu, E.H.; Qi, L.W.; Peng, Y.B.; Cheng, X.L.; Wu, Q.; Li, P.; Li, C.Y. Rapid
D.A. Qualitative evaluation and quantitative determination of 10 major ac- separation and identification of 54 major constituents in Buyang Huanwu de-
tive components in Carthamus tinctorius L. by high-performance liquid coction by ultra-fast HPLC system coupled with DAD-TOF/MS. Biomed.
chromatography coupled with diode array detector. J. Chromatogr. A, 2009, Chromatogr., 2009, 23, 828-842.
1216, 2063-2070. [107] Wang, Y.Q.; Guo, Z.M.; Jin, Y.; Zhang, X.L.; Wang, L.; Xue, X.Y.; Liang,
[88] Lin, J.T.; Liu, S.C.; Tsay, G.J.; Yang, D.J. Composition of flavonoids and X.M. Identification of prenyl flavonoid glycosides and phenolic acids in
phenolic acids in Glycin tomentella Hayata cultivated in various soils. Food Epimedium koreanum Nakai by Q-TOF-MS combined with selective en-
Chem., 2010, 121, 659-665. richment on “click oligo (ethylene glycol)” column. J. Pharm. Biomed.
[89] Kite, G.C.; Veitch, N.C.; Boalch, M.E.; Lewis, G.P.; Leon, C.J.; Simmonds Anal., 2010, 51, 606-616.
M.S.J. Flavonol tetraglycosides from fruits of Styphnolobium japonicum [108] Deng, X.Y.; Gao, H.H.; Zheng, S.N.; Li, F.M. Qualitative and quantitative
(Leguminosae) and the authentication of Fructus Sophorae and Flos Sopho- analysis of flavonoids in the leaves of Isatis indigatica Fort. by ultra-
rae. Phytochemistry, 2009, 70, 785-794. performance liquid chromatography with PDA and electrospray ionization
[90] Horwath, A.B.; Grayer, R.J.; Keith-Lucas M.; Simmonds M.S.J. Chemical tandem mass spectrometry detection. J. Pharm. Biomed. Anal., 2008, 48,
characterisation of wild populations of Thymus from different climatic re- 562-567.
gions in southeast Spain. Biochem. Syst. Ecol., 2008, 36, 117-133. [109] Nagy, K.; Redeuil, K.; Bertholet, R.; Steiling, H.; Kussman, M. Quantifica-
[91] Yang, R.Z.; Wei, X.L.; Gao, F.F.; Wang, L.S.; Zhang, H.J.; Xu, Y.J.; Li, tion of anthocyanins and flavonols in milk-based food products by ultra per-
C.H.; Ge, Y.X.; Zhang, J.J.; Zhang, J. Simultaneous analysis of anthocyanins formance liquid chromatography-tandem mass spectrometry. Anal. Chem.,
and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liq- 2009, 81, 6347-6356.
uid chromatography-photodiode array detection/electrospray ionization mass [110] Ortega, N.; Macià, A.; Romero, M.R.; Trullols, E.; Morello, J.R.; Anglès, N.;
spectrometry. J. Chromatogr. A, 2009, 1216, 106-112. Motilva M.J. Rapid determination of phenolic compounds and alkaloids of
[92] Lin, L.Z.; Mukhopadhyay S.; Robbins R.J.; Hamly J.M. Identification and Carob Flour by improved liquid chromatography tandem mass spectrometry.
quantification of flavonoids of Mexican oregano (Lippia graveolens) by LC- J. Agric. Food Chem., 2009, 57, 7239-7244.
DAD-ESI/MS analysis. J. Food Compos. Anal., 2007, 20, 361-369. [111] Hanhineva, K.; Rogachev, I.; Kokko, H.; Mintz-Oron, S.; Venger, I; Kären-
[93] Lin, L.Z.; James, M.H. Identification of the phenolic components of Collard lampi, S.; Aharoni, A. Non-targeted analysis of spatial metabolite composi-
Greens, Kale, and Chinese Broccoli. J. Agric. Food Chem., 2009, 57, 7401- tion in strawberry (Fragaria x ananassa) flowers. Phytochemistry, 2008, 69,
7408. 2463-2481
[94] Gardana, C.; Scaglianti, M.; Pietta, P.; simonetti, P. Analysis of the polyphe- [112] Zheng, X.T.; Shi, P.Y.; Cheng, Y.Y.; Qu, H.B. Rapid analysis of a Chinese
nolic fraction of propolis from different sources by liquid chromatography- herbal prescription by liquid chromatography-time-of-flight tandem mass
tandem mass spectrometry. J. Pharm. Biomed. Anal., 2007, 45, 390-399. spectrometry. J. Chromatogr. A, 2008, 1206, 140-146.
[95] Li, H.L.; Song, F.R.; Xing, J.P.; Tsao, R.; Liu, Z.Q.; Liu, S.Y. Screening and [113] Repollés, C.; Herrero-Martiinez, J.M.; Ràflos, C. Analysis of prominent
structural characterization of -glucosidase inhibitors from Hawthorn leaf flavonoid aglycones by high-performance liquid chromatography using a
flavonoids extract by ultrafiltration LC-DAD-MSn and SORI-CID FTICR monolithic type column. J. Chromatogr. A, 2006, 1131, 51-57.
MS. J. Am. Soc. Mass. Spectrom., 2009, 20, 1496-1503. [114] Alaerts, G.; Matthijs, N.; Smeyers-Verbeke, J.; Heyden, Y.V. Chroma-
[96] Clarkson, C.; Starerk, D.; Hansen, S.H.; Jaroszewski, J.W. Hyphenation of tographic fingerprint development for herbal extracts: A screening and opti-
solid-phase extraction with liquid chromatography and nuclear magnetic mization methodology on monolithic columns. J. Chromatogr. A, 2007,
resonance: application of HPLC-DAD-SPE-NMR to identification of con- 1172, 1-8.
stituents of Kanahia laniflora. Anal. Chem., 2005, 77, 3547-3553. [115] Vukics, V.; Ringer, T.; Kery, A.; Bonn, G.K.; Cuttman, A. Analysis of
[97] Schmidt, B.; Jaroszewski, W.J.; Bro, R.; Witt, M.; Staerk, D. Combining heartsease (Viola tricolor L.) flavonoid glycosides by micro-liquid chroma-
PARAFAC analysis of HPLC-PDA profiles and structural characterization tography coupled to multistage mass spectrometry. J. Chromatogr. A, 2008,
using HPLC-PDA-SPE-NMR-MS experiments: commercial preparations of 1206, 11-20.
St. John’s Wort. Anal. Chem., 2008, 80, 1978-1987. [116] Wang, Y.; Lu, X.; Xu, G.W. Simultaneous separation of hydrophilic and
[98] Goulas, V.; Papoti, V.T.; Exarchou, V.; Tsimidou, M.Z.; Gerothanassis, I. hydrophobic compounds by using an online HILIC-RPLC system with two
Contribution of flavonoids to the overall radical scavenging activity of olive detectors. J. Sep. Sci., 2008, 31, 1564-1572.
(Olea europaea L.) leaf polar extracts. J. Agric. Food Chem., 2010, 58, [117] Zhang, H.; Guo, Z.M.; Zhang, F.F.; Xu, Q.; Liang, X.M. HILIC for separa-
3303-3308. tion of co-eluted flavonoids under RP-HPLC mode. J. Sep. Sci., 2008, 31,
[99] Qi, L.W.; Yu, Q.T.; Li, P.; Li, S.L.; Wang, Y.X.; Sheng, L.H.; Yi, L. Quality 1623-1627.
evaluation of Radix Astragali through a simultaneous determination of six [118] Zhang, H.; Guo, Z.M.; Li, W.; Feng, J.T.; Xiao, Y.S.; Zhang, F.F.; Xue,
major active isoflavonoids and four main saponins by high-performance liq- X.Y.; Liang, X.M. Purification of flavonoids and triterpene saponins from
uid chromatography coupled with diode array and evaporative light scatter- the licorice extract using preparative HPLC under RP and HILIC mode. J.
ing detectors. J. Chromatogr. A, 2006, 1134, 162-169. Sep. Sci., 2009, 32, 526-535.
Extraction, Separation, Detection, and Structural Analysis of Flavonoids Current Organic Chemistry, 2011, Vol. 15, No. 15 2565
[119] Yanagida, A.; Murao, H.; Ohnishi-Kameyama, M.; Yamakawa, Y.; Shoji, dem mass spectrometry. Rapid Commun. Mass Spectrom., 2006, 20, 994-
A.; Tagashira, M.; Kanda, T.; Shindo, H.; Shibusawa, Y. Retention behavior 1000.
of oligomeric proanthocyanidins in hydrophilic interaction chromatography. [143] Portet, B.; Fabre, N.; Rozenberg, R.; Habi-Jiwan, J.L.; Moulis, C.; Quetin-
J. Chromatogr. A, 2007, 1143, 153-161. Leclercq, J. Analysis of minor flavonoids in Piper hostmannianum var. ber-
[120] Ikegami, T.; Tomomatsu, K.; Takubo, H.; Horie, K.; Tanaka, N. Separation bicense using liquid chromatography coupled with atmospheric pressure
efficiencies in hydrophilic interaction chromatography. J. Chromatogr. A, chemical ionization mass spectrometry. J. Chromatogr. A, 2008, 1210, 45-
2008, 1184, 474-503. 54..
[121] SeQuant, http://www.sequant.de/products/products sequant.shtml. [144] Oliveira, M.C.; Esperança, P.; Ferreira M.A.A. Characterisation of anthocya-
[122] Jandera, P.; Vyuchalová, K.; Hájek, T.; esla, P.; Vohralik, G. Characteri- nidins by electrospray ionization and collision-induced dissociation tandem
zation of HPLC columns for two-dimensional LC x LC separations of pheno- mass spectrometry. Rapid Commun. Mass Spectrom., 2001, 15, 1525-1532.
lic acids and flavonoids. J. Chemom., 2008, 22, 203-217. [145] Ye, M.; Guo, D.A.; Ye, G.; Huang, C.G. Analysis of homoisoflavonoids in
[123] Wang, Y.; Kong, L.; Lei, X.Y.; Hu, L.H.; Zou, H.F.; Welbeck, E.; Bligh, Ophiopogon japonicus by HPLC-DAD-ESI-MSn. J. Am. Soc. Mass Spec-
S.W.A.; Wang, Z.T. Comprehensive two-dimensional high-performance liq- trom., 2005, 16, 234-243.
uid chromatography system with immobilized liposome chromatography [146] Suryawanshi, S.; Mehrotra, N.; Asthana, R. K.; Gupta, R.C. Liquid
column and reversed-phase column for separation of complex traditional chromatography/tandem mass spectrometric study and analysis of xanthone
Chinese medicine Longdan Xiegan Decoction. J. Chromatogr. A, 2009, and secoiridoid glycoside composition of Swertia chirata, a potent
1216, 2185-2191. antidiabeticy. Rapid Commun. Mass Spectrom., 2006, 20, 3761-3768.
[124] Zhou, Y.; Wang, Y.; Wang, R.F.; Guo, F.; Yan, C. Two-dimensional liquid [147] Abad-García, B.; Garmón-Lobato, S.; Berrueta, L.A.; Gallo, B.; Vicente, F.
chromatography coupled with mass spectrometry for the analysis of Lobelia New features on the fragmentation and differentiation of C-glycosidic fla-
chinensis Lour. using an ESI/APCI multimode ion source. J. Sep. Sci., 2008, vone isomers by positive electrospray ionization and triple quadrupole mass
31, 2388-2394. spectrometry. Rapid Commun. Mass Spectrom., 2008, 22, 1834-1842.
[125] De Souza, L.M.; Cipriani, T.R.; Sant’Ana, C.F.; Iacomini, M.; Gorin, P.A.J.; [148] Geng, P.; Sun, J.; Zhang, R.; He, J..; Abliz, Z. An investigation of the frag-
Sassaki, G.I. Heart-cutting two-dimensional (size exclusion
reversed phase) mentation differences of isomeric flavonol-O-glycosides under different col-
liquid chromatography-mass spectrometry analysis of flavonol glycosides lision-induced dissociation based mass spectrometry. Rapid Commun. Mass
from leaves of Maytenus ilicifolia. J. Chromatogr. A, 2009, 1216, 99-105. Spectrom., 2009, 23, 1519-1524.
[126] Theodoridis, G.; Lasáková, M.; Veronikákeíková; Tegou, A.; Giantsiou, N.; [149] Zhang, Y.F.; Zhang, P.; Cheng, Y.Y. Structural characterization of isopreny-
Jandera, P. Molecular imprinting of natural flavonoid antioxidants: Applica- lated flavonoids from Kushen by electrospray ionization multistage tandem
tion in solid-phase extraction for the sample pretreatment of natural products mass spectrometry. J. Mass Spectrom., 2008, 43,1421-1431.
prior to HPLC analysis. J. Sep. Sci., 2006, 29, 2310-2321. [150] Davis, B.D.; Brodbelt, J.S. An investigation of the homolytic saccharide
[127] Tiberti, L.A.; Yariwake, J.H.; Ndjoko, K.; Hostettmann, K. Identification of cleavage of deprotonated flavonol 3-O-glycosides in a quadrupole ion trap
flavonols in leaves of Maytenus ilicifolia and M. aquifolium (Celastraceae) mass spectrometer. J. Mass Spectrom., 2008, 43, 1045-1052.
by LC/UV/MS analysis. J. Chromatogr. B, 2007, 846, 378-384. [151] Yan, C.Y.; Liu, S.Y.; Zhou, Y.H.; Song, F.R.; Cui, M.; Liu, Z.Q. A study of
[128] Davis, B.D.; Brodbelt, S. LC-MSn methods for saccharide characterization of isomeric diglycosyl flavonoids by SORI CID of Fourier transform ion cyclo-
monoglycosyl flavonoids using postcolumn manganese complexation. Anal. tron mass spectrometry in negative ion mode. J. Am. Soc. Mass. Spectrom.,
Chem., 2005, 77, 1883-1890. 2007, 18, 2127-2136.
[129] Exarchou, V.; Fiamegos, Y.C.; van Beek, T.A.; Nanos, C.; Vervoort, J. [152] Berardini, N.; Fezer, R.; Conrad, J.; Beifuss, U.; Carle, R.; Schiever, A.
Hyphenated chromatographic techniques for the rapid screening and identifi- Screening of mango (Mangifera indica L.) cultivars for their contents of fla-
cation of antioxidants in methanolic extracts of pharmaceutically used plants. vonol O- and xanthone C-glycosides, anthocyanins, and pectin. J. Agric.
J. Chromatogr. A, 2006, 1112, 293-302. Food Chem., 2005, 53, 1563-1570.
[130] Cuyckens, F.; Claeys, M. Mass spectrometry in the structural analysis of [153] Ding, S.; Dudley, E.; Plummer, S.; Tang, J.; Newton, R.P.; Brenton, A.G.
flavonoids. J. Mass Spectrom., 2004, 39, 1-15. Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-
[131] March, R.; Brodbelt, J. Analysis of flavonoids: Tandem mass spectrometry, MS/MS. Phytochemistry, 2008, 69, 1555-1564.
computational methods, and NMR. J. Mass Spectrom., 2008, 43, 1581-1617. [154] Cavaliere, C.; Cucci, F.; Foglia, P.; Guarino, C.; Samperi, R; Laganà, A.
[132] Vukics, V.; Guttman, A. Structural characterization of flavonoid goycosides Flavonoid profile in soybeans by high-performance liquid chromatogra-
by multi-stage mass spectrometry. Mass Spectrom. Rev., 2010, 29, 1-16. phy/tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2007, 21,
[133] March, R.E.; Lewars, E.G.; Stadey, C.J.; Miao, X.S.; Zhao, X.M.; Metcalre, 2177-2187.
C.D. A comparison of flavonoid glycosides by electrospray tandem mass [155] Suzuki, H.; Sasaki, R.; Ogata, Y.; Nakamura, Y.; Sakurai, N.; Kitajima, M.;
spectrometry. Int. J. Mass Spectrom., 2006, 248, 61-85. Takayama, H.; Kanaya, S.; Aoki, K.; Shibata, D.; Saito, K. Metabolic profil-
[134] De Rijke, E.; Zappery, H.; Ariese, F.; Gooijer, C.; Brinkman, U.A.T. Liquid ing of flavonoids in Lotus japonicus using liquid chromatography Fourier
chromatography with atmospheric pressure chemical ionization and electros- transform ion cyclotron resonance mass spectrometry. Phytochemistry, 2008,
pray ionization mass spectrometry of flavonoids with triple-quadrupole and 69, 99-111.
ion-trap instruments. J. Chromatogr. A, 2003, 984, 45-58. [156] Lai, J.P.; Lim, Y.H.; Su, J.; Shen, H.M.; Ong, C.N. Identification and charac-
[135] Yang, M.; Sun, J.H.; Lu, Z.Q.; Chen, G.T.; Guan, S.H.; Liu, X.; Jiang, B.H.; terization of major flavonoids and caffeoylquinic acids in three Compositae
Ye, M.; Guo, D.A. Phytochemical analysis of traditional Chinese medicine plants by LC/DAD-APCI/MS. J. Chromatogr. B, 2007, 848, 215-225.
using liquid chromatography coupled with mass spectrometry. J. Chroma- [157] Lin, L.Z.; Chen, P.; Marnly, J.M. New phenolic components and chroma-
togr. A, 2009, 1216, 2045-2062. tographic profiles of green and fermented teas. J. Agric. Food Chem., 2008,
[136] Prasain, J.; Wang, C.C.; Barnes, S. Mass spectrometric methods for the 56, 8130-8140.
determination of flavonoids in biological samples. Free Radical Biol. Med., [158] Zhao, H.Y.; Sun, J.H.; Fan, M.X.; Fan, L.; Zhou, L.; Li, Z.; Han, J.; Wang,
2004, 37, 1324-1350. B.R.; Guo, D.A. Analysis of phenolic compounds in Epimedium plants using
[137] Xing, J.; Xie, C.F.; Lou, H.X. Recent applications of liquid chromatography- liquid chromatography coupled with electrospray ionization mass spectrome-
mass spectrometry in natural products bioanalysis. J. Pharm. Biomed. Anal., try. J. Chromatogr. A, 2008, 1190, 157-181.
2007, 44, 368-378. [159] Ye, M.; Han, J.; Chen, H.B.; Zheng, J.H.; Guo, D.A. Analysis of phenolic
[138] Li, H.J.; Deinzer, M.L. Tandem mass spectrometry for sequencing proantho- compounds in rhubarbs using liquid chromatography coupled with electros-
cyanidins. Anal. Chem., 2007, 79, 1739-1748. pray ionization mass spectrometry. J. Am. Soc. Mass Spectrom., 2007, 18,
[139] March. R.E.; Miao, X.S. A fragmentation study of kaempferol using elec- 82-91
trospray quadrupole time-of-flight mass spectrometry at high mass resolu- [160] Ye, M.; Yan, Y.N.; Guo, D.A. Characterization of phenolic compounds in
tion. Int. J. Mass Spectrom., 2004, 231, 157-167. the Chinese herbal drug Tu-Si-Zi by liquid chromatography coupled to elec-
[140] Es-Safi, N.-E.; Kerhoas, L.; Einhorn, J.; Ducrot, P.-H. Application of trospray ionization mass spectrometry. Rapid Commun. Mass Spectrom.,
ESI/MS, CID/MS and tandem MS/MS to the fragmentation study of eriodic- 2005, 19, 1469-1484.
tyol 7-O-glucosyl-(12)-glucoside and luteolin 7-O-glucosyl-(12)- [161] Appeldoorn, M.M.; Vincken, J.P.; Sanders, M.; Hollmna, P.C.H.; Gruppen,
glucoside. Int. J. Mass Spectrom., 2005, 247, 93-100. H. Combined normal-phase and reversed-phase liquid chromatography/ESI-
[141] Zhou, D.Y.; Xu, Q.; Xue, X.Y.; Zhang, F.F.; Jing, Y.; Liang, X.M. Rapid MS as a tool to determine the molecular diversity of A-type procyanidins in
qualitative and quantitative analyses of flavanone aglycones in Fructus au- peanut skins. J. Agric. Food Chem., 2009, 57, 6007-6013.
rantii by HPLC ion-trap MS. J. Sep. Sci., 2007, 30, 858-867. [162] Sun, B.S.; Fernandes, T.A.; Spranger, M.I. A new class of anthocyanin-
[142] Tai, Y.P.; Pei, S.F.; Wan, J.P.; Cao, X.J.; Pan, Y.J. Fragmentation study of procyanidin condensation products detected in red wine by electrospray ioni-
protonated chalcones by atmospheric pressure chemical ionization and tan-
2566 Current Organic Chemistry, 2011, Vol. 15, No. 15 Qiao et al.
zation multi-stage mass spectrometry analysis. Rapid Commun. Mass Spec- [183] Yao, J.C.; Zhang, L.Y.; Zhao, X.Y.; Hu, L.; Jiang, Z.Z. Biol. Pharm. Bull.,
trom., 2010, 24, 254-260. 2006, 29, 1483-1486..
[163] Li, H.J.; Deinzer, M.L. The mass spectral analysis of isolated hops A-type [184] Nirogi, R.V.S.; Kandikere, V.N.; Shukla, M.; Mudigonda, K.; Maurya, S.;
proanthocyanidins by electrospray ionization tandem mass spectrometry. J. Komarneni, P. Rapid Commun. Mass Spectrom., 2006, 20, 3030-3038.
Mass Spectrom., 2008, 43, 1353-1363. [185] Raynie, D.E. Modern extraction techniques. Anal. Chem., 2006, 78, 3997-
[164] Michodjihoun-Mestres, L.; Souquet, J.M.; Fulcrand, H; Bouchut, C.; Reynes, 4004.
M.; Brillouet, J.M. Monomeric phenols of cashew apple (Anacardium occi- [186] Wang, Y.G.; Wang, S.Q.; Liu, Y.X; Yan, L.P; Dou, G.F.; Gao, Y. Charac-
dentale L.). Food Chem., 2009, 112, 851-857. terization of metabolites and cytochrome P450 isoforms involved in the mi-
[165] Suárez, M.; Macià, A.; Romero, M.P.; Motilva, M.J. Improved liquid chro- crosomal metabolism of aconitine. J. Chromatogr. B, 2006, 844, 292-300.
matography tandem mass spectrometry method for the determination of phe- [187] Haginaka, J. Molecularly imprinted polymers as affinity-based separation
nolic compounds in virgin olive oil. J. Chromatogr. A, 2008, 1214, 90-99. media for sample preparation. J. Sep. Sci., 2009, 32, 1548-1565.
[166] Rybak, M.E.; Parker, D.L.; Pfeiffer, C.M. Determination of urinary phytoes- [188] Abdel-Rehim, M. Recent advances in microextraction by packed sorbent for
trogens by HPLC-MS/MS: A comparison of atmospheric pressure chemical bioanalysis. J. Chromatogr. A, 2010, 1217, 2569-2580.
ionization (APCI) and electrospray ionization (ESI). J. Chromatogr. A, 2008, [189] Gray, M.J.; Chang, D.; Zhang, Y.; Liu, J.; Bensoussana, A. Development of
861, 145-150. liquid chromatography/mass spectrometry methods for the quantitative
[167] Surowiec, I.; Szostek, B; Tronanowicz, M. HPLC-MS of anthraquinoids, analysis of herbal medicine in biological fluids: a review. Biomed. Chroma-
flavonoids, and their degradation products in analysis of natural dyes in ar- togr., 2010, 24, 91-103.
cheological objects. J. Sep. Sci., 2007, 30, 2070-2079. [190] Prasain, J.K.; Barnes, S. Metabolism and bioavailability of flavonoids in
[168] Tolonen, A.; Uusitalo, J. Fast screening method for the analysis of total chemoprevention: current analytical strategies and future prospectus. Mol.
flavonoid content in plants and foodstuffs by high-performance liquid chro- Pharm., 2007, 4, 846-864.
matography/electrospray ionization time-of-flight mass spectrometry with [191] Guo, J.F.; Zhao, Y.M.; Zhao, L.; Zhang, W.Q.; Zhang, A.J.; Xu, B. Simulta-
polarity switching. Rapid Commun. Mass Spectrom., 2004, 18, 3113-3122. neous quantification of CTN986 and its deglycosylation products in rat se-
[169] Pang, T.; Yuan, Z.Y.; Dai, Y.S.; Wang, C.; Yang, J.; Peng, L.M.; Xu, G.W. rum using liquid chromatography/tandem mass spectrometry. Rapid Com-
Identification and determination of glycosides in tobacco leaves by liquid mun. Mass Spectrom., 2006, 20, 1701-1708.
chromatography with atmospheric pressure chemical ionization tandem mass [192] Ichiyanagi, T.; Shida, Y.; Rahman, M.M.; Hatano, Y.; Konishi, T. Extended
spectrometry. J. Sep. Sci., 2007, 30, 289-296. glucuronidation is another major path of cyanidin 3-O--D-glucopyranoside
[170] Inbaraj, B.S.; Lu, H.; Kao, T.H.; Chen, B.H. Simultaneous determination of metabolism in rats. J. Agric. Food Chem., 2005, 53, 7312-7319.
phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC- [193] Tian, Q.G.; Giusti, M.M.; Stoner, G.D.; Schwartz, S.J. Urinary excretion of
DAD-ESI-MS. J. Pharm. Biomed. Anal., 2010, 51, 549-556. Black Raspberry (Rubus occidentalis) anthocyanins and their metabolites. J.
[171] Ortega, N.; Romero, M.P.; Macia, A.; Reguant, J.; Angles, N.; Morelló, J.R.; Agric. Food Chem., 2006, 54, 1467-1472.
Motilva, M.J. Obtention and characterization of phenolic extracts from dif- [194] Liu, R.X.; Wang, W.; Wang, Q.; Bi, K.S.; Guo, D.A. Identification and
ferent Cocoa sources. J. Agric. Food Chem., 2008, 56, 9621-9627. determination of major flavonoids in rat urine by HPLC-UV and HPLC-MS
[172] Bolling, B.W.; Dolnikowski, G.; Blumberg, J.B.; Chen, C.Y.O. Quantifica- methods following oral administration of Dalbergia odorifera extract. Bio-
tion of almond skin polyphenols by liquid chromatography-mass spectrome- med. Chromatogr., 2006, 20, 101-108.
try. J. Food Sci., 2009, 74, C326-C312. [195] Guo, J.M.; Shang, E.X.; Duan, J.A.; Tang, Y.P.; Qian, D.W.; Su, S.L. Fast
[173] Cavaliere, C.; Foglia, P.; Gubbiotti, R.; Sacchetti, P.; Samperi, R.; Laganà, and automated characterization of major constituents in rat biofluid after oral
A. Rapid-resolution liquid chromatography/mass spectrometry for determi- administration of Abelmoschus manihot extract using ultra-performance liq-
nation and quantitation of polyphenols in grape berries. Rapid Commun. uid chromatography/quadrupole time-of-flight mass spectrometry and Me-
Mass Spectrom., 2008, 22, 3089-3099. taboLynx. Rapid Commun. Mass Spectrom., 2010, 24, 443-453.
[174] Chen, Y.; Guo, Z.P.; Wang, X.Y.; Qiu, C.G. Sample Preparation. J. Chroma- [196] Zhao, X.J.; Zhang, Y.; Meng, X.L.; Yin, P.Y.; Deng, C.; Chen, J.; Wang, Z.;
togr. A, 2008, 1184, 191-219. Xu, G.W. Effect of a traditional Chinese medicine preparation Xindi soft
[175] Chang, M.S.; Ji, Q.; Zhang, J.; Ei-Shourbagy, T.A. Historical review of capsule on rat model of acute blood stasis: A urinary metabonomics study
sample preparation for chromatographic bioanalysis: pros and cons. Drug based on liquid chromatography-mass spectrometry. J. Chromatogr. B.,
Dev. Res., 2007, 68, 107-133. 2008, 873, 151-158.
[176] Nováková, L.; Vlková, H. A review of current trends and advances in [197] Li, L.; Liang, S.P.; Du, F.F.; Li, C. Simultaneous quantification of multiple
modern bio-analytical methods: Chromatography and sample preparation. licorice flavonoids in rat plasma. J. Am. Soc. Mass. Spectrom., 2007, 18,
Ana. Chim. Acta, 2009, 656, 8-35. 778-782.
[177] Ma, J.; Shi, J.; Le, H.; Cho, R.; Huang, J.C.; Miao, S.; Wong, B.K. A fully [198] Yáñeza, J.A.; Remsberg, C.M.; Miranda, N.D.; Vega-Villa, K.R; Andrews,
automated plasma protein precipitation sample preparation method for LC- P.K.; Davies, N.M. Pharmacokinetics of selected chiral flavonoids: hes-
MS/MS bioanalysis. J. Chromatogr. B, 2008, 862, 219-226 peretin, naringenin and eriodictyol in rats and their content in fruit juices.
[178] Kostiainen, R.; Kotiaho, T.; Kuuranne, T.; Auriola, S. Liquid chromatogra- Biopharm. Drug Dispos., 2008, 29, 63-82.
phy/atmospheric pressure ionization-mass spectrometry in drug metabolism [199] Campanero, M.A.; Escolar, M.; Perez, G.; Garcia-Quetglas, E.; Sadaba, B.;
studies. J. Mass Spectrom., 2003, 38, 357-372. Azanza, J.R. Simultaneous determination of diosmin and diosmetin in human
[179] Wang X.J.; Jin Y.X.; Ying J.Y.; Zeng S.; Yao T.W. Determination of rutin plasma by ion trap liquid chromatography-atmospheric pressure chemical
deca(H-) sulfate sodium in rat plasma using ion-pairing liquid chromatogra- ionization tandem mass spectrometry: Application to a clinical pharmacoki-
phy after ion-pairing solid-phase extraction. J. Chromatogr. B, 2006, 833, netic study. J. Pharm. Biomed. Anal., 2010, 51, 875-881.
231-235. [200] Zhao, Y.; Sun, Y.; Li, C. Simultaneous determination of ginkgo flavonoids
[180] Shen, Y.; Feng, F. Sensitive liquid chromatography-tandem mass spectrome- and terpenoids in plasma: ammonium formate in LC mobile phase enhancing
try method for the determination of scutellarin in human plasma: application electrospray ionization efficiency and capacity. J. Am. Soc. Mass. Spectrom.,
to a pharmacokinetic study. J. Chromatogr. B, 2006, 830, 1-5. 2008, 19, 445-449.
[181] Kataoka, H. New trends in sample preparation for clinical and pharmaceuti- [201] Urpi-Sarda, M.; Monagas, M.; Khan, N.; Lamuela-Raventos, R.M.; Santos-
cal analysis. Trends. Anal. Chem., 2003, 22,232-244 Buelga, C.; Sacanella, E.; Castell, M.; Permanyer, J.; Andres-Lacueva, C.
[182] Hennion, M.C. Solid-phase extraction: method development, sorbents, and Epicatechin, procyanidins, and phenolic microbial metabolites after cocoa in-
coupling with liquid chromatography. J. Chromatogr. A, 1999, 856, 3-54. take in humans and rats. Anal. Bioanal. Chem., 2009, 394, 1545-1556.