Journal of Chromatography A, 1112 (2006) 92–102

Review

Pressurized hot water extraction of bioactive or marker compounds

in botanicals and medicinal plant materials
Eng Shi Ong ∗ , Jane Si Han Cheong, David Goh
Applied Science School, Temasek Polytechnic, 21 Tampines Avenue 1, Singapore 59757, Singapore
Available online 18 January 2006

Abstract
To reduce the use of organic solvent, pressurized hot water extraction (PHWE) has been shown to be a feasible option for the extraction of
bioactive and marker compounds in botanicals and medicinal plants. The parameters that may affect the extraction efficiencies in PHWE include
temperature, extraction time and addition of small percentage of organic solvent or surfactants. Currently, applications of PHWE for the extraction
of thermally labile compounds in botanicals are still rather limited. PHWE with and without the additional of a small percentage of organic solvent
such as ethanol is highly suited for the chemical standardization and quality control of medicinal plants. At the same time, it can be applied at the
pilot scale as a manufacturing process for medicinal plants. Surfactant assisted PHWE was found to enhance the extraction of thermally labile and
more hydrophobic species in medicinal plants at a lower temperature. The addition of small amount of surfactants in PHWE is highly suited for
the determination of bioactive or marker compounds in medicinal plants. With proper optimization, PHWE was observed to have good extraction
efficiency and precision when compared to other reference methods of extraction.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Pressurized hot water extraction; Medicinal plants; Botanicals; Surfactant assisted PHWE; Extraction techniques

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
2. Pressurized hot water extraction (PHWE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3. Instrumentation and basic experimental set-up for PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4. Extraction steps for PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
5. Physicochemical consideration for enhanced extraction steps in PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6. Parameters affecting the extraction process in PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.2. Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.3. Extraction time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.4. Addition of organic solvent in PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.5. Surfactant assisted PHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7. The need for clean-up and pre-concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

1. Introduction
monographs of medicinal plants can be found in the United
States Pharmacopeia [1], Chinese Pharmacopeia [2], WHO
Botanicals and medicinal plants in their natural and pro-
monographs for medicinal plants [3,4], Japanese Pharmacopeia
cessed form are widely used in traditional medicine. Currently,
[5] and herbal medicine (expanded Commission E monographs)
[6]. Other information on the chemical substances in medicinal
∗ Corresponding author. plants and their pharmacological properties can be found in the
E-mail address: esong@tp.edu.sg (E.S. Ong). following books [7–10].

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2005.12.052
 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
According to the guidelines stated by the United States Food
and Drug Administration (USFDA) [11] and The European
Agency for the evaluation of medicinal products [12], various
aspects of analysis must be performed for the purpose of qual-
ity control of botanical drugs and herbal preparations. For the
analysis of botanicals and herbal preparations, a holistic solu-
tion involving the combination of the extraction methods and
analytical techniques such as separation tools are required. The
whole analytical process can be wasted if an unsuitable sam-
ple preparation has been employed before the botanical extracts
reach the chromatograph. The various approaches for the prepa-
ration of samples and assay of bioactive or marker compounds
in botanical extracts and herbal preparations are stated in a few
previous review papers [13–18]. In the monograph stated in the
pharmacopeias, methods of extraction such as sonication, heat-
ing under reflux, Soxhlet extraction and others are commonly
used [1,2,5]. However, such methods can be time consuming,
require the use of large amount of organic solvent and may have
lower extraction efficiencies. Thus, this will lead to higher cost
involved during the extraction process.
From the guidance document from USFDA, other than qual-
ity control tests, a description of the manufacturing process for
the botanical drug substance is required [11]. The quality of the
botanical products can be affected by the type and concentration
of extraction solvent used and the type of manufacturing proce-
dures. These may include steady state extraction (maceration)
or exhaustive extraction (percolation) [19]. Hence, the extrac-
tion of bioactive or marker compounds play an important role in
the manufacturing process, batch-to-batch consistency and qual-
ity control of botanical products. A highly desirable method of
extraction is one that is simple, rapid, and reproducible, with
high extraction efficiency and does not require the extensive use
of organic solvent.
2. Pressurized hot water extraction (PHWE)
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), super-
critical fluid extraction (SFE), accelerated solvent extraction
(ASE) or pressurized liquid extraction (PLE) and pressurized hot
water extraction have been introduced for the extraction of ana-
lytes present in plant materials [14–16]. Water is non-flammable,
non-toxic, readily available and an environmentally acceptable
solvent. It has not yet received much attention as an analytical
extraction solvent for plant materials because water is too polar
to efficiently dissolve most organics that are associated with
botanicals. It has been demonstrated that raising the tempera-
ture with enough pressure to maintain the liquid state allows
it to quantitatively extract a wide variety of organic solutes
from may different matrixes. The ability of water to extract
non-polar organics such as polycyclic aromatic hydrocarbons
(PAH) and polychlorinated biphenyl (PCB) is linked to the fact
that the dielectric constant (polarity) of water can be reduced
significantly with increasing temperature. The dielectric con-
stant (ε) of water at 25 ◦ C is approximately 80, making it an
extremely polar solvent. However, sub-critical water at 250 ◦ C
93
and 50 bar has ε = 27 which allowed quantitative extraction of
PAH in 15 min from soil and urban air particulates. At 250 ◦ C
with enough pressure to maintain the liquid state, it reduces
ε to approximately 27, which falls between those of methanol
(ε = 33) and ethanol (ε = 24) at 25 ◦ C [20–22]. Thus, under these
conditions, water has the properties resembling certain organic
solvents.
Various aspects of sub-critical or superheated water extrac-
tion in solid environmental samples and plant materials were
discussed in a review paper [23]. The early applications of
PHWE involved the extraction of organic pollutants such as
PAHs, PCBs, phenols, pesticides, herbicides, phenolic com-
pounds and others from solid environmental samples such as
sediment, soil, air particulates and others [20–28]. The retention
behavior of phenols, anilines and alkylbenzenes in liquid chro-
matographic separation using sub-critical water as mobile phase
had been reported [29]. At the same time, sub-critical water
extraction was coupled on-line and off-line to HPLC for the
analysis of organic pollutants and flavones [30–32]. However,
at the temperature used for the extraction of organic pollutants,
most bioactive or marker compounds present in botanicals would
have degraded. With certain fine tuning, a method using a lab-
oratory made PHWE system was applied to the extraction of
thermally labile and non polar to polar components in botani-
cals. These included berberine in Coptidis rhizoma, glycyrrhizin
in Radix glycyrrhizae (liquorice), baicalein in Scutellariae radix,
marker compounds in Radix codonopsis pilosula and tanshinone
I and IIA in Salvia miltiorrhiza [33–35]. As for the extraction
of volatile components such as essential oils in plant materi-
als with PHWE, further information can be found in the review
papers [23,36]. Other applications of PHWE in medicinal plant
materials can be found in Table 1. From Table 1, the extraction
recoveries and precision of PHWE are comparable to other refer-
ence methods of extraction. To our best knowledge, applications
of PHWE for the extraction of thermally labile constituents in
medicinal plants are still rather limited.
3. Instrumentation and basic experimental set-up for
PHWE
The experimental set-up in most of the papers cited was per-
formed using a laboratory made system [21–31,33–35]. The
differences in the design of the various instrumentations used can
be seen in Fig. 1. As seen in Fig. 1A and B, some set-up requires
the use of gases, cooling coils and a second high pressure pump
compared to the alternative system proposed by Ong and Len
[33–35]. Some groups have proposed coupling on-line PHWE
with microporous membrane liquid–liquid extraction (MMLLE)
and gas chromatography (GC) [28]. Instrumentation for sub-
critical water extraction with an on-line solid-phase extraction
(SPE) or sorbent trap was proposed [30–32].
All the set-ups consist of a stainless steel preheating coil
to ensure that the water is at the operating temperature prior
to entering the extraction cell. The stainless steel tubings used
were 1/16 in. O.D. and 0.18 mm I.D. For the experimental set-
up in Fig. 1B, the backpressure was generated using a back
pressure regulator by VICI Jour Research (Onsala, Sweden).
 94
Table 1
Applications of PHWE for the extractions of bioactive or marker compounds from botanicals and medicinal plants
Medicinal plant Chemical Temperature Pressure Flow-rate Extraction time for Reference Sample Method of Extraction References
constituents (◦ C) (ml/min) static extraction method(s) pre-treatment analysis recoveries and
with ASE or after precision (%)
dynamic extraction extraction

Piper methysticum Kava lactones 175 60–70 bar 1.0 20–40 min (1) Boiling LLE GC–MS 85.0–100.0 [37]
(Kava Kava) (2) Soxhlet apparatus (RSD: less
(3) Sonication than 12.0)
Morinda citrifolia Anthraquinones 100,170, 220 7 MPa 2.0,4.0,6.0 180 min Organic solvent Nil UV NA [38]
extraction
American ginsengb Ginsenosides Rg1, 50,90,120 1500 psi Nil 10 mina Ultrasonic-assisted Nil HPLC 99.0–139.0 [39]
Panax Re, Rb, Rc, Rd extraction (RSD: less
quinquefolium than 10.0)
Spirulina platensis Antioxidants 60, 115, 170 1500 psi Nil 3, 9,15 mina Nil Nil Anti-oxidant Nil [40]
activity

E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102

determination
Spirulina platensis Antioxidants 115, 170 1500 psi Nil 9, 15 mina Nil Nil MEKC–DAD Nil [41]
Grapes Phenolic 40, 100 40, 150 atm Nil 3 min × 10 mina Spiked standards SPE HPLC 26.0–51.0 [42]
compounds (RSD: Nil)
Tea leaves, grape Catechin, 100–200 1500 psi Nil 5, 10 mina Ultrasound-assisted Nil HPLC 6.8–12.5 [43]
seeds epicatechin extraction (RSD: Nil)
Dried red grape skin Anthocyanins, 20–140 10.1 MPa Nil 15 mina Soxhlet extraction Nil HPLC–MS 105.0–116.0 [44]
total phenolics (RSD: NA)
Juniperus Cedarwood oil 50, 100, 150, 500, 750, Nil 15 mina Liquid and LLE SFC NA [45]
virginianna 200 1500, supercritical carbon
3000 psi dioxide extraction
Fructus amomi Essential oil 150 50 bar 1.0 5 min Recovery and SPME GC–MS ∼90.0 (RSD: [46]
repeatability 4.3–9.8)
Acorus tatarinowii Essential oil 150 50 bar 1.0 5 min Steam distillation SPME GC–MS ∼92.0 (RSD: [47]
Schott. less than 13.0)
Fructus amomi Essential oil 160 60 bar 1.0 5 min Steam distillation LPME GC–MS 50.0–120.0 [48]
(RSD: less
than 12.0)
Laurel leaves Laurel essential 150 ∼50 bar 2.0 15 min Hydrodistillation LLE GC–FID, Greater than [49]
oil GC–MS 100.0 (RSD:
less than 15.0)
Marjoram leaves Marjoram 150 50 bar 2.0 15 min Hydrodistillation LLE GC–FID, Greater than [50]
essential oil GC–MS 100.0 (RSD:
less than 7.5
Veronica longifolia Iridoid glycosides 100 145 atm Nil 30 min Maceration with Nil CE 83.0–92.0 [51]
leaves (Catalpol, various organic (RSD: 22 to 8)
Aucubin) solvents
Rosemary plants Antioxidant 25, 100, 150, 40–70 bar 1.0 30 min Nil Nil LC–MS Nil [52]
compounds 200
Dioscorea alata 1, 1-Diphenyl-2- 100 1.342 Mpa 10.0 <180 min Nil Nil HPLC Nil [53]c
picrylhydrazyl
Free Radical
Scavenging
Compounds
Rosmarinus Rosemary 125–175 ∼20 bar 2.0 30 min Steam distillation LLE GC Greater than [54]
officinalis oxygenated 100.0 (RSD:
fragrance and 3.4–34.0
flavour
compounds
Orange peel Flavone, 100, 150 Nil 0.5 Nil Sample spiking SPE SBWC/UV, 69.0–92.0 [31]
7-hydroxyflavone GC (RSD: 10–17)
Coptidis rhizome Berberine 95–140 10–20 bar 1.0 40 min Soxhlet extraction Nil HPLC 77.7–90.8 [33]c
(RSD:
2.4–4.5)
Radix glycyrrhizae Glycyrrhizin 95–140 10–20 bar 1.0 40 min Sonication Nil HPLC 99.4–130.4 [33]
(RSD:
3.5–7.4)
Scutellariae radix Baicalein 95–140 10–20 bar 1.0 40 min Soxhlet extraction Nil HPLC 89.6–99.5 [33]c
(RSD:
7.3–11.1)
Radix codonopsis Tetradeca-4E, 95 10–20 bar 1.0 40 min Soxhlet extraction SPE HPLC 90.0–110.0 [34]c
pilosulab 12E-diene-8, (RSD:

E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102

10-diyne-1,6, 2.8–7.7)
7-triol,
Tetradeca-4E,
12E-diene-8,
10-diyne-1, 6,
7-triol-O-ß-d-
glucoside
Salvia miltiorrhiza Tanshinone I and 95–140 10–20 bar 1.0 20, 40 min Soxhlet extraction SPE HPLC, 84.6–113.0 [35]c
IIA LC–MS (RSD:
2.4–9.0)
Origanum onites Borneol, 100,125,150,175 60 bar 2.0 30 min Steam distillation, SPE GC × GC/ 50.0–110.0 [55]
terpinen-4-ol, Soxhlet extraction TOFMS (RSD: Nil)
carvacrol
Origanum micrathum Essential oils 100,125,150,175 40–80 bar 1.0–3.0 30 min Nil SPE GC × GC/ 100.0 [56]
TOFMS
Ziziphora taurica pulegone, 150 60 bar 2.0 30 min Steam distillation, SPE GC × GC/ 90.0–110.0 [57]
subsp. Taurica terpinen-4-ol, direct thermal TOFMS (RSD:
cis-carveol, desorption 0.8–4.4)
trans-carveol, and
verbenone
Defatted soybean Total iso-flavones 110 641 psig nil 2.3 h Soxhlet extraction SPE HPLC 43.8–93.6 [58]
flakes (RSD: Nil)
Satureja hortensis Oxygenated and 100–175 60 bar 1.0 12–40 min Hydrodistallation, LLE GC 70.0–100.0 [59]
(Savory), Mentha non-oxygenated SFE (RSD:
piperita flavor & fragrance 1.0–11.0)
(peppermint) compounds
Rosmarinus Antioxidant 60–100 1500 psi Nil 25 mina Nil Nil CE–ESI–MS Nil [76]
officinalis L. compounds
(rosemary) (rosmarinic acid
& carnosic acid)
Dried red grape skin Anthocyanins and 100–160 Nil Nil 40 sa Conventional hot Nil HPLC 90.0–100.0 [78]
phenolics water, aqueous 60 % (RSD:
methanol extraction 1.0–9.0)

95
96 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
chromatography, SPE, solid-phase extraction; SMPE, solid-phase microextraction; UV, ultraviolet; GC × GC/TOFMS, comprehensive gas chromatography/time of flight mass spectrometry; CE–ESI–MS capillary
Abbreviations: CE, capillary electrophoresis; DAD, diode array detection; FID, flame ionization detector; GC, gas chromatography; HPLC, high performance liquid chromatography; LLE, liquid–liquid extraction;
LPME, liquid phase microextraction; MEKC, Miceller electrokinetic chromatography; LC–MS, liquid chromatography–mass spectrometry; SBWC, sub-critical water chromatography; SFC, supercrtical fluid
References

[81]c
[79]

[80]

[82]
recoveries and
precision (%)

102.0–155.0
90.0–106.0
(RSD: 7.9)

(RSD: 8.0)
Extraction

80.0–98.0

0.9–2.9)
(RSD:
Nil
Anti-oxidant activity
determination
Method of

GC–FID
GC–MS
analysis

HPLC
pre-treatment

extraction
Sample

SPME
after

LLE
Nil

Nil
Ultra-sonication with

methanol/water (3:7)
Anti-oxidant activity
Spiking of standards

Solvent boiling

Fig. 1. (A) Schematic diagram of PHWE equipment with a cooling coil and a
determination

methods with

second pump (rinse solvent) delivering dichloromethane [26] and (B) Schematic
method(s)
Reference

methanol

diagram of an alternative laboratory made PHWE system proposed by other

group [33–35].

The system consists of stainless steel extraction cells with

dynamic extraction
Extraction time for

10 mm I.D. × 150 mm (approximately 10 ml) or larger. The

static extraction

extraction cell was heated in a gas chromatograph oven and a

3, 9, 15 mina
with ASE or

10–30 mina

pump was used to pump the fluid into the extraction cell. By pres-
30 min

30 min

surizing the sample cell, it was possible to keep the fluid in liquid
phase at the high extraction temperatures used (up to 200 ◦ C)
[29–31]. For some work, a restrictor was used to generate the
Flow-rate
(ml/min)

electrophoresis–electrospary–mass spectrometry; ASE accelerated-solvent extraction.

back pressure. For certain set-ups, in-order to prevent deposi-

1.5–2.0

1.5–2.0

tion of the solutes from the water that cools during collection, a
Nil
1.0

second pump is used to inject chloroform/dichloromethane into

a fused silica-lined tee placed in the oven between the satura-
Pressure

1500 psi

tion cell and collection valve (Fig. 1A). A cooling trap is used
100 atm
100 bar
40 bar

to cool the fluid coming out from the extraction cell to room
temperature (Fig. 1A) [21–27]. From the systems that are built,
c Modifiers such as ethanol or other organic solvent added.

care must be taken to avoid narrow bore tubing that might be

a Extraction performed using the Dionex ASE 200 unit.
Temperature

60, 115, 170

blocked by a precipitate when the botanical extracts leave the

25– 100

20–140

oven.
(◦ C)

150

At the same time, sub-critical water extraction using the

Dionex ASE 200 unit was reported [39–45].
pseudo-hypericin
Hypericin and
Antioxidants

4. Extraction steps for PHWE

constituents

Ligustilides

trilactones
Chemical

Terpene

b Surfactant assisted PHWE.

All experiments with PHWE are performed in a dynamic

mode at a constant flow or static extraction with the Dionex
ASE 200 unit. Before loading the plant materials into the extrac-
Table 1 (Continued )

Angelica sinensis
Spirulina platensis
chuanxiong and

tion cell, the samples are often pre-treated in some ways. It will
Medicinal plant

St. John’s Wort

Ginkgo biloba

involve either plain air- or freeze-drying. The drying step is

Ligusticum

followed by grinding of the sample to a size in the range of

100–1000 ␮m and sieving with 2 mm sieve. Plant materials that
are ground to smaller sizes might be advantageous in facilitat-
 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
ing analyte transport to particle surfaces, possibly due to shorten
diffusion paths.
For PHWE, the extraction of plant materials normally
involves the following steps.
• Before PHWE extraction in the dynamic mode, the medici-
nal plant samples are weighed directly into a glass tube and
mixed thoroughly with a high proportion of sand. The sand
and plant material mixture are transferred into the extraction
cell for PHWE. The extraction cells are finally filled with
sand to avoid any void spaces. This step is required as plant
materials have a strong tendency to adsorb water in the course
of extraction. Hence, the ground plant materials must be dis-
pensed evenly with sand to prevent any blockage of the system
[33–35]. However, this step was not required for PLE using
organic solvent such as methanol.
• Plant materials are loaded into the extraction cell.
• Extraction cell is filled with the water and the cell is set to
certain values.
• Extraction cell is heated in an oven to the set values.
• Extracting the plant materials for a certain time with dynamic
or static extraction. For dynamic extraction, the pump will be
set at a constant flow rate of 0.5–2.0 ml/min.
• In dynamic extraction, the fluid coming out of the extraction
cell is collected in the collection vial as seen in Fig. 1. For
static extraction with the Dionex ASE 200 unit, the fluid is
purged from the extraction cell to the collection vial using a
suitable gas.
• Suitable pre-concentration and clean-up procedures are
selected for the aqueous extract from PHWE.
5. Physicochemical consideration for enhanced
extraction steps in PHWE
The extraction process in PHWE can be proposed to have
three sequential steps. First, the solutes must diffuse from the
core of the plant materials to the surface. Next, they should be
transferred from the surface into the extraction fluid. Finally,
the solutes are eluted out of the extraction cell. A schematic rep-
resentation of the three sequential steps can be seen in Fig. 2.
The extraction rate is limited by the slowest of these three steps
[60,61]. Extraction with pressurized hot water can be fitted
using the simple thermodynamic model defined by the distri-
bution ratio (KD ) in which KD = (concentration of analyte in
the matrix)/(concentration of analyte in the extraction fluid) at
equilibrium. For this model, it is assumed that the kinetics of the
initial desorption step and subsequent fluid–matrix partitioning
are rapid, and thus do not significantly affect the extraction rate.
The mass of analyte in each unit mass of extraction fluid and
the mass of analyte remaining in the matrix at that period in the
entire extraction time is based on the KD value determined for
each compound. The thermodynamic elution of analytes from
matrix was the prevailing mechanism in PHWE as evidenced by
the fact that extraction rate increased proportionally with the hot
water flow rate [62].
The main reasons why extraction fluids at elevated temper-
atures and pressures give enhanced performance compared to
97
Fig. 2. A proposed schematic presentation of the extraction steps in PHWE: (1)
rapid fluid entry; (2) desorption of solutes from matrix active sites; (3) diffusion
of solutes through organic materials; (4) diffusion of solutes through static fluid
in porous materials; (5) diffusion of solutes through layer of stagnant fluid outside
particles; and (6) elution of solutes by the flowing bulk of fluid.
extraction at lower temperature and atmospheric pressure are:
(i) solubility and mass transfer effects and (ii) disruption of sur-
face equilibria. First, the use of higher temperatures modify the
properties of water. It increases the capacity of the fluid to sol-
ubilize analytes. At the same time, a faster diffusion rate occurs
as a result of increasing the temperature of the extraction. How-
ever, it can be difficult to obtain an exact relationship for the
effect of temperature on diffusion rate, especially with finite con-
centrations and a multi-component system. If fresh fluid were
introduced during a static or dynamic extraction in PHWE, this is
analogous to what occurs in traditional methods such as Soxh-
let extraction. It improves mass transfer and, hence, increases
extraction rate. The introduction of fresh fluid will increase the
concentration gradient between the solution extraction cell and
surface of the sample matrix.
Both temperature and pressure play a significant role in the
disruption of surface equilibria. The increased temperatures can
disrupt the strong solute–matrix interaction caused by van der
Waals forces, hydrogen bonding, dipole attraction of the solutes
molecules and active sites in the matrix. The thermal energy
supplied can overcome cohensive (solute–solute) and adhesive
(solute–matrix) interaction by decreasing the activation energy
required for the desorption process. The higher temperature will
also decrease the viscosity of the liquid used, thus allowing better
penetration of matrix particles and enhancing extraction.
A sufficient pressure is required to be exerted on the fluid
used when temperature above the boiling point is used. The use
of elevated temperatures and the associated advantages would
be precluded without the use of elevated pressure to maintain the
fluid as liquid. The use of pressure can facilitate extraction from
samples in which the analytes have been trapped in matrix pores.
The pressure forces the fluid into areas of the matrixes that would
not normally be contacted by fluid under atmospheric conditions
[63].
98 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102

6. Parameters affecting the extraction process in PHWE
The parameters that may affect the extraction efficiencies
in PHWE include temperature, extraction time and addition of
organic solvent or surfactants. The geometry of the extraction
cell or vessel and the flow direction of the water had only minor
effect on the recoveries of the target analytes in the solid sam-
ples [64]. In the validation of methods using PHWE, it is often
required to compare the extraction efficiencies with reference
methods such as Soxhlet extraction, sonication and others. In
botanicals, the bioactive or marker compounds are present natu-
rally and significant analyte–matrix interaction will be present.
Hence, spiking of the target compounds into the plant matrix will
not mimic the real environment [33–35,65–68]. A comparison of
the chromatographic fingerprint of the extracts from Scutellariae
radix obtained from Soxhlet extraction and PHWE Fig. 3A
and B. The chromatographic profile obtained from PHWE
was similar to that obtained from extraction with an organic
solvent.
6.1. Temperature
Temperature is one of the most important factors contributing
to the increased extraction efficiencies in PHWE and other meth-
ods of extraction such as MAE, SFE and others. An important
operational difference between PHWE and Soxhlet extraction is
the extraction temperature. In PHWE, the applied temperature
for extraction may be well above the normal boiling point of the
fluid used. Contrarily, in Soxhlet extraction, the extraction tem-
perature is limited by the boiling point of the solvent used. In the
extraction of organic pollutants from solid environmental sam-
ples with PHWE, the extraction temperature had been reported
to affect the recoveries. For low polarity organic pollutants, a
higher temperature (at 300 ◦ C) will enhance the solubility and
increase the extraction efficiencies [20,22–27].
For bioactive and marker compounds in botanicals and
medicinal plants, the extraction temperature used in PHWE
has an important impact on the extraction efficiencies
[33–38,41–54,55–57,59]. Increasing the extraction temperature
from 125 to 180 ◦ C will result in higher recoveries for the extrac-
tion of volatile components such as kava lactones and essential
oils from plant materials [33,46–50,55–57,59]. The bioactive
and marker compounds in the medicinal plants may be non-
polar to polar and thermally labile. In order to extract non-polar
components efficiently, it may be necessary to increase the
extraction temperature from 150 to 250 ◦ C. However, an increase
in the extraction temperature will cause the analytes present
to degrade. In the extraction of naturally occurring substances
such as berberine, strychnine, aristolochic acids, baicalein, gly-
cyrrhizin, tanshinone I and IIA and others in medicinal plants,
increasing the applied temperature to a certain point would
result in higher recoveries, after which the compounds will
start to degrade [33–35,65–68]. A similar trend was observed
when using pressurized liquid extraction for the extraction of
essential oils in medicinal plants, antioxidant compounds from
rosemary plants and catechin and epicatechin in tea leaves and
grape seeds [33,43,46–50,52–57,59]. The effects of increasing
the extraction temperature from 80 to 160 ◦ C on the amount
of marker compounds in Radix codonopsis pilosula by PHWE
were investigated. The data reported showed that a downward

Fig. 3. Chromatogram obtained for baicalin and baicalein in Scutellariae radix by: (A) Soxhlet extraction and (B) PHWE. Mobile phase of (A) 0.1% formic acid
in water and (B) 0.1% formic acid in acetonitrile. Initial condition was set at 20% of (B) gradient up to 100% (B) in 20 min before returning to initial condition for
10 min. Detection was at 254 nm. Oven temperature was at 40 ◦ C and flow rate was 1.0 ml/min.
 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
trend was observed for the amount of the three marker com-
pounds extracted with increasing temperature. However, the
three marker compounds were found to be stable up to 160 ◦ C
when methanol was used as the extraction solvent [34]. The
applied temperature and the pressurized hot water may produce
undesired effects for the target compounds. Hence, the effects
of the extraction temperature must be thoroughly investigated
when using PHWE for the extraction of constituents present in
botanicals and medicinal plants.
6.2. Pressure
Elevated pressures from 10 to 80 bar are mainly used to
keep the fluid from boiling in PHWE. A certain minimum
pressure is required to maintain the extraction fluid as a liq-
uid at the extraction temperature. As seen in the extraction of
organic pollutants in solid environmental samples, the pressure
is of minor importance for the resulting recoveries in PHWE
[20,22,27]. Similarly, varying the pressure did not result in any
changes in the recoveries for the extraction of essential oils
from medicinal plants and ginsenosides from American ginseng
[39,47,48].
6.3. Extraction time
PHWE can be performed in the static mode such as using
the Dionex ASE 200 unit and dynamic mode with the labo-
ratory made system. As seen in Table 1, the extraction times
in PHWE using static and dynamic mode were very short
compared to those in conventional solid–liquid extraction tech-
niques. An advantage of PHWE in the dynamic mode is that
the fluid is continuously refreshed during the whole course of
extraction. Evidently, PHWE in dynamic mode will require a
larger volume of fluid compared to static extraction. On the
other hand, PHWE using static mode may lead to incomplete
extraction because of the limited volume of fluid used. In
Table 1, a single static extraction of 10–15 min, 2 min × 10 min
or lesser can be used for quantitative recoveries. For PHWE in
the dynamic mode, an extraction time of 5 min–2 h will result
in good recoveries for medicinal plants and solid environmen-
tal samples. It was observed that an extraction time of 40 min
with PHWE in the dynamic mode will give acceptable recov-
eries for a number of compounds present in medicinal plants
[33–35]. Hence, the time for extraction or volume of fluid
required for PHWE has to be determined during the validation
process.
6.4. Addition of organic solvent in PHWE
In the extraction of catechins in tea leaves and grape seeds
and phenolic compounds in grapes with pressurized liquid, it
was observed that water gave the lowest recoveries compared
to other organic solvents such as methanol, ethanol and ethyl
acetate [42,43]. Similarly, it was observed that PHWE will give a
lower recovery with reference to Soxhlet extraction for the deter-
mination of marker compounds in Radix codonopsis pilosula
and tanshinone IIA in Salvia miltiorrhiza [34,35]. For tanshi-
99
none IIA in Salvia miltiorrhiza, it was observed that increasing
the extraction temperature did not result in higher extraction
efficiencies while an increasing amount of ethanol added in the
water (0–30%) will enhance the extraction efficiencies [35]. A
similar trend was observed in the determination of berberine in
Coptidis rhizome [33].
The addition of organic solvent such as ethanol into the
extraction fluid in PHWE will result in higher recoveries for the
target compounds in medicinal plants [33–35]. It was observed
that the presence of ethanol in the water (0–20%) will increase
the capacity of the fluid to solubilize the naturally occurring
compounds in plant materials. Compared to traditional meth-
ods of extraction, the consumption of organic solvent will be
reduced significantly. A similar strategy was applied for the
analysis of benzalkonium chloride and anthropogenic waste
indicator compounds in sediment samples. PLE with a mixture
of acetonitrile/water (6:4 or 7:3) and water/isopropanol (50:50)
was observed to give acceptable recoveries [69,70]. The use of
mixtures such as water/isopropanol in PLE will reduce sam-
ple preparation, solvent consumption and minimize background
interferences [70].
6.5. Surfactant assisted PHWE
Even with the addition of a low percentage of organic sol-
vent such as ethanol, it was observed that PHWE did not result in
extraction efficiencies that were comparable to Soxhlet extrac-
tion for hydrophobic marker compounds in Radix codonopsis
pilosula and tanshinone IIA in Salvia miltiorrhiza [34,35]. The
difficulties in extracting certain naturally occurring compounds
in medicinal plants with PHWE were demonstrated. Micelle
formation for the improvement of sub-critical water extraction
had been applied for the removal of polycyclic aromatic hydro-
carbons in soil and environmental samples. The samples were
subjected to static-dynamic extraction with sodium dodecyl sul-
fate (SDS)—water at a pressure of 50 bar and a temperature of
150–225 ◦ C. The instrument setup was similar to those shown
in Fig. 1 [71,72].
To improve the recoveries of marker compounds in Radix
codonopsis pilosula, a method combining surfactants and
PHWE with an applied temperature at 95 ◦ C was developed.
With surfactant-assisted PHWE, the effects of different added
surfactants such as sodium dodecyl sulfate and Triton X-100
were studied. Surfactant assisted PHWE with Triton X-100
proved to be at least equivalent or better compared to Soxhlet
extraction in terms of quantitative analysis of marker compounds
in Radix codonopsis pilosula [34]. The feasibility of employ-
ing aqueous non-ionic surfactants solutions as an alternative
solvent system in pressurized liquid extraction was demon-
strated by another research group using the root of American
ginseng as model solid samples. When compared to ultrasonic
extraction with an organic solvent, the presence of a common
non-ionic surfactant (Triton X100) in water at a concentration
above its critical micelle concentration was shown to give com-
parable amount of pharmacological active compounds extracted
from ginseng roots [39]. For surfactant assisted PHWE, other
than the temperature and time of extraction, the parameter that
100 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
needs optimization is the concentration of surfactants used. In
both instances, it was shown that changing the concentration
of surfactants such as Triton X100 above a certain concen-
tration at a certain temperature was found to be not signifi-
cant in affecting the amount of marker compounds extracted
[34,39].
For surfactant assisted PHWE in a dynamic mode and PLE
using non-ionic surfactant solution, the presence of surfactant
such as Triton X-100 in the water enhances the solubility of
the target compounds and forces the target analytes in the sam-
ple matrix in the mobile phase to completeness with the fresh
liquid pumped through the sample continuously. As the tar-
get compounds were naturally occurring in Radix codonopsis
pilosula and American ginseng, strong analyte–matrix inter-
action will be present, the addition of surfactants in the fluid
was proposed to disrupt the strong analyte–matrix interac-
tion present naturally in the medicinal plants and improve the
extraction efficiencies. At the same time, extraction can be
carried out at a lower temperature without a significant com-
promise in extraction efficiencies. The degradation of ther-
mally unstable ingredients in medicinal plants can also be
avoided. The addition of surfactants in PHWE was found to
enhance the extraction of more hydrophobic species in medicinal
plants [34,39].
7. The need for clean-up and pre-concentration
The extract from PHWE is a relatively dilute aqueous solution
and it is possible for sample loss by re-adsorption onto the orig-
inal matrix. The dilute aqueous extract from PHWE although
free of the matrix often required concentration or extraction
before any subsequent assay step with separation tools. The
aqueous extract is a relatively clean matrix, sample handling
and pre-treatment is much easier than from the original sample
materials. One of the approaches was to evaporate the excess
liquid collected from PHWE with a rotary evaporator under vac-
uum to 10–15 ml. The recoveries were found to be comparable
with those of other methods of extraction such as sonication
and Soxhlet extraction [33]. Another common method to pre-
concentrate the aqueous extract from PHWE is to extract the
aqueous solution with a small amount of immiscible organic
solvent [20–22,23,27]. Depending on the target analyte, PHWE
with liquid–liquid extraction (LLE) may result in some sample
loss but will give a more concentrated solution for subsequent
analysis. Ideally, an internal standard should be employed.
To minimize the use of organic solvent in the clean-up step,
methods that are solvent free or require minimal solvent such
as solid-phase extraction have been used (Table 1). The SPE
cartridges introduced two important features into the method;
it includes standardization and hence greater reproducibility
for the extraction. The very wide range of SPE phases (C18,
C8, SCX, SAX and others) means that polarity, hydrophobic-
ity or ionization may be used as trapping mechanism. In the
case where the solid samples are extracted with a mixture of
acetonitrile/water (6:4 or 7:3) and water/isopropanol (50:50), a
suitable amount of water can be added to the aqueous extract
before the clean-up step with SPE [69,70]. SPE was found to be
exceptionally useful when used together with surfactant assisted
PHWE and samples with high salt content. The salt and sur-
factant present may pose a problem when used with analytical
tools such as LCMS. The limitations of SPE are the degree of
pre-concentration, which is imposed by the breakthrough vol-
ume of the cartridge and the overloading of the cartridge by
other sample components. The more polar constituents present
in the medicinal plants may pose a problem when using SPE as
adsorption on the SPE cartridge may occur. At the same time,
packing materials that have larger particle size will be favored
as it can minimize blockage from the precipitates that are often
formed from the aqueous extracts from medicinal plants from
PHWE.
Solid-phase microextraction (SPME) is a technique that
requires no solvent or complicated apparatus. It can concen-
trate volatile and non-volatile compounds such as essential
oils from medicinal plants, in both liquid and gaseous sam-
ples for analysis by GC, GC–MS or HPLC (Table 1). The
pre-concentration of aqueous solution from PHWE with SPME
from dynamic or static extraction followed by GC analysis
was introduced [23,46,47]. It had been shown that the method
using PHWE–SPME with final analysis by GC or GC–MS gave
good repeatability and recovery. It is an alternative method for
the determination of essential oils and volatile components in
medicinal plants and can be used as a powerful tool for quality
assessment [46,47].
Microporous membrane liquid–liquid extraction is a promis-
ing new technique for extracting, cleaning and concentrating
aqueous samples and it can be used in PHWE in place of the
solid-phase trap. For MMLLE, in addition to the LLE process,
size exclusion also takes place thereby increasing the selectivity
of the extraction. Methods using PHWE with MMLLE was used
for the analysis of pesticides in grapes and other organic pollu-
tants in solid environmental samples [25,26,28] and is suitable
for the analysis of bioactive and marker compounds in medicinal
plants.
8. Conclusions
With an increasing interest in avoiding organic solvents in the
extraction of active or marker compounds from medicinal plants,
PHWE has been shown to be a feasible alternative approach.
The equipment required can be relatively simple and avoids the
need for high pressures employed in SFE. Even with the addi-
tional sample preparation steps, methods using PHWE have been
shown to give good repeatability and recovery.
Based on the works cited in this review paper, PHWE with
and without the addition of a small percentage of organic solvent
such as ethanol is highly suited for the chemical standardization
and quality control of medicinal plants. Currently, sub-critical
water apparatus at the pilot scale using 4 kg of sample had been
proposed [73]. Surfactant assisted PHWE was found to enhance
the extraction of more hydrophobic species in medicinal plants
at a lower temperature and is highly suited for the determination
of bioactive or marker compounds in medicinal plants.
The analytical method will not be complete without the
use of separation tools for the final analysis of the botanical
 E.S. Ong et al. / J. Chromatogr. A 1112 (2006) 92–102
extracts. At this moment, CE with an aqueous buffer solu-
tion is used for the separation and determination of thermally
labile bioactive or marker compounds present in plant materials
[16,51,67,68,74–77]. Hence, the combination of PHWE with
microbore HPLC or CE will be an attractive option to mini-
mize or eliminate the use of organic solvent for the chemical
standardization of medicinal plants.
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