2013

NPC Natural Product Communications Vol. 8

No. 12
Methods for Extraction and Determination of Phenolic Acids in 1821 - 1829
Medicinal Plants: A Review
Agnieszka Arceusz, Marek Wesolowski* and Pawel Konieczynski

Department of Analytical Chemistry, Medical University of Gdansk, 80-416 Gdansk, Poland

[email protected]

Received: July 18th, 2013; Accepted: October 8th, 2013

Phenolic acids constitute a group of potentially immunostimulating compounds. They occur in all medicinal plants and are widely used in phytotherapy and
foods of plant origin. In recent years, phenolic acids have attracted much interest owing to their biological functions. This paper reviews the extraction and
determination methods of phenolic acids in medicinal plants over the last 10 years. Although Soxhlet extraction and ultrasonic assisted extraction (UAE) are
commonly used for the extraction of phenolic acids from plant materials, alternative techniques such as supercritical fluid extraction (SFE), and accelerated
solvent extraction (ASE) can also be used. After extraction, phenolic acids are determined usually by liquid chromatography (LC) owing to the recent
developments in this technique, especially when it is coupled with mass spectrometry (MS). Also detection systems are discussed, including UV-Vis, diode
array, electrochemical and fluorimetric. Other popular techniques for the analysis of this group of secondary metabolites are gas chromatography coupled with
mass spectrometry (GC-MS) and capillary electrophoresis (CE).

Keywords: Phenolic acids, Medicinal plants, Extraction, Chromatographic techniques, Capillary electrophoresis, Secondary metabolites.

Introduction and in esters as depsides and depsidones [11]. In addition, in plant

tissues, compounds of phenolic acids with other natural constituents
Vascular plants synthesise many organic compounds, often called
have been identified, for instance, with flavonoids, fatty acids, sterols
secondary metabolites [1]. These low-molecular-weight compounds
and cell wall polymers.
determine the basic life processes of plants. It has been estimated that
about 8000 compounds naturally occurring in plants are phenols.
The contents of phenolic acids in plant raw materials, as well as of
Their characteristic structural feature is an aromatic ring with at least
other phenolic compounds, depend on many factors. The biggest
one hydroxyl substituent. Many phenolic compounds occur
impacts come from climatic conditions, cultivation, fertilization and
constitutively, but some stress factors contribute to increases in or
time of harvest [2,12,13]. Also important are storage conditions of the
de novo synthesis of phenolics, such as infection, plant tissue
plant materials and the methods of preparation [10]. It is known that
damage, UV radiation and elevated temperature [2].
plants from the Lamiaceae family contain especially large amounts of
phenolic acids [14]. These compounds can also be found in large
The popularity and consumption of medicinal plants have grown
amounts in fruits and vegetables. The main source of phenolic acids is
significantly in recent years. This phenomenon has led to a faster and
herbal infusions; they also occur in black and green tea, and coffee
better evaluation of the quality of plant products. One of the tools
[15]. In food products, phenolic acids may influence the color, flavor,
playing a crucial role as an element controlling the quality of plant
fragrance and oxidation stability of food. In this case, their level
material used in medicine is the so-called fingerprint analysis [3-5].
depends, among other factors, on the type of technological process
applied to a given plant material [10].
Determination of phenolic compounds can be very helpful in
estimation of pharmacological activity of medicinal plants [6]. Within
this huge group of compounds, a significant role is played by Source and roles of phenolic acids in human
phenolic acids, aromatic secondary metabolites widespread in the Phenolic acids constitute an important group of compounds,
plant kingdom. These compounds contain both hydroxyl and characterised by a wide spectrum of pharmacological activity. They
carboxyl groups. Phenolic acids include hydroxyl derivatives of are responsible for free radical scavenging, metallic ions chelation,
benzoic and cinnamic acids (Table 1). Much attention has been and changing enzymatic activity. Moreover, these compounds exhibit
focused on gallic, vanillic, salicylic, caffeic and p-coumaric acids, antiviral activity, for instance rosmarinic acid, anti-inflammatory
which are active constituents of many plants [7,8]. Examples include activity, for example a mixture of esters of benzoic and cinnamic
thyme, whose phenolic acids comprise gallic, caffeic and rosmarinic acids; diuretic, anti-allergic, as well as cholagogic and choleretic
acids, also sage containing ferulic, gallic, rosmarinic, vanillic, and activities [15-17]. Phenolic acids participate in regeneration and
caffeic acids, as well as rosemary, characterised by a high content of adaptation processes in humans and are used in prevention against
vanillic, caffeic and rosmarinic acids [7]. many diseases [18,19]. It was proved that they prevent coronary
disease, inflammation, type-2 diabetes, as well as aiding in the
Source and roles played by phenolic acids in plants treatment of cancer. Several of these compounds, for instance ferulic
and caffeic acids, are called cancer growth inhibitors. Furthermore,
Phenolic acids are secondary metabolites and constitute an important
recent studies have also shown pro-coagulant activity of phenolic
group of hydrophilic compounds in plant tissues [9,10]. They seldom
acids isolated from Blumea riparia DC [20] and possible degradation
occur in the free form and, therefore, they appear in low
pathway of salvianolic acid B in water solution and simulated gastric
concentrations. In plants, phenolic compounds can appear mainly in
and intestinal fluids [21].
their bound forms, for example as glycosides (phenolic glycosides)
1822 Natural Product Communications Vol. 8 (12) 2013 Arceusz et al.

Table 1: Structural formulas of phenolic acids.

Hydroxybenzoic acids R1 R2 R3 R4
Benzoic acid H H H H
p-Hydroxybenzoic acid H H OH H
Vanillic acid H OCH3 OH H
Gallic acid H OH OH OH
Syringic acid H OCH3 H OCH3
Protocatechic acid H OH OH H
Gentisic acid OH H H OH
Veratric acid H OCH3 OCH3 H
Salicylic acid OH H H H
Hydroxycinnamic acids R1 R2 R3 R4
Cinnamic acid H H H H
o-Coumaric acid OH H H H
m-Coumaric acid H OH H H
p-Coumaric acid H H OH H
Ferulic acid H H OH OCH3
Caffeic acid H OH OH H

The availability of phenolic acids in humans is mostly influenced by between the concentrations of analyte in the cell solution and in the
their chemical form, as well as the morphological part of the plant solvent is maintained throughout, which leads to total extraction of a
[22]. More hydrophilic compounds are characterised by a higher plant material.
bioavailability and are more readily absorbed in the upper part of the
digestive system. On the other hand, substances in their bound form Multiple extraction with the use of a Soxhlet apparatus was applied
are absorbed after enzymatic hydrolysis, which is mediated by for extraction of phenolic acids from plant material (Sambucus nigra
intestinal microflora. L., Polygonum aviculare). Methanol was a solvent and the digestion
process lasted 15 h. Application of this modification of extraction in
The biological significance of phenolic acids calls for the need of the case of wild common lilac allowed a high extraction yield of p-
elaboration of appropriate analytical methods enabling their hydroxybenzoic, vanillic and ferulic acids [30]. Soxhlet extraction
monitoring in drugs and foods of plant origin, as well as in plant raw was also used for qualitative and quantitative analysis of phenolic
materials used for their production. Bearing all this in mind, the aim compounds occurring in Salvia halophila and S. virgata [31]. To
of this work is to present an up-to-date review of the most often used optimize the extraction conditions, n-hexane, ethyl acetate, methanol
methods for extraction and determination of phenolic acids in plant and 50% aqueous methanol solution were used during 8 h. It was
materials. found that the highest extraction yield of total phenolics was obtained
when 50% methanol was used, and the lowest by using n-hexane.
Extraction methods Koşar et al. [32] tested the same solvents when they investigated
extraction of S. halophila.
One of the main sources of errors committed during analytical
procedure is the stage of sample preparation. As this stage determines
Soxhlet extraction was also used by Karasová and Lehotay [33], who
the time of analysis, it is crucial to shorten the procedure and to
reported on the isolation of benzoic acid derivatives from Melissa
enhance its accuracy and selectivity while identifying the majority of
officinalis. Different extraction times were tested (1 h, 4 h, 8 h), as
chemical compounds [23]. In order to separate the analytes from solid
well as the solvent, which was a mixture of methanol with water at
samples, the most often used solvents are those with low evaporation
different volume ratios (60:40 and 80:20). Eventually, for separation
heats and low boiling points. The solvents also have to be non-toxic,
of the derivatives, the 80:20 mixture of methanol and water was
non-flammable and chemically neutral, and they should not influence
applied, and the extraction was performed during 1 h. It has been
negatively the instrumentation and stability of the analysed
concluded that the time of extraction had no influence on the results.
substances. Solvents such as methanol, ethanol, acetone, ethyl acetate
The only exception was gallic acid, because, after extending the
and their mixtures with water are commonly used [6,10,24,25].
extraction time, the lowest concentrations of this compound were
obtained.
Phenols and phenolic acids also exist as insoluble bound complexes,
which are coupled to cell wall polymers through ester and
The main disadvantages of Soxhlet extraction are that it is a time-
glycosidic links and they are not extractable by organic solvents
consuming process and uses costly solvents, which must be of
[11,26]. Bound phenolic acids are typically liberated using base
appropriate quality. For these reasons, in recent years other methods
hydrolysis, acid hydrolysis, or both before extraction [27-29]. Many
have been preferred that are less time-consuming and require smaller
extraction procedures incorporate the use of an antioxidant as
volumes of solvents [34]. Among these methods are ultrasound-
stabilizer; compounds that have been used for this purpose include
assisted extraction (UAE), supercritical fluid extraction (SFE), and
butylated hydroxyanisole, tert-butylhydroquinone and ascorbic acid.
accelerated solvent extraction (ASE). These methods can be
successfully used for extraction of phenolic compounds from plant
Soxhlet extraction
materials. A comparison of these methods is presented in Table 2.
Extraction is a first stage of the process leading to isolation of
secondary metabolites from plant materials. A classical method used Ultrasound-assisted extraction (UAE)
for this purpose is Soxhlet extraction. This method ensures multiple
Ultrasound-assisted extraction (UAE) is based on the action of
digestion of a plant material and continuous extraction, while a fresh
ultrasonic vibrations directed toward an extracted sample, which
portion of a solvent is delivered. Thanks to this, the largest difference
enhance efficacy of penetration of a sample by solvent. This method
Extraction and determination of phenolic acids Natural Product Communications Vol. 8 (12) 2013 1823

Table 2: Comparison of extraction techniques.

Parameter Soxhlet UAE MAE SFE ASE

Costs low low average high high
Time of extraction 6-48 h < 30 min < 30 min < 60 min < 30 min
Solvent volume to be used [mL] 200-600 < 50 < 40 <10 < 100

is characterized by high speed, simplicity, and usually takes several short extraction time, possibility of automation, as well as off-line and
minutes. Apart from the type of a solvent, sample size, pH of extract, on-line coupling with a majority of chromatographic techniques (GC,
temperature and pressure, factors such as particle size, duration of HPLC). The solvent used in SFE is a fluid in a supercritical state,
sonification and its amplitude, also have an impact on the efficacy of usually carbon dioxide, owing to its low price and low toxicity.
the extraction process [35,36]. Regardless of these factors, the method However, in the case of phenolic compounds, it is not the best
is considered as the simplest one possible to perform in a laboratory solvent, since its polarity is low in comparison with that of phenolics
[37,38]. One of its advantages is the possibility to carry out the [44]. In spite of this, Castro-Vargas et al. [45] used carbon dioxide for
extraction of several samples simultaneously within a relatively short isolation of phenolic fractions from the seeds of Psidium guajava L.,
time. However, it is necessary to decant the extract or to filter it family Myrtaceae.
through appropriate paper filters. On the other hand, an opaque
solution after extraction could get stuck on an HPLC column. In Many parameters influence the efficacy of extraction by supercritical
practice, during the UAE process disruption of plant cell walls takes fluids. They can be divided into two groups [46]. The first
place, this enabling the effective extraction of metabolites contained encompasses parameters related to conducting the extraction, such as
in the cells. This method was applied by Pawar et al. [39], who pressure, temperature, time, sample weight, and flow intensity. The
analysed different species of ginger. The UAE process was conducted other group involves parameters connected with the matrix of a
for 30 min, using methanol as a solvent. sample, such as its form, homogeneity, solubility and desorption
ability of analytes. Apart from this, the SFE process depends on the
Ultrasound-assisted extraction is not the sole process used for pH. Its variations can influence the extraction yield and the speed of
extraction. Sometimes it is preceded by another procedure in order to extraction of the analyte from an aqueous phase.
prepare adequately the sample, for example to moisten it with a
solvent. Such a procedure has been proposed by You et al. [29], who SFE is commonly applied in the pharmaceutical industry, as well as in
extracted the fruits of blackberry with an 80:20 (v/v) methanol/water the food and cosmetic areas [38]. Supercritical fluid extraction serves
mixture, with addition of acetic acid, during 16 h in a dark place. for the isolation of biologically active compounds from plant
Then, sonication was performed and the analysed samples were materials, mainly for those which cannot be separated by the use of
centrifuged twice. Benetis et al. [40] have also used ultrasound for the simple solvent extraction.
analysis of phenolic compounds in Achillea millefolium L.
Preliminary studies conducted with the use of ethanol/water solutions Accelerated solvent extraction (ASE)
of concentrations from 40% to 96% (v/v), and applying different
The accelerated solvent extraction method is a relatively new
extraction times of 5, 10, 20, 30 and 60 min, have shown that the best
technique developed and distributed mainly by Dionex (Dionex
results were obtained with the 70% ethanol solution. An extraction
Corporation, Sunnyvale, CA). In the literature, there are alternative
profile in relation to ultrasonication time has shown that by extending
names for this technique, such as pressurized fluid extraction (PFE)
the time of extraction the yield increased, but after 60 min a decrease
and pressurized liquid extraction (PLE).
in yield was noticed. Finally, for further experiments, a 70% ethanolic
solution was chosen as solvent and a 30-min ultrasonication used,
In the ASE technique the same solvents are used as those in classical
with a 0.25g sample and 25 mL of solvent.
methods, but a higher pressure (about 3.3-20.3 MPa) and elevated
temperature, about 40-200°C, are applied [38]. A sample extracted by
Among numerous references describing application of UAE to
this technique is placed in an extraction vessel made of stainless steel.
sample preparation, there is one, which compares the efficacy of this
The time of analysis is short, within the range of 5-15 min [47].
method with the classical liquid-liquid extraction [41]. For both
processes, the following solvents were used: a 60% methanolic
Basic advantages of ASE are: the possibility to extract samples with
solution, a 60% acetone solution, water, and a 60:30 (v/v) mixture of
high humidity, short extraction time, better penetration of a sample by
water and ethyl acetate. The analysed material was aromatic plants,
solvent, good extraction kinetics, automation of the process and
including Rosmarinus officinalis and Origanum majorana. Classical
relatively easy use of instrumentation [38]. Also, lesser volumes of
digestion was performed in a water bath at 90°C for 2 h, whereas
solvents are needed. For example, during ASE extraction lasting
ultrasonication was performed for 1 h at 25° and 60°C. Both
about 20 min, only 10-50 mL of solvent were used, whereas in
processes were carried out in triplicate using each of the above
classical methods, such as Soxhlet extraction, extraction time is
mentioned solvents. The methanolic solvent appeared to be the best,
usually from 10 to 48 h, and the volume of solvent exceeds 400 mL
and the temperature of 60°C. Under these conditions, the highest yield
[47].
of extraction was achieved.
Shake extraction
Supercritical fluid extraction (SFE)
Beside the above-mentioned methods of sample extraction used for
This type of extraction often precedes analysis by HPLC [42]. the analysis of plant material, extraction with various shaking devices
Supercritical fluid extraction is a modern technique, which has many has been performed. The process of shaking is aimed at enlarging the
advantages over the classical extraction methods [38,43]. Among surface area where the solvent interacts with plant material, and in this
these are low temperatures, which is a positive feature in the case of way enhancing efficacy of the whole process and shortening its time.
analysis of thermally labile compounds. Other advantages are high In this method, a sample is suspended in a certain volume of solvent
selectivity, significant reduction in solvent volumes used for and then shaken at a set stirrer speed. The method was applied by Li
extraction, low mass of sample for extraction (around several mg), et al. [48] for the analysis of phenolics in Phalaris canariensis L. The
1824 Natural Product Communications Vol. 8 (12) 2013 Arceusz et al.

procedure was as follows. To the analysed sample, an 85:15 (v/v) material prior to analysis, as well as on the determination method and
mixture of 95% ethanol and 1 M hydrochloric acid was added, and the presence of interfering agents, such as fats, terpenes and
the suspension was shaken on an orbital (rotary) shaker for 1 h at chlorophyll [10]. Usually, chromatographic methods are applied, of
30°C. After centrifugation of the extract, the plant material was re- which high-performance liquid chromatography [62,63] is the most
extracted under the same conditions, and finally the combined filtrates common. In this method, different types of columns, mobile phases,
were evaporated under reduced pressure. For further analysis, the dry column temperature and, to a smaller extent, the flow rate of the
residue was used after dissolving it in a 50% methanolic solution. mobile phase, have been tested. Water, methanol and acetonitrile are
the most common constituents of the mobile phase. Sometimes it is
Fernand et al. [49] have also applied the shaking process for the necessary to add modifiers, which facilitate the resolution of the
extraction of a plant material. An ethanolic solution was used as the components. The most popular modifiers are formic acid, ammonium
extractant, whereas the dry residue, after evaporation of the solvent, acetate and acetic acid, the presence of which prevents tailing in the
was dissolved in a 1:1 (v/v) mixture of ethanol and water. In the next chromatograms. As far as the time of analysis is concerned, it is not a
step, solid-phase extraction was used to remove from the extract fixed parameter, because by modification of the flow rate of the
constituents interfering with phenolic components. mobile phase it is possible to extend or to shorten it. In the HPLC
technique it is possible to use detection systems such as a UV-Vis
Santos-Gomes et al. [50] conducted their investigation in a similar spectrometer with either single wavelength or diode-array
way. However, instead of an ethanolic solution they used acetone and capability, chemiluminescence detector (CL), coulometric electrode
a shortened extraction time. A triple extraction was applied and array system (CEAD) and mass spectrometer (MS).
simultaneous reduction of the solvent’s volume: 50 mL over 15 min,
50 mL over 10 min and 25 mL over 5 min. Then, the residue left after Reversed-phase, high-performance, liquid chromatography with UV-
concentration of the extracts under reduced pressure was dissolved in Vis detection was used by Waksmundzka-Hajnos et al. [30] for the
a methanolic solution, which was analysed by HPLC. Extraction by analysis of the inflorescence of Sambucus nigra L. and the foliage of
shaking was also applied for the analysis of rosmarinic acid in the Polygonum aviculare L. Isocratic elution was applied with the use of
leaves of lemon balm [51]. Aqueous methanol solutions were used as two mobile phases. The first, used for S. nigra, was a 22:78 (v/v)
solvents, with methanol/water ratios of 40, 60 and 80% (v/v). Also, mixture of methanol and orthophosphoric acid, and the other, applied
the impact of extraction time (30, 60 and 90 min) and temperature for P. aviculare L., was a methanol/water (25:75 v/v) mixture, with
(25, 40 and 55ºC) on the process was studied. The optimum addition of 1% of acetic acid. Phenolic compounds were detected at
extraction conditions were obtained when the process was conducted 520 nm.
during 60 min with use of 60:40 (v/v) methanol/water solution.
Santos-Gomes et al. [50] applied HPLC for the analysis of phenolics
Solid-liquid extraction carried out in a separation funnel is also in several plant species of Lamiaceae. The mobile phase was a
considered as one of the shaking methods, but extraction yields are mixture of acetonitrile, water and acetic acid (15:84:0.85 v/v) as
more variable because shaking speed and strength cannot be solvent A and methanol as solvent B. Gradient elution was applied
controlled with great accuracy. In the case of analysis using gas and the flow rate was set at 0.8 mL/min. The analytical wavelength
chromatography (GC), the most often applied extraction processes was 280 nm, and identification of particular phenolic compounds was
are: solid-phase micro-extraction (SPME), headspace single-drop performed based on comparison of the retention times of the
micro-extraction (HSDME) and microwave-assisted extraction compounds with those of the standards.
(MAE) [52,53]. The last is commonly used for isolation of active
substances from plant material [54]. The parameters most affecting its Benetis et al. [40] have used HPLC with UV-Vis detection for the
efficacy, are the following: the type and volume of solvent, radiation analysis of phenolic compounds in Achillea millefolium L. Gradient
potential, and extraction time and temperature of the process [53,55- elution was applied with a mobile phase of acetonitrile/water, and
58]. Usually organic solvents are used, and their volume depends on trifluoroacetic acid (TFA) as a modifier to prevent ionization of
the kind and mass of a sample [53,58]. In comparison with Soxhlet phenolic groups. Before performing the analysis, two types of
extraction (Table 2), lesser amounts of solvent are needed, and the columns were tested: Xterra RP18 (Waters) and Ascentis RP-Amide
extraction time is shorter. The root of Salvia miltiorrhizae was Supelco; the latter column was chosen. The phenolic compounds
extracted by the MAE technique in order to determine selected were analysed at 25°C, and a 10 µL sample was injected into the
phenolic acids [59], as well as dry roots of Eucommia ulmodies, in column with a flow rate of 1.5 mL/min and detection at 360 nm.
which chlorogenic acid, among others, was determined by using a
methanol and water mixture and conducting the extraction at 40ºC Liquid chromatography with UV detection was also applied for the
[60]. A slightly lower temperature, 30ºC, was used for determining determination of phenolic acids in Echinacea purpurea [64], the
phenolic compounds in the herbs of Hypericum perforatum and leaves of lemon balm [65], aqueous extracts of Hypericum
Thymus vulgaris. HCl solution was used as the extractant [61]. perforatum [66], and in 32 medicinal plants growing in Poland [67].
In the first case, the derivatives of caffeic acid were determined at 330
Methods for the determination of phenolic acids nm. In the second case, caffeic, ferulic and p-coumaric acids were
determined at 325 nm, gallic, vanillic and syringic acids at 280 nm,
For separation, purification and identification of phenolic compounds
and in the third case, all phenolic acids at 210 nm.
in plant materials numerous chromatographic methods have been
applied [10]. Moreover, chromatographic techniques are also used for
Liquid chromatography with diode array detection (DAD), in
investigation of interactions of phenolic compounds with other food
comparison with single wavelength UV/Vis detection, enables
constituents.
registration of the absorption spectrum of a compound over a wide
range of wavelengths and determination of the absorbance maxima.
High-performance liquid chromatography (HPLC)
This type of detection was applied for establishing the chemical
Quantitative analysis of phenolic compounds in plant material composition of extracts of Salvia halophila [32], for identification and
depends on the chemical nature of the constituents, the method of quantification of p-hydroxybenzoic acid derivatives in lemon balm
extraction, particle size, time and conditions of storage of the plant (Melissa officinalis) [33], phenolic acids in the roots of Salvia
Extraction and determination of phenolic acids Natural Product Communications Vol. 8 (12) 2013 1825

Table 3: Comparison of LC-MS with GC-MS for the analysis of phenolic compounds in medicinal plants [77].
Parameters LC-MS GC-MS
Time of sample preparation 20 min 180 min
Time of analysis 60 min 50 min
Range of linearity limited good
Selectivity good high
Limit of detection 5-15 ng/mL 10-80 ng/mL
mass spectral library for large group of compounds; fragmentation
Identification possibility of calculation of empiric equation
enables evaluation of molecular structure
Ruggedness of the system satisfactory very good

miltiorrhiza [68], in preparations of Chinese plants [69], and in plant- possible to analyse only several small molecule phenolic acids (below
derived foods [27]. Another example of UV detection of polyphenolic 600 D) [66,77]. Moreover, GC requires high temperatures, which can
compounds in HPLC was the use of a photodiode array detector by lead to sample decomposition [78].
scanning between 200 and 400 nm, with a resolution of 1.2 nm [28].
In recent years, a growing interest has been gained in HPLC with For the analysis of phenolic acids in plant materials, GC coupled with
chemiluminescent detection, the advantage of which is high mass spectrometry (GC-MS) is very often used. In comparison with
sensitivity and selectivity. However, this method also has some LC-MS, this method allows better selectivity, precision and accuracy,
drawbacks. First is its limited application, due to the small number of especially when small amounts of compounds have to be analysed in
HPLC-CL chemiluminescent reactions. There are several chemical plant material [77].
agents enabling derivatization, and in spite of the fact that they
broaden the identification range for constituents not connected with GC-MS was applied by Fiamegos et al. [79], who determined the
chemiluminescent reactions, their excess or lack of integration can contents of phenolic acids in medicinal plant raw materials and their
interfere with identification of analytes. Another disadvantage appears infusions. They used GC-MS instrumentation with SIM (selective ion
when the sample constituents are detected at a slow flow rate, and the monitoring). Owing to this modification of the method, the currents
mobile phase is incompatible with the chemiluminescent reactions; were registered only for selected ions with characteristic masses,
this precluding identification of these constituents. Another drawback typical of the studied analyte. The method with SIM is useful for
of the method is that the intensity of chemiluminescence depends on determining constituents occurring at low concentrations in complex
such environmental factors as the kind of solvent, pH and ionic mixtures.
strength [70]. Despite those disadvantages, Cui et al. [70] decided to
use this method in their research. They performed both isocratic and Proestos et al. [41] have also applied GC-MS for the determination of
gradient elution at 25°C and a flow rate of 1.0 mL/min. The mobile phenolic acids in aromatic plants, such as nettle and common rue.
phase was chosen out of three mixtures: acetonitrile-orthophosphoric They used capillary gas chromatography, silylation as a derivatization
acid-water, methanol-acetic acid-water, and phosphate-acetonitrile- procedure, and N,O-bis(trimethylsilyl)-trifluoro-acetamide and
methanol. It was found that when the concentration of acetic acid was trimethylchlorosilane as silylating agents. Along with capillary gas
1.5%, the intensity of chemiluminescence was highest. Similar effect chromatography (CGC-MS), they also used HPLC with UV detection
was obtained with a 35% methanolic solution and, accordingly, the at 280 nm.
35:65 (v/v) of methanol-1.5% acetic acid mixture was selected as the
mobile phase for the analysis. After completion of these A comparison of GC-MS with LC-MS (Table 3) has shown that the
measurements, it was found that peaks in the chromatograms first method requires a time-consuming sample preparation step prior
registered using CL detection were higher than those obtained by the to analysis, but the time of analysis is shorter, and the linearity and
DAD detection system. selectivity of the method is much better than that in the case of
LC/MS [77]. The sample preparation step includes dynamic
A less commonly used type of detection system is coulometric sonication-assisted extraction of herbal samples, followed by the
electrode array detector (CEAD). This detector was applied for extracts being further treated by liquid-liquid extraction and
determining ferulic and p-coumaric acids in the seeds of common flax derivatization. In GC-MS the compounds are identified by
and in the roots of nettle [71]. HPLC-CEAD is characterised by high comparison of their spectra with those available in the mass spectra
sensitivity and precision, based on redox activity of the analyte [72]. library.

In some laboratories, HPLC coupled with mass spectrometry (MS) is It is also worth mentioning that, among the chromatographic methods,
also used [73]. Guo et al. [74] have applied the LC-MS technique for high-speed, counter-current chromatography (HSCCC) [80] was used
the simultaneous determination of six phenolic acids in rat plasma by Yang et al. [81] for isolation and purification of phenolic acids
after intravenous administration of a traditional Chinese medicinal originating from a Chinese medicinal plant, Smilax china.
preparation Guanxinning in a form of a lyophilised powder containing
Salvia miltiorrhiza Bge. The same type of detection was used for Capillary electrophoresis (CE)
determining caffeic acid in products from Echinacea sp. [45], Recently, capillary electrophoresis (CE) has achieved growing
chlorogenic acid in the leaves of lemon balm [65], and caffeic and significance in the analysis of phenolic compounds [82]. This method
protocatechuic acids in the roots of sage [75]. Furthermore, LC-MS allows the separation and determination of polar substances of both
and NMR techniques were also used for identification and structure ionic and non-ionic character, as well as non-polar, non-ionic
establishment of eighteen phenolic acids and mono- and diglycosidic substances. Advantages of CE are a small volume of electrolytes,
flavonoids [76]. short analysis time, high resolution potential and small sample weight
[83-86]. In order to achieve proper resolution by this method, it is
Gas chromatography (GC) necessary to optimize such parameters as the type of buffer, its pH
Gas chromatography is one of the less common techniques used for and concentration, the type of capillary and its volume, temperature of
the determination of phenolic acids. Using this technique, it is electrophoresis, voltage and the way of injection of a sample [87].
1826 Natural Product Communications Vol. 8 (12) 2013 Arceusz et al.

Each of these parameters will depend on CE methodology and on the useful when other analytical techniques do not provide reliable
chemical properties of the studied phenolic acid and its matrix. results. Moreover, the CE-MS combination opens more analytical
options, such as increase in sensitivity and specificity of the method.
Before analysis by capillary electrophoresis, the active compounds Application of this hyphenated method does not require using
are extracted into solution, usually by supported liquid extraction complicated sample preparation procedures and in comparison with
(SLE) [37,44,88-92], supercritical fluid extraction (SFE) [93,94], and the chromatographic methods, it is a good alternative for
liquid-liquid extraction (LLE) [9,25]. The less common methods are simultaneous analysis of phenolic compounds, because it provides
microwave-assisted solvent extraction (MAE), taking only several rapid and efficient separations and uses reduced sample and solvent
minutes [84], and Soxhlet extraction [95]. consumption [103].

Apart from CE, another popular technique applied for the Concluding remarks
determination of phenolic compounds, is capillary zone
Phenolic acids are secondary metabolites widespread in the plant
electrophoresis (CZE) [96,97]. This method was used, among others,
kingdom. Because of their wide spectrum of biological activity,
for the determination of rosmarinic acid in commercial sage tea-bags
isolation, quantification and structure establishing of these
[85]. Of the tested solvents, such as methanol, acetone and
compounds is still a crucial matter. On the one hand, there are
acetonitrile, the best was methanol. As the extraction technique,
methods of isolation of phenolic acids from medicinal and dietary
ultrasonication was used, and for detection, UV spectrometry at 210
plants, on the other, analytical techniques for quantitation of these
nm. Resolution of the compounds was performed in a quartz
secondary metabolites. Literature screening has shown that although
capillary, 50 µm in diameter, and a solution of borate as a separating
Soxhlet and ultrasound-assisted (UAE) extractions are commonly
buffer was used. Beside CE and CZE, sometimes micellar
used for isolation of polyphenols from plant matrices, alternative
electrokinetic chromatography (MEKC) is also used. By using this
techniques of extraction, such as supercritical fluid (SFE) and
technique, phenolic acids were determined in the roots and pods of
accelerated solvent (ASE) are more and more often used. Non-
Echinacea purpurea [9], as well as in the herb of Artemisia capillaris
questionable advantages of Soxhlet extraction are its low cost and
[98]. Capillary electrochromatography (CEC) was applied for the
good recovery, but there are some disadvantages, such as large
analysis of extracts obtained from the flowers of chamomile [82].
volume of solvent, long time of extraction and tedious handling
procedure. Recently introduced advanced techniques of extraction,
As detection methods in capillary electrophoresis and its
such as SFE and ASE, are not as profitable as initially expected.
modifications, optical methods are used (fluorimetric,
Supercritical fluid extraction takes place under subcritical
phosphorimetric, chemiluminescent, UV-Vis, IR and Raman
conditions, and this time-consuming and expensive procedure is
spectrometries, and refractometry), as well as electrochemical
limited to compounds of low or medium polarity. On the other
methods (conductometric, potentiometric, amperometric and
hand, accelerated solvent extraction is performed with high
voltamperometric), and other, such as mass spectrometry and
extraction temperatures that may lead to degradation of
radiometry. The most frequently used detectors are those based on
thermolabile compounds. Furthermore, this technique is considered
UV, MS and amperometric methods. They assure high sensitivity and
as a potential alternative to SFE for extraction of polar compounds.
selectivity.
Quantitation of phenolic acids is usually performed by liquid
CE with UV detection is commonly used for the analysis of phenolic
chromatography (LC) owing to the recent developments in this
acids in plant materials, and the most often applied electrolyte is a
technique, especially when it is coupled with mass spectrometry
borate buffer of pH 9.2 [99]. This buffer was used for the
(MS). Other advanced techniques used for quantitation of the
determination of phenolic acids in Strobili lupuli [91], Cortex fraxini
secondary metabolites are gas chromatography coupled with mass
[92], and the fruits of sea buckthorn berries [100]. Less frequently, a
spectrometry (GC-MS) and capillary electrophoresis (CE). All the
MOPSO buffer solution is used; this was applied together with Tris
above techniques have some limitations. For example, LC with
solution and boric acid, pH 8.3, for the determination of chlorogenic
DAD and MS provided short time analysis with less sample
acid in Hypericum perforatum [101]. Also a phosphate buffer with
preparation, while GC method, which is used very often with MS
acetonitrile, pH 2.8, was used for the determination of caffeic and
detection, required a relatively high temperature, which can lead to
chlorogenic acids in Matricaria chamomilla L. [82].
sample decomposition. Moreover, GC can be used only for several
small molecule phenolic acids (below 600 D). Recently, CE has
MS detection is useful for identification of chemical compounds in
achieved growing significance in quantitation of phenolic acids.
mixtures as it can elucidate chemical and structural information about
This technique provides short analysis time and small volume of
molecules from their molecular weight and distinctive fragmentation
electrolytes, and the most common detectors are UV, MS and
patterns [102]. In the CE method coupled with MS, resolution of the
amperometric.
constituents can be regulated by changing the pH. This technique is

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