Journal of Chromatography B, 812 (2004) 2333

Review

Extraction methods and chemical standardization of botanicals

and herbal preparations
Eng Shi Ong
Applied Science School, Temasek Polytechnic, 21 Tampinese Avenue 1, Singapore 529757, Republic of Singapore
Received 4 April 2004; accepted 20 July 2004
Available online 11 September 2004

Abstract
Botanicals and herbal preparations are medicinal preparations, containing a single or two or more medicinal plants. The focus of this review
paper is on the analytical methodologies, which included the combination of sample preparation tools and chromatographic techniques for the
chemical standardization of marker compounds or active ingredients in botanicals and herbal preparations. The common problems and key
challenges in the chemical standardization of botanicals and herbal preparations were discussed. As sample preparation is the most important
step in the development of analytical methods for the analysis of constituents present in botanicals and herbal preparations, the strength and
weakness of different extraction techniques are discussed. For the analysis of compounds present in the plant extracts, the applications of
common chromatographic techniques, such as HPLC, CE, HRGC/MS, HPLC/MS and HPLC/MS/MS are discussed. The strength, weakness
and applicability of various separation tools are stated. Procedures for the identification of marker or active compounds in plant extracts, using
HPLC/MS, were proposed. Finally, the effects of batch-to-batch variation of the medicinal plants are investigated and discussed.
2004 Elsevier B.V. All rights reserved.
Keywords: Sample preparation; Extraction; Botanicals; Herbal preparations

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Preparation of sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Analysis of botanicals and herbal preparation by liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Analysis of botanicals and herbal preparations by capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Hyphenation procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Botanicals and herbal preparations are medicinal preparations, containing a single plant or a mixture of two or more
E-mail address: esong@tp.edu.sg.
1570-0232/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2004.07.041

23
25
27
28
29
32
32
32

different types of medicinal plants. Monographs of medicinal

plants can be found in the United States Pharmacopeia [1],
Chinese Pharmacopeia [2], WHO monographs for medicinal plants [3,4], Japanese Pharmacopeia [5] and Herbal
Medicine (Expanded Commission E Monographs) [6]. Other
information on the chemical substances in medicinal plants
and their pharmacological properties can be found in the

24

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

Table 1
Some of the common herbal products, their uses and side effects [3,6]
Botanicals

Common medicinal uses

Side effects

Aloe vera

Short-term treatment of occasional constipation

Abdominal spasms and pain may occur after even single dose.
Overdose can lead to colicky abdominal spasms and pain, as
well as the formation of thin, watery stools.
Overdose will result in electrolyte imbalance.

Echinacea

To support and promote the natural powers of resistance of the

body, especially in infectious conditions, such as influenza and cold
in the nose and throat.
External uses include promotion of wound healing and treatment of
inflammatory skin conditions.
For psychovegetative disturbances, depressive moods, anxiety and
nervous unrest
For symptomatic treatment of disturbed performance in organic
brain syndrome within the regimen of a therapeutic concepts, with
the following principal symptoms: memory deficients, disturbances
in concentration, depressive emotional conditions, dizziness,
tinnitus and headache.
As a support to dietary measures at elevated levels of lipids in the
blood and as a measure for age dependant vascular changes.

Chills, short-term fever reaction and nausea and vomiting may

occur.

A tonic for invigoration and fortification times of fatigue and

debility or declining capacity for work and concentration.
For bronchitis, peptic ulcer, chronic gastritis, rheumatism and
adrenocorticoid insufficiency.

None

St. Johns Wort

Gingko biloba

Galic
Ginseng
Liquorice root

books cited [710]. A list of commercially available products,

containing medicinal plants, their common medicinal uses
and side effects are tabulated in Table 1. The major compound types in Chinese medicinal plants include alkaloids,
saponins, flavonoids, anthraquinones, terpenoids, coumarins,
lignans, polysaccharides, polypeptides and proteins.
Botanicals, which show promising results included the
use of Ginkgo biloba in which the gingkolides have antioxidant, neuroprotective and chlolinergic activities relevant
to Alzeimers disease [1113]. The in vivo neuromodulatory effects of G. biloba was studied, using a product that
is a standardized leaf extract (EGb761) reported to contain
24% flavone glycosides (primarily composed of quercetin,
kaempferol and isorhamnetin) [13]. The inhibitory effects of
cancer cell proliferation and anti-tumor effects of the total extracts from Scutellariae radix were well studied. In an in vivo
experiment, significant inhibition of tumor growth had been
observed. Scutellaria baicalensis inhibited cyclooxygenase2 (COX-2) expression and a 66% reduction in tumor mass
was observed in the nude mice. S. baicalensis selectively and
effectively inhibits cancer cell growth in vitro and in vivo
and can be an effective chemotherapeutic agent for head and
neck squamous cell carcinoma. It was proposed that the inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anticancer activity [14,15].
The pharmacological basis of Yang-invigoration in herbs,
such as Herba epimedii, Rhizoma drynariae and others were
discussed [16]. On the other hand, problems with regard to
the usage of herbs were encountered. Chinese herb nephropathy or end stage renal failure was reported in patients taking
weight reducing pills containing Chinese herbs, because of a

Photosensitization is possible, especially in fair skin individuals.

Very seldom, cases of stomach or intestinal upset, headaches or
skin allergic skin reaction.

In rare instances, there may be gastrointestinal symptoms,

changes to the flora of the intestine, or allergic reaction.

On prolong use and higher doses, sodium and water retention

and potassium loss may occur, accompanied by hypertension,
edema, hypokalemia and in rare cases, myoglobinuria.

manufacturing error, one of the medicinal plants in the pills

was replaced with Aristolochia fangchi where the main components are aristolochic acids [17]. In Singapore, undeclared
drugs, such as chlorpheniramine, paracetamol and others can
be found in health supplements and herbal preparations [18].
At the same time, works with some botanicals have progressed to clinical trials. A multicentric, open, prospective,
observational and non-randomized clinical trial with 190 patients for the efficacy and safety of a standardized phytoestrogen preparation derived from Glycine max (L.) Merr in
Climacteric symptomatology was carried out [19]. A randomized controlled trial with 428 patients on the effects
of standardized Hypericum perforatum (St. John Wort) in
major depressive disorder was reported [20]. A Phase II clinical trial on botanical explored standardized green teas antineoplastic effects in patients with androgen independent
prostate carcinoma [21]. Other Phase II and III clinical trials
sponsored by National Center for complementary and alternative medicine (NCCAM) are progressing at the moment
(http://nccam.nih.gov).
For botanicals and herbal preparations, there is a need to
approach scientific proof and clinical validation with chemical standardization, biological assays, animal models and
clinical trials. Quality assurance of botanicals and herbal
preparations is the prerequisite of credible clinical trials. According to the draft guidelines stated by the United States
Food and Drug Administration (USFDA) [22] and The European Agency for the Evaluation of Medicinal Products [23],
various aspects of analysis must be performed for the purpose
of certification of botanical drugs and herbal preparations.
For medicinal plants, the tests include identification, water

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

content, chemical assay of active ingredients, inorganic impurities (toxic metals), microbial limits, mycotoxins, pesticides
and others. For herbal preparations, in addition to the tests
mentioned above, other tests include disintegration, dissolution, hardness/friability and uniformity of dosage unit should
be available. However, due to the unique nature of botanicals
and herbal preparations, the USFDA found it appropriate to
apply regulatory policies that was different from those applied to synthetic, semi-synthetic, or otherwise highly purified or chemically modified drugs. Clinical studies may be
initiated at phase II for common botanicals. The continuing drive to optimize health in todays society has created a
huge market for dietary supplements where there is a growing
need to validate methods of analysis in botanicals and herbal
preparation [24].
The focus of this review paper is on the analytical methodologies for the chemical standardization of marker compounds or active constituents in botanicals and herbal preparations. According to draft guidelines stated by the USFDA,
a marker compound is a chemical constituent of a botanical
raw material, drug substance, or drug product that is used
for identification and/or quality control purposes, especially
when the active constituents are not known or identified. The
active constituent is responsible for the intended pharmacological activity or therapeutic effects. Chemical standardization often involves chemical identification by spectroscopic
or chromatographic fingerprint and chemical assay (assay)
for active constituents or marker compounds if available. The
key challenges in the development of analytical methods for
botanicals and herbal preparations are: (1) analysis of marker
or active compounds in a complex and sometimes unknown
environment, (2) target analytes may be polar and thermally
labile, (3) lack of chemical reference substances and certified reference materials, (4) selection of extraction method
and (5) batch-to-batch variation of the composition of the
plant materials obtained. The analytical methods developed
should be stability indicating, be used for chemical fingerprinting and assaying of marker or active compounds. With
the chemical fingerprint obtained, the method should be able
to perform composition analysis and monitor the batch-tobatch variation of the plant materials obtained for use. Due
to the number of parameters required, methods that are simple, rapid and environmentally friendly are attractive options
for regulatory laboratories, testing laboratories and laboratories supporting manufacturing activities. For the analysis
of botanicals and herbal preparations, a holistic solution involving the combination of the methods of extraction and
analytical techniques, such as separation tools are required.
In this article, recent developments and applications of
modern sample preparation techniques and separation tools
for final analysis in plant extracts are reviewed. Emphasis
is placed on the brief description of the unique capabilities
and advantages and disadvantages of the approaches used.
The common problems and challenges in the analysis of target components in botanicals and herbal preparations will be
discussed. More detailed description of the basic principles

25

of these modern sample preparation tools, chromatographic

techniques and hyphenated procedures, can be found in the
review articles and other papers cited in the respective sections.

2. Preparation of sample
Sample preparation is the most important step in the development of analytical methods for the analysis of botanicals
and herbal preparations. Review papers on sample preparation techniques for the extraction plant materials are available
[25,26]. A brief summary of the experimental conditions for
the various methods of extraction stated in this paper can be
found in Table 2. The basic operation included steps, such as
pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the
kinetics of analyte extraction. For the monograph stated in
the pharmacopeia, methods, such as sonication, heating under reflux, Soxhlet extraction and others are commonly used
[1,2,5]. However, such methods can be time consuming, required the use of large amount of organic solvent and may
have lower extraction efficiencies. As the target compounds
may be non-polar to polar and thermally labile, the suitability
of the methods of extraction must be considered. The other
problem for the selection of methods of extraction is that the
active or marker compounds are present naturally, significant
analytematrix interaction will be present, hence spiking of
the target compounds into the plant matrix will not mimic
the real environment. Depending on how the method is validated, it may be possible to have a method with high recovery but lacks accuracy. From the various botanicals studied
in our laboratory, different methods of extraction and differing conditions may often be required for the extraction
of marker compounds from different plant materials. Even
with the same technique of extraction, for different marker
compounds in different plant materials, different operating
conditions, such as the solvent use, temperature applied and
others may be required.
In the move to reduce or eliminate the use of organic
solvent and improve the extraction processes, newer sample preparation methods, such as microwave assisted extraction (MAE), supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE) or pressurized liquid extraction
(PLE) have been introduced for the extraction of analytes
present in plant materials. Using MAE, the microwave energy is used for solution heating and results in significant
reduction of extraction time (usually in less than 30 min).
Other than having the advantage of high extraction speed,
MAE also enables a significant reduction in the consumption of organic solvents [2527]. A novel MAE method had
been developed for the extraction and determination of tanshinones (tanshinone IIA, cryptotanshinone and tanshinone I)
from the root of Salvia miltiorrhiza with analysis by HPLC
[28,29]. Other methods, such as the use of SFE that used
carbon dioxide and some form of modifiers had been used

4050 min
4045
[45,48]
4050 min
4045
[38,4146]
2040 min
2030
[3440]
2040 min
2040
[25,26,32]
30100 min
NA
[25,26,30,31]

1020 bar
1050 bar
1020 bar
100 bar
250450 atm
NA

1h
50100
[25,26]

Pressure applied

Time required
Volume of solvent required (ml)
Refs.

318 h
150200
[25,26]

80150
Can be heated
Temperature ( C)

Depending on
solvent used
NA

Depending on if it
is closed or
opened vessel
extraction
1040 min
2050
[2529]

80200

80200

80300

Water with
surfactants, such
as Triton X100 or
SDS
80200
Water or water
with 1030 %
ethanol
Methanol
Methanol

Carbon dioxide or
carbon dioxide
with modifiers,
such as methanol
40100
Methanol, ethanol,
or mixture of
alcohol and water
Methanol, ethanol,
or mixture of
alcohol and water
Methanol, ethanol,
or mixture of
alcohol and water
Common Solvents used

Sonication

Soxhlet extraction

Microwave
assisted extraction
(MAE)

Supercritical Fluid
extraction (SFE)

Accelerated
Solvent extraction,
static (ASE)

Pressurized
Liquid Extraction,
dynamic (PLE)

Pressurized Hot
Water Extraction
(PHWE)

Surfactant assisted
PHWE

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

Table 2
A brief summary of the experimental conditions for various methods of extraction, such as sonication, Soxhlet extraction, MAE, supercritical extraction, ASE, PLE, PHWE and surfactant assisted PHWE for
medicinal plants

26

in the extraction of compounds present in medicinal plants

[25,26,30,31].
In the move to reduce the use of solvent and time in the
methods of extraction, our laboratory have developed analytical methods for botanicals and herbal preparations using
PLE. Methods using PLE, have been commonly used for the
extraction of persistent organic pollutants in environmental
samples. The technique uses organic solvent at elevated temperature and pressure, which drastically improves the speed
of the extraction process. The parameters that have a significant effect on the extraction efficiencies of marker compounds in botanicals and herbal preparations by PLE are the
time for extraction/volume of solvent, applied temperature
and the nature of solvent used. The time for extraction was set
at 20 min as it was found that a significant portion of the target analytes would be extracted. The pressure was observed
to have little effect on the extraction efficiency as it was applied to keep the solvent in the liquid phase. Application of
PLE, using commercially available systems based on static
extraction to medicinal plants, was reported [32,33]. Our laboratory have reported the used of a laboratory made dynamic
PLE system for the extraction of aristolochic acids, berberine,
strychnine, ginsenosides, glycyrrhizin, baicalein and others
in medicinal plants and herbal preparations [3438]. It was
observed that the extraction efficiency of marker compounds
in botanicals and herbal preparations by PLE was comparable or higher than Soxhlet extraction. The main reasons for
the enhanced performance when using PLE over Soxhlet extraction and other conventional methods of extraction are the
higher solubility of analytes in solvent and higher diffusion
rate as a result of higher temperature. At higher temperature,
the strong solutematrix interaction caused by van der Waals
forces, hydrogen bonding and dipole attractions between solute molecules and active sites on the matrix were disrupted.
The used of pressurized solvent extraction of polar steroids,
such as withanolides from the leaves of Lochroma gesnerioides and cocaine and benzylecgonine from coca leaves had
been reported [39,40].
Water has similar properties as methanol at an applied
pressure of 50 bar and a temperature between 150 and 200 C.
However, it was observed that a number naturally occurring substances may decompose, using the stated temperature. To reduce the use of organic solvent, pressurized hot
water extraction (PHWE) was used for the extraction of essential oil components from plant materials was developed
[41,42]. Sub-critical water under pressure and between 125
and 175 C had been used to rapidly extract oxygenated fragrance and favor compounds from rosemary [43]. A laboratory made system using sub-critical water was developed
for the extraction of iridoid glycosides in plant matrix with
final determination by micellar-electrokinetic capillary chromatography [44]. In our laboratory, PHWE was applied for
the extraction of thermally labile and reasonably polar components, such as berberine in Coptidis rhizoma, glycyrrhizin
in Radix glycyrrhizae (liquorice), baicalein in S. radix and
marker compounds in Radix codonopsis pilosula [38,45]. An

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

instrumental set-up similar to PLE can be used for PHWE.

The parameters that would have a significant effect on the
extraction efficiencies of marker compounds in botanicals,
using PHWE, are the applied temperature, pressure and percentage of organic modifiers. The non-significant effect of
the pressure was observed.
However, due to the heat sensitivity and unique properties
of certain marker compounds in botanicals, the extraction
efficiencies of PHWE may not be as comparable to conventional methods, such as Soxhlet extraction. For components,
such as tanshinone I and IIA in S. miltiorrhiza, PLE using
methanol was found to give comparable or higher extraction
efficiencies compared to PHWE with reference to Soxhlet
extraction [46]. For the analysis of marker compounds in R.
codonopsis pilosula (DangShen), it was observed that PHWE
give lower extraction efficiencies compared to Soxhlet extraction [45]. To improve the extraction efficiencies of methods
using PHWE, surfactants, such as Triton X-100 and sodium
dodecyl sulfate (SDS) may be added into the water to enhance
the solubility of target compounds present in the botanicals.
In this model of extraction, a high dilution of the extract was
produced as higher extractant volume was used. With surfactant assisted PHWE in a dynamic mode, the presence of
surfactant, such as Triton X-100 in the water was found to
enhance the solubility of the target compounds and pushed
the target analytes in the sample matrix in the mobile phase to
completeness with the fresh liquid pumped through the sample continuously [45]. Hence, micelle-mediated extraction
and preconcentration of ginsenosides from Chinese herbal
medicine had been introduced. When compared to methanol
and water, an aqueous surfactant solution containing 10% Triton X-100 yielded faster kinetics and higher recovery for the
extraction of various ginsenosides [47]. A method, using the
combination of surfactant and PWHE with an applied temperature below the boiling point and lower pressure from 10 to
20 bars was developed for the analysis of marker compounds
that are reasonably hydrophobic, such as tetradeca-4E, 12Ediene-8,10-diyne-1,6,7-triol and tetradeca-4E, 12E-diene8-10-diyne-1,6,7-triol-O--d-glucoside in R. codonopsis
pilosula (DangShen). The type of surfactants, such as Triton
X-100, sodium dodecyl sulfate (SDS) and others added to the
water will affect the extraction efficiencies of the methods using surfactant assisted PHWE [45]. Similarly, PLE with nonionic surfactants solution had been applied for the extraction
of ginsenosides from the roots of American ginseng as model
solid samples. The combination of PLE and cloud point extraction was shown to be a new and effective approach for
the rapid sample pre-concentration of herbal materials prior
to analysis by HPLC [48].

3. Analysis of botanicals and herbal preparation by

liquid chromatography
Liquid chromatography with a isocratic/gradient elution
remain to be the method of choice in the pharmacopeia and

27

based on the number papers cited in this review for the analysis of marker compounds that are thermally labile in botanicals and herbal preparations. In most of the papers cited, the
reversed octadecyl silica (C-18) is most commonly used. In
the course of our experiments, we found that columns with
smaller inner diameter, such as 1.0 or 2.1 mm i.d. were well
suited to the analysis of components present in botanicals. For
columns with smaller inner diameter, it was observed that the
system precision for the retention time and peak area/height
were comparable to analytical columns with 4.6 mm i.d. [46].
Most important of all, methods using columns with smaller
inner diameter and the right mobile phase can be readily
adopted to mass spectrometry. The most common mode of
detection remains to be ultraviolet detection. Applications of
chromatographic techniques to medicinal plants and Chinese
traditional medicines are outlined [49]. Methods using gradient elution HPLC with reversed phase columns had been
applied for the analysis of multiple constituents present in
medicinal plants and herbal preparations [5052]. Gradient
elution HPLC with ultraviolet detection, using a C18 reversed
phase column had been used to profile components present
in C. rhizoma, Radix aristolochiae, ginseng, R. glycyrrhizae
(liquorice), S. radix, R. codonopsis pilosula and S. miltiorrhiza [34,36,38,45,46]. The advantages of liquid chromatography include its high reproducibility, good linear range, ease
of automation and its ability to analyze the number of constituents in botanicals and herbal preparation. However, for
the analysis of marker compounds in herbal preparations with
two or more medicinal plants, co-eluting peaks were often
observed in the chromatograms obtained due to the complexity of the matrix [54]. The complexity of matrix may
be reduced with additional sample preparation steps, such as
liquid-liquid partitioning, solid phase extraction, preparative
LC and TLC fractionation.
One of the challenges for the analytical methods developed was to study the effects of batch-to-batch variations in
the medicinal plants. It was reported that HPLC analysis of
marker compounds in Platycodi radix of Playcodon grandiforum showed variation in the composition from different
sources [55]. With HPLC methods for simultaneous analysis of cichoric acids and alkamides in Echinacea purpurea
plant and products, a selection of international herbal products available on the Danish market show surprising variable quality, not necessary reflecting the product information
given on the labels [56]. A reversed phase HPLC method
with ultraviolet detection was used to identify ginsenosides
in products classified as health supplements where the results
did not reflect what is given on the label [36]. Similarly, in
one product labeled as containing pure Panax ginseng, no ginsenosides could be detected [8]. For ginseng, the variety and
the method of cultivation are very important. It was observed
that various species of ginseng, from a morphological point
of view, are similar, but as far as their content of substances
is concerned they are not [8].
To investigate the batch-to-batch variations of the plant
materials obtained, a method using PLE with liquid

28

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

Table 3
Comparison of Radix scutellariae extracted from different batches of medicinal plants by PLE at 120 C with HPLC [31]
Batch No.

Amount of Baicalein (mg/g)

EC50

1
2
3
4

20.65 0.20
21.47 0.28
12.80 0.16
19.34 0.16

18.13 0.75
17.63 1.65
11.88 0.25
17.00 0.58

For the determination of EC50, sample stock solutions were diluted to five
different concentrations in ethanol. One ml of a 0.3 mM DPPH ethanol solution was added to 2.5 ml of sample solutions of different concentration
and allowed to react at room temperature. After 30 min the absorbent values
were measured at 518 nm, using a Jasco V-530 LSE 1335 UV Spectrophotometer (Japan) and converted into the percentage antioxidant activity (AA),
using the following formula: AA% = 100 {[(Abssample Absblank )
100]/Abscontrol }. Ethanol (1.0 ml) plus plant extract solution (2.5 ml) was
used as a blank. DPPH solution (1.0 ml; 0.3 mM) plus ethanol (2.5 ml) was
used as a negative control. The positive controls were those using the standard solutions. EC50 represents the level where 50% of the radical were
scavenged by test sample.

chromatography with ultraviolet detection [38] was used to

assay the content of baicalein in S. radix from four different
sources labeled as Batch 14 in Table 3. As shown in Table 3,
batch-to-batch variations were observed with the amount of
baicalein varied from 12.8 to 21.47 mg/g. A chromatographic
fingerprint of various components present in S. radix was
shown in Fig. 1. A similar chemical fingerprint as in Fig. 1
was obtained for all the four different batches of S. radix profiled. On top of the chemical fingerprint, the antioxidant activity, using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical
photometric assay in the process guided by its discoloration,
was measured. However, the level of baicalein and the antioxidant activities was found to vary in the four different
batches of S. radix. The anti-oxidant activities measured were

Fig. 1. HPLC Chromatogram obtained for baicalein in S. radix by PLE.

Mobile phase of (A) 25 mM NaH2 PO4 at pH 2.5 and (B) acetonitrile. Initial
condition was set at 30% of B, gradient up to 100% B in 15 min before
returning to initial condition for 10 min. C-18 reversed phase HPLC column (4.6 mm i.d. 150 mm length, 5 . Detection was at 254 nm. Oven
temperature was at 40 C and flow rate was 1.0 ml/min.

found to correlate with the amount baicalein and other components present in S. radix. Hence, the assayed of marker or
active compounds together with chemical fingerprinting, using HPLC, will be able to provide further information about
the quality of the botanicals and herbal preparations.

4. Analysis of botanicals and herbal preparations by

capillary electrophoresis
Capillary electrophoresis (CE) proved to be a powerful alternative to HPLC in the analysis of polar and thermally labile
compounds. Reviews on the analysis of natural medicines
or natural products in complex matrix by CE are well reported [56,57]. Many publications showed that all aspects of
CE, such as capillary zone electrophoresis (CZE), micellarelectrokinetic capillary chromatography (MEKC) and capillary isoelectric focusing (cIEF) have been used for the
separation of natural products. The separation in CZE is based
on the differences in the electrophoretic mobilities resulting
in different velocities of migration of ionic species in the electrophoretic buffer in the capillary. For MEKC, the main separation mechanism is based on solute partitioning between the
micellar phase and the solution phase. Factors that are known
to affect separation in CZE and MEKC include pH of running
buffer, ionic strength, applied voltage and concentration and
type of micelle added.
From the review articles, CE had been used to determine
the amount of catechin and others in tea composition, phenolic acids in coffee samples and flavonoids and alkaloids in
plant materials [56,57]. In our laboratory, analytical methods
using CE, included the determination of aristolochic acids in
R. aristolochiae, strychnine in Strychnos nux-vomica, berberine in Rhizoma coptidis (Huang-lian) and glycyrrhizin in
R. glycyrrhizae (liquorice) [35,37,53]. CZE was found to
be a method for the rapid separation and determination of
four phenylpropanoid glycosides from T. chamaedrys [58].
Methods using CZE and MEKC have been proved to be
a powerful technique in the chemical profiling and assaying of marker compounds present in extracts from medicinal
plants.
As herbal preparations are products that were often observed to contain two more different types of medicinal
plants, it had always been a challenge for the analysis
of target compounds in herbal preparations and products
that are classified as, Traditional Chinese Medicine (TCM)
or herbal remedies. It is certain that the presence of the
number of medicinal plants used in the herbal preparations will increase the complexity of the sample matrix obtained. In the analysis of marker compounds in complex
environment, methods using MEKC had been applied for
the determination of icariin, rhein, chrysophanol, physcion,
glycyrrhetic acid and glycyrrhizic acid in traditional Chinese herbal preparations [59]. The use of MEKC with
electrochemical or ultraviolet detection had been described
for the determination of hesperidin, naringin, puerarin and

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

daidzein in medicinal preparations [60,61]. To differentiate

between medicinal plant, such as S. radix from Astragali
radix, CE with electrochemical detection was employed
[62].
For the determination of berberine, aristolochic acids I
and II in herbal preparations, we reported that it was possible
to assay the amount of marker or active compounds with a
single step extraction followed by CZE. This was due to the
fact that separation by CZE is mainly based on the different
electrophoretic mobility of charged solutes in solution in relation to their different molecular weight, size and charge at
a given pH. It is also possible for both cations and anions
to be separated in a single run which provides specific selectivity for the separation method. It was observed that it
can be difficult to assay the amount of marker compounds
in herbal preparations using HPLC as co-eluting peaks were
often present. With CZE and under the conditions used for
aristolochic acids, it was found that most of the positively
charged and neutral species migrate close or with the electroosmotic flow (EOF). The amount of aristolochic acid I and
II present in herbal preparations, that was in a complex environment, could be determined with a single step extraction
followed by CZE [35,53].
The advantages of methods using CE for botanicals
and herbal preparations are: (1) the high selectivity and
ability to analyze target compounds in complex environment, such as herbal preparations without additional sample preparation steps, (2) significant reduction or complete
elimination of organic solvent for the electrolyte solution
or running buffer, (3) good linear range, (4) ease of automation and (5) speed of analysis. Using ultraviolet detection, on-column detection was often used in CE. Without
using bubble capillary columns and applying any specific
pre-concentration techniques, this will result in lower sensitivity compared to methods using HPLC. To improve the
reproducibility of migration time and peak area with methods using CE, internal standards are often added. However,
the system precision for the relative migration time and peak
area will always be higher in methods using CE compared
to HPLC. It was observed in our laboratory that run failures were encountered in CZE and MEKC where standard
solutions were prepared in high percentage of methanol.
The effect was due to a disruption of the conductivity inside the capillary caused either by the presence of a nonconductivity plug or the result of the out-gassing in the
injection zone. As the sample was extracted with methanol,
10% water in methanol was added to standard solutions and
sample extracts to counter run failures [35,37,53]. Our observations on run failure, was consistent with other reports
[63,64].
Despite of some the weakness described for methods using
CE, the enantioseparation of naturally occurring substances,
such as atropine and flavonoids, such as medicarpin and vestitone in plant materials by CE with chiral selectors remained
to be the method of choice [6567]. From our works with
plant materials, the applicability of methods using CE, will

29

be more difficult for compounds that are essentially non-polar

present in some medicinal plants.

5. Hyphenation procedures
The use of chromatographic separation with mass spectrometry for the chemical characterization and composition
analysis of botanicals has been growing rapidly in the recent years. Reviews on the use of mass spectrometry and
high-performance liquid chromatography mass spectrometry (HPLC/MS) on botanicals had been reported [68,69].
The use of hyphenated techniques, such as high resolution gas chromatography mass spectrometry (HRGC/MS),
high performance liquid chromatography/mass spectrometry (HPLC/MS), liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and tandem mass spectrometry
(MS/MS) to perform on line composition and structural analyses provide rich information that is unsurpassed by other
techniques.
The use of HRGC/MS, remains the method of choice for
the analysis of volatile and semi-volatile components, such
as essential oil and others in botanicals and herbal preparations. With high resolution separation with capillary column coupling with mass spectrometry using electron impact
ionization (EI), undeclared drugs, such as chlorpheniramine,
paracetamol and others that may be present or added in health
supplements and herbal preparations can be detected easily
[18]. Other than undeclared drugs, marker compounds, such
as tetrahydrompalmatine in Corydalis yanhusuo can be identified using HRGC/MS with the chromatograms and the mass
spectra as shown in Fig. 2. The power of HRGC/MS is the
ability to rapidly detect compounds that are volatile or semivolatile present in a complex environment, such as extracts
from herbal preparations using a single step extraction without any additional clean-up steps.
For the analysis of components present in botanicals,
HPLC/MS has been playing a increasingly significant role
as the technique is capable of characterizing compounds
that are thermally labile, ranging from small polar molecules
to macromolecule, such as peptides/proteins, carbohydrates
and nucleic acids. The most common mode of ionization in
HPLC/MS included electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Mass analyzer,
such as single quadruple, triple quadruple, ion-trap, time-of
flight, quadruple time-of-flight (Q-TOF) and others are used.
With tandem mass spectrometry, additional structural information can be obtained for the target compounds. However,
methods using HPLC/MS is still limited to conditions that
are suitable for MS operations. There are restrictions on pH,
solvent choice, solvent additives and flow rate for LC in-order
to achieve optimal sensitivity.
In our laboratory and other report, HPLC/MS had been
used to characterize ginsenosides in ginseng, tanshinone I,
tanshinone II and cryptotanshinone present in S. miltiorrhiza
[46,68] and marker compounds present in R. codonopsis pilo-

30

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

Fig. 2. A Total ion chromatograms (TIC) obtained for marker compounds, such as tetrahydrompalmatine in C. yanhusuo with HRGC/MS and 2B) mass spectra
with electron impact (EI) ionization obtained for tetrahydropalmatine. Injector: splitless at 250 C. Carrier: helium. Oven: initial at 80300 C at 15 C/min.
Detector: Mass selective detector, 280 C, full scan: 40600 amu. Column: HP5, 30 m 0.25 mm i.d., 0.25 m.

sula [45]. The presence of non-volatile components, such as

tanshinone I, tanshinone II and cryptotanshinone in S. miltiorrhiza can be easily identified by positive ion ESI/MS. Direct
sample introduction by ESI or separation with reversed phase
column resulted in the abundant of [M + H]+ , [M + Na]+ and
[M + M + Na]+ for tanshinone I, tanshinone II and cryptotanshinone [46,68]. For compounds, such as tetradeca-4E, 12Ediene-8,10-diyne-1,6,7-triol and tetradeca-4E, 12E-diene8-10-diyne-1,6,7-triol-O--d-glucoside in R. codonopsis
pilosula (DangShen), [M + Na]+ was observed to be the most
abundant ion [45]. With ESI/MS, there is a great tendency for
certain compounds in medicinal plants to form adducts, such
as [M + Na]+ or [M + K]+ .
Methods using HPLC/ESI/MS had been applied for the
identification components present in Betula pubescen and

Peschiera fuschiaefolia. [70,71]. For the characterization

of hydrolysable tannins from leaves of B. pubescen, the
main peak in the mass spectra of an individual hydrolysable
tannins in the negative ion ESIMS, was the deprotonated
molecule [M H] [70]. The alkaloids, such as voacamine, voacamidine, vobasine and voachalotine present in
P. fuschiaefolia were determined using external standard
calibration with acceptable precision and accuracy. The presence of [M + H]+ , [M + 2H]2+ and other adduct ions were
observed in the positive electospray ionization mode [71].
Using APCI interface with mass analyzer, such as ion trap
or triple quadruple instruments, main constituents present in
Rhodiola rosea extracts and tropane alkaloids from Erythroxylum vacciniifolium can be characterized [72,73]. With internal standard calibration, constitutents, such as salidroside,

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

31

Fig. 3. A Total ion chromatogram (TIC) obtained for glycyrrhizin in the

methanolic extract R. glycyrrhizae using HPLC/ESI/MS and 3B MS/MS
of glycyrrhizin with precursor ion m/z: 839.1 amu. The gradient elution,
using a mobile phase consisting of (A) 0.1% formic acid in water and (B)
0.1% formic acid in acetonitrile. The initial condition was set at 20% of B,
gradient up to 100% in 20 min and returning to initial condition for 6 min.
Oven temperature was set at 40 C and flow rate was set at 0.2 ml/min. For all
experiments, 5 l of standards and sample extract were injected. The column
was a reversed phase C18 Luna, 150 mm 2.0 mm, 5 m (Phenomenex,
USA). The ESI-MS was acquired in the positive ion mode. The scanning
mass range was from 100 to 1000 amu. The heated capillary temperature was
maintained at 350 C, the drying gas and nebulizer nitrogen gas flow rates
were 10 l/min and 50 psi, respectively. Data was acquired using automated
MS/MS.

rosavin and benzyl-O--glucopyranoside in R. rosea extracts

was determined [73]. Similarly, the content of ginsenosides,
such as Rb1, Rc and Re in plant extracts from P. ginseng and
Panax quinquefolius was determined by HPLC/ESI/MS with
external standard calibration. The [M + H]+ and [M + Na]+
ions were observed for ginsenosides standards (Rb1, Rb2, Rc,
Rd, Re, Rf and Rg1) [74]. In our earlier work, it was observed
that it would be difficult to differentiate between P. ginseng
and P. quinquefolius using reversed phase HPLC with ultraviolet detection [36]. With HPLC/ESI/MS, the presence and
ratio of ginsenosides Rf and 24-(R)-pseudoginsenosides F11
in the plant extracts can be used to differentiate between P.
ginseng and P. quinquefolius [75,76].
From Figs. 3A and 4A, the total ion chromatograms
(TIC) of glycyrrhizin and baicalein in the methanolic extracts from R. glycyrrhizae and S. radix were obtained, using
HPLC/ESI/MS. With tandem mass spectrometry (MS2 ), significant fragmentation pattern was observed for glycyrrhizin
and baicalein as in Figs. 3B and 4B. In most instances, significant fragmentation patterns can be obtained for a number of
compounds in plant materials with MS2 . However, for certain
compounds, such as baicalin in S. radix, significant fragmentation pattern was not observed with MS2 as in Fig. 4C. As
baicalin and baicalein differs from each other by one side

Fig. 4. A Total ion chromatogram (TIC) obtained for baicalein in the

methanolic extracts from S. radix using LC/ESI/MS and 4B) MS/MS of
baicalein with precursor ion m/z: 270.7 amu, 4C) MS/MS spectra of baicalin
with precursor ion m/z: 446.6 amu. Conditions used were the same as in
Fig. 3.

chain, MS/MS of balcalin with precursor ion m/z: 470.7 gave

the molecular ion of baicalein as in Fig. 4C. With MS3 , the
fragmentation pattern for baicalein as in Fig. 4B was observed
for baicalin. The current example showed the ability of MS2
and MS3 in the structural elucidation of naturally occurring
compounds in botanicals. For certain compounds, MS2 may
not be able to provide as much information compared to MS3 .
The strength of HPLC/MS and HPLC/MSn remained in
its ability to determine the presence of target compounds in
complex environment, such as in herbal preparations. This
was seen in the evaluation of major active components in
St. Johns Wort dietary supplements with HPLC/ESI/MS.
The content of the target components, such as rutin, hyperosides, quercetin and others were determined using internal standard calibration. The presence of [M + H]+ and
[M + Na]+ ions were observed for mass spectra obtained
for rutin and hyperosides [77]. As a result of end stage

32

E.S. Ong / J. Chromatogr. B 812 (2004) 2333

renal failure reported in patients taking weight reducing pills

containing Chinese herbs that was found to contain aristolochic acids, the presence of aristolochic acids in a number
of herbal remedies was screened and detected with methods using HPLC/ESI/MS and HPLC/APCI/MS [78]. Other
groups have also used HPLC/MS to confirm the presence of
aristolochic acids in Chinese phytomedicines and dietary supplements used as slimming regiments [79,80]. Using selective
ion monitoring (SIM) or extracted ion chromatogram at a particular m/z, the mass analyzer can become a highly selective
detector for the detection of target components in complex
environment, such as herbal preparations. An HPLC/ESI/MS
method had been developed for an estimation of the saponin
content in food supplement containing Tribulus terrestis L.
[81]. In conjunction with an AOAC task group on dietary
supplement, a method using liquid chromatography/tandem
mass spectrometry was validated for measurement of six major alkaloids in raw Ephedra sinica herb, Ephedra extracts
and health supplement products [82]. In both methods, internal standard was added to determine the content of target
compounds present.
For the identification of substances present in botanicals
and herbal preparations using HRGC/MS or HPLC/MS, the
authors find that the following conditions are useful when
standards are available. These include: (1) a suspect peak
has to show retention time similar to average retention time
of the pure standard or control sample and (2) mass spectra
for the suspect peaks has to show relative abundance 10%
(arithmetic difference) of the relative abundance of standard
analyzed that day [83]. With HPLC/MS, applying the right
separation, with the right ionization interface and mass analyzer, significant information can be obtained with regards to
the target compounds. However, for the quantitation of compounds in plant materials, the system precision will be higher
compared to HPLC with ultraviolet detection. For the analysis of target compounds in botanical extracts, systems that
allowed for gradient elution HPLC are often required. For
on-line HPLC/MS, the internal diameter of the column selected will be an important consideration. The used of 1.0
or 2.1 mm i.d. columns with flow rate at 100200 l/min
or lower is optimal for most ESI/MS operation. Post column splitter is often needed to reduce LC flow into the mass
spectrometer when using 4.6 mm i.d. columns with flow rate
at 1.0 ml/min. On top of that, the common buffers, such
as phosphate buffer and others used in most HPLC methods cannot be adopted for methods using HPLC/MS. The
choice of buffers and ion pairing reagents that can be used
in the proposed methods using HPLC/MS are more limited.
For further information of the suitable of solvents and additives in HPLC/MS, the readers can refer to an excellent Ref.
[84]. From our experiences, samples that are found to contain
high salt content will create problems for the mass analyzer
in most HPLC/MS system. Finally, interpretations of mass
spectra from ESI/MS will be more tricky in the presence of
adduct ions, such as [M + Na]+ , [M + 2H]2+ , dimmers and
others.

6. Conclusions
Chemical analysis of extracts from plant material will
play a central role in the development and modernization of
Chinese medicines, botanicals and herbal preparations. The
method used is required to identify the active or marker compounds, composition analysis and fingerprinting purposes.
For the chemical standardization of botanicals and herbal
preparations, the method involved sample preparation procedures, such as techniques of extraction and other analytical techniques, such as the separation tools. However, due
to complexity of the plant materials obtained, different methods using different methods of extraction and separation tools
may be needed. It is certain that methods that are simple, rapid
and environmentally friendly will be preferred and is likely to
play an important role in the effort in providing high quality
products to consumers worldwide. Just as for pharmaceutical products, a well-validated method will help in monitoring
the stability of the botanicals and herbal preparations over a
period of time. It is likely that the combination of chemical standardization with biological assay will provide further
knowledge about therapeutic effects of the medicinal plant.
The use of validated methods in the chemical standardization
of botanicals and herbal preparations will enhance the quality of the products, assist in pharmacological studies, perform
credible clinical trials and propel the move towards evidence
based medicine.

Acknowledgements
The author would like to thank A/P Swee Ngin Tan of
National Institute of Education, Nanyang Technological University for reading the manuscript.

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