J. Sep. Sci.

2011, 34, 27–36 27

Tomasz Tuzimski Research Article

Department of Physical
Chemistry, Chair of Chemistry,
Faculty of Pharmacy with Medical
Analytics Division, Medical
University of Lublin, Lublin,
Poland
Received August 4, 2010
Revised October 9, 2010
Accepted October 10, 2010
Determination of analytes in medical
herbs extracts by SPE coupled with
two-dimensional planar chromatography
in combination with diode array scanning
densitometry and HPLC-diode array detector
The purpose of this study is to demonstrate an application of 2-D high-performance
planar chromatography-diode array detector (DAD) and HPLC-DAD after solid-phase
extraction (SPE) for identification and quantitative analysis of pesticides (isoproturon,
aziprotryne, hexazinone, flufenoxuron, methabenzthiazuron, procymidone, and a-cyper-
methrin) in Melissa officinalis L. (Labiatae) samples. The procedure described for the
determination of compounds is inexpensive and can be applied to routine analysis of
analytes in medical herbs’ samples after preliminary cleanup and concentration by SPE.
Average recoveries on C18 SPE cartridges of pesticides eluted with 5 mL tetrahydrofuran
by the proposed HPLC-DAD method, before and after 2-D-high-performance planar
chromatography separation of analytes from M. officinalis L. samples spiked with pesticide
at a concentration level of 10 mg/g in plant material are presented. Method validation
parameters for the quantification of pesticides by the proposed HPLC-DAD after SPE
method are also presented.

Keywords: Diode array scanning densitometry / Medical herbs extracts /

Pesticides / Solid-phase extraction (SPE) / Two-dimensional high-performance
planar chromatography
DOI 10.1002/jssc.201000582

1 Introduction extraction of acephate, chlorpropham, pirimicarb, bifen-

Hundreds of pesticides are widely used in current
agricultural practices around the world, and it is not
uncommon for residues of these pesticides to occur in food
products, fruits, vegetables, herbs, and especially medical
herbs. Simultaneous determination of 102 pesticide resi-
dues in Chinese teas by gas chromatography-mass spectro-
metry (GC-MS) was described [1]. A multiresidue method
for the determination of 234 pesticides in Korean herbs
(Acanthopanax senticosus, Morus alba L., and Hovenia dulcis)
using GC-MS was described [2]. Solid-phase microextraction
coupled with GC-MS was used to determine pesticide
residues in Chinese herbal formulations [3]. The two-
dimensional (2-D) coordination polymer was tested for the
Correspondence: Dr. Tomasz Tuzimski, Department of Physical
Chemistry, Chair of Chemistry, Faculty of Pharmacy with
Medical Analytics Division, Medical University of Lublin, 6
Chodźki Street, 20-093 Lublin, Poland
E-mail: tomasz.tuzimski@umlub.pl
Fax:148-81-5357350
Abbreviations: DAD, diode array detector; 2-D-HPTLC, 2-D
high-performance planar chromatography; MDPC, multi-
dimensional planar chromatography; MeCN, acetonitrile;
NP, normal phase
thrin, tetradifon, and phosalone from the medicinal plant
Cordia salicifolia, whose extracts are commercialized in
Brazil as diuretic, appetite suppressant, and weight loss
products, using GC/MS, SIM [4]. Extraction and cleanup
methods for the determination of organochlorine pesticide
residues in medicinal plants was described [5]. Behavior of
some organophosphorus pesticide residues in peppermint
tea during the infusion process was reported [6]. Determi-
nation of multipesticide residues in Mentha piperita L. was
described [7]. Residue analysis of multiclass pesticides in
watermelon (Citrullus vulgaris Schrad.) by LC-MS/MS was
reported [8]. High-throughput analysis for the identification
and quantification of 150 pesticides in tomato, strawberry,
potato, orange, and lettuce samples using quick, easy,
cheap, effective, rugged and safe (QuEChERS) methodology
and low-pressure GC-time-of-flight (TOF) MS was also
described [9]. In another article, the authors demonstrated
the dramatical improvement of separation in comparison
with conventional GC by employing GC  GC-TOFMS with
cryogenic modulator. All analyzed 58 pesticides in food
extracts could be identified using their full-scan mass
spectra, which was not possible when using 1-D-GC-
TOFMS [10].
Sometimes, the monodimensional chromatographic
separation cannot be sufficient to resolve all of the

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
28 T. Tuzimski J. Sep. Sci. 2011, 34, 27–36

Table 1. The pesticides investigateda)

No. Pesticide Structure Log P

1 Isoproturon 2.31
H CH3
H3C N C N
CH CH3
H3C O
2 Aziprotryne 2.99
CH3
S

N N
CH3
N3 N
HN HC
CH3
3 Hexazinone 2.37
O

N N
CH3
O N N
CH3
CH3
4 Flufenoxuron 4.83
F F
O Cl F
O F
N F

F H N O
H
5 Methabenzthiazuron 1.89
CH3
N
N C NH CH3
S
O
6 Procymidone 3.44
Cl O CH3

N CH2

Cl O CH3
7 a-Cypermethrin 5.53
CH3
Cl
C CH CO2 CN
Cl C O
H HH
CH3

a) Values of Log P of pesticides were calculated in HyperChem program.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2011, 34, 27–36
Table 2. Method validation parameters for the quantification of pesticides by the proposed HPLC-DAD after SPE method
No. Pesticide tr (min 1)
1 Isoproturon 9.501–9.575
2 Aziprotryne 15.764–15.858
3 Hexazinone 4.696–4.843
4 Flufenoxuron 30.991–31.083
5 Methabenzthiazuron 7.618–7.966
6 Procymidone 19.060–19.540
7 a-Cypermethrin 34.826–34.942
components of interest of multicomponent mixtures.
Problem of peak overlapping may occur, and a presepara-
tion of the sample is often necessary. This preseparation
aims at reducing the complexity of the original matrix, by
separating several simpler fractions of the original matrix.
The fractions should contain the same amounts of the
analytes as in the whole sample, ready for analysis and free
from substances that can interfere during the chromato-
graphic analysis. Fractionations of complex mixtures of
analytes by solid-phase extraction (SPE) combined with
HPLC-diode array detector (DAD) and TLC-DAD for the
determination of pesticides in water were described [11–15].
The most popular solvents used to elute the analytes from
sorbents in SPE are methanol, acetonitrile (MeCN), and
tetrahydrofuran (THF). Each of these solvents possesses
different properties that are related to molecular interactions
between the solvent used to elute (methanol, MeCN, or
THF) and remaining components of the chromatographic
system (water, analytes, hydrocarbon chains, and silanol
groups). The purpose of the published study [15] was to
demonstrate an application of TLC-DAD and HPLC-DAD
after SPE on four types of adsorbents (C18, C18 Polar Plus,
C18/SDB-1, and CN) with these three solvents for identifi-
cation and quantitative analysis of 25 pesticides in water
samples. Average recoveries for these four SPE cartridges
and three solvents by the proposed HPLC-DAD method
after SPE were reported [15]. In another article [16], SPE was
used not only to preconcentrate the analytes, but also for
partial purification of the samples. The application descri-
bed relates to a method for determination of clofentezine in
medical herbs with various solvents with C18/SDB-1
cartridges which permits to refine, reduce impurities, and
purify the matrix in the first step and to elute the analytes in
the second step of SPE experiments. For the identification of
clofentezine in Herba Thymi, the methanol and THF
eluates concentrated after SPE were injected into a C18
column and analyzed by HPLC-DAD. Impurities were
eluted by means of methanol and then the analytes were
eluted with THF. The methanol eluates contained traces of
clofentezine (o0.09%). THF eluates were also analyzed by
multidimensional planar chromatography with DAD
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography 29
222 nm
LOD (mg/mL) LOQ (mg/mL) Range (mg/mL) r
0.03 0.10 0.15–100 1.0000
0.44 1.35 0.1–50 0.9999
0.05 0.15 0.3–50 1.0000
0.05 0.15 0.3–50 1.0000
0.10 0.31 0.3–100 1.0000
0.03 0.10 1.2–100 1.0000
0.09 0.28 0.3–100 1.0000
(MDPC-DAD). The heart-cut spots of clofentezine (filtered
and concentrated to 1 mL acetone solution) from the
stationary phase were injected into a C18 column and
analyzed by HPLC-DAD [16]. Multidimensional chromato-
graphy including MDPC has important applications in
environmental analysis [17, 18]. The best combination
for MDPC is the parallel combination of stationary and
mobile phases. As described earlier [19, 20], the mode
of MDPC for the separation of multicomponent mixtures
was realized on multiphase plates. The largest differences
were obtained by combination of normal-phase (NP)
systems of the type silica/nonaqueous eluent in the first
step of MDPC and reversed-phase (RP) systems of the
type octadecyl silica/water 1 organic modifier (methanol,
MeCN, dioxane, and THF) in the next steps of MDPC on
multiphase plates, e.g. with a narrow zone of SiO2 and a
wide zone of RP-18 (or vice versa) which are commercially
available from Whatman (Multi K SC5 or CS5 plates)
[17]. Application of MDPC-DAD with NP system on silica
and HPLC-DAD with RP system on octadecyl silica was
described for correct identification of pesticides in plant
extracts on silica plates [17, 21]. The application of MDPC
combined with different separation systems and modes of
chromatogram development is often necessary for
performing the separation of more complicated multi-
component mixtures. High separation efficiency can be
obtained by a combination of NP and RP systems on silica
plates. Application of MDPC with different systems, e.g.
hydrophilic interaction chromatography and adsorption
chromatography can be useful for correct identification of
pesticides in complicated mixtures.
Medical plants are liable to contain pesticide residues,
which accumulate from agricultural practices, such as
spraying, treatment of soil during cultivation, and admin-
istration of pesticides during storage. Although many
countries have issued restricting regulations concerning the
use of pesticides, pesticide residues may still contaminate
crude herbal drugs. The procedure described in this article
for the separation of complex mixtures of compounds,
isolation of analytes from difficult matrix, and qualitative
determination in Melissa officinalis L. (Labiatae) samples is
www.jss-journal.com
30 T. Tuzimski J. Sep. Sci. 2011, 34, 27–36

inexpensive and can be applied to routine analysis of
pesticides in the samples of natural origin, after preliminary
cleanup and concentration, e.g. by SPE.
2 Materials and methods
2.1 Pesticide standards
The standards of the investigated pesticides (Table 1) were
obtained from the Institute of Organic Industry (IPO,
Warsaw, Poland). All standards were dissolved in methanol.
2.2 Solvents and mobile-phase solution
MeCN, methanol, and THF were pro chromatography grade
from E. Merck (Darmstadt, Germany). Aqua purificata from
F.P.P.A.H. PROLAB (Nak"o, Poland) was used.
2.3 Sampling and sample preparation
Medical herbs (M. officinalis L. (Labiatae)) were obtained
from the purchasing centre of medical herbs (Fajs"awice,
Southeastern Poland) in the summer of 2010. Ultrasound-
assisted solvent extraction from 2 g of M. officinalis
L. (Labiatae) by 15 mL MeCN/acetic acid (9:1, v/v) was
performed in ultrasonic bath (Unimasz UM-4, Koszalin,
Poland) at elevated temperature (501C) for 15 min. The
procedure was repeated twice. The extracts obtained were
isolated, loaded on 4 g anhydrous Na2SO4, and then filtered.
The herbs extract sample was passed through 0.45 mm
membrane filters (Millipore, Bedford, MA, USA). Next, the
sample was evaporated and concentrated to 0.5 mL MeCN
volume, and water (aqua purificata) was added to 100 mL, so
that the concentration of MeCN in sample was not higher
than 1%. The samples once again were passed through
0.45 mm membrane filters (Millipore). SPE was performed
with Bakerbond (J. T. Baker, Deventer, The Netherlands):

Table 3. Average recoveries on C18 SPE cartridges of pesticides eluted with 5 mL THF by the proposed HPLC-DAD method, before and
after 2-D-HPTLC separation of analytes from M. officinalis L. samples spiked with pesticide at a concentration level of 10 mg/g in
plant material

No. Pesticide l (nm) Recovery7SD (%)

Before 2-D-HPTLC separation After 2-D-HPTLC separation

1 Isoproturon 202 57.0 7 4.0a) 17.5 7 1.5

2 Aziprotryne 222 48.5 7 2.5 16.3 7 2.0
3 Hexazinone 240 40.3 7 3.0a) 10.0 7 1.0
4 Flufenoxuron 222 4.1 7 1.5 0
5 Methabenzthiazuron 222 16.0 7 1.0a) 10.0 7 1.0
6 Procymidone 202 36.0 7 1.5a) 9.0 7 2.0
7 a-Cypermethrin 202 11.0 7 2.0 0

a) Peak impure.

Figure 1. Chromatogram obtained by HPLC-DAD after SPE from M. officinalis L. (Labiatae).

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2011, 34, 27–36 Liquid Chromatography 31

octadecyl cartridges (C18 2000 mg/6 mL, no. 7020–08).
Before use, each cartridge was conditioned with 3  2 mL
MeCN and 3  2 mL water. After being loaded with the
extract of herbs’ samples (100 mL, flow rate 10 mL/min, and
pressure 85 mm Hg), the SPE columns (C18) were eluted
with 5 mL THF.
2.4 2-D high-performance planar chromatography
on Multi-K plates
The eluates (100 mL) were applied with Camag Linomat 5
applicator (Muttenz, Switzerland) as bands no longer than
0.5 cm to the narrow zone on Multi-K multiphase plates.
Multiphase plates with a narrow zone of silica and
a wide zone of RP-18 (or vice versa) are commercially
available from Whatman (Multi-K SC5 or CS5 plates). The
eluates (M. officinalis L. (Labiatae) extract and standards) were
applied to the opposite corners of the narrow zone. The
Multi-K SC5 or CS5 plates were developed to a distance
of 10 cm in a horizontal Teflon DS chamber [22]. The
Multi-K CS5 plates were developed with 80:20 v/v
methanol–water as mobile phase (step A). After drying in
air for 30 min, the plates were turned by 901 (so that the
initially separated mixture of compounds was the start of the
next step (step B)). Next, the Multi-K CS5 plates were
developed with 20:80 v/v dioxane–n-heptane as mobile phases
(step B). The Multi-K CS5 or SC5 plates were scanned in
the wavelength range of 200–400 nm (TLC-DAD scanner,
J&M, Aalen, Germany).

Figure 2. Left column: Comparisons of the UV spectra of standards (library) and spectra found in M. officinalis L. (Labiatae); right column:
purities of peaks found in M. officinalis L. (Labiatae) by HPLC experiments before 2-D-HPTLC.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
32 T. Tuzimski
2.5 HPLC procedure
For the identification of pesticides in M. officinalis L.
(Labiatae), the eluates concentrated after SPE were injected
on a C18 column and analyzed by HPLC-DAD.
For the identification of pesticides in M. officinalis L.
(Labiatae), the eluates concentrated after SPE, and heart-cut
spots of pesticides (filtered and concentrated to 0.5 mL
solution in acetone (hexazinon, methabenzthiazuron,
procymidone, and a-cypermethrin) or to 0.5 mL methanol
solution (isoproturon, aziprotryne, and flufenoxuron)) from
the stationary phase after 2-D-TLC experiments on Multi-K
plates were also injected on a C18 column and analyzed by
HPLC-DAD. Eluates and heart-cut spots of pesticides were
analyzed at 221C using an Agilent Technologies 1200 series
chromatograph equipped with a quaternary gradient pump
with a degasser set at a flow rate of 1 mL/min, and with a
DAD. Eluates were injected into the eluent with a Rheodyne
20 mL injector. The HPLC apparatus was equipped with a
ZORBAX Eclipse XDB-C18 150 mm  4.6 mm column,
5-mm particle size (Agilent Technologies, Wilmington, DE,
USA). The gradients applied were start – 30%B, 0–30 min –
linear to 76%B, 30–35 min – linear to 100%B, 35–45 min –
isocratic 100%B (A – H2O, B – MeCN).
Calibration was based on the peak areas obtained from
pesticide standards prepared as solutions in methanol at
seven concentrations (0.3–50 mg/mL (hexazinon, flufenoxur-
on), 0.3–100 mg/mL (methabenzthiazuron, a-cypermethrin),
0.15–100 mg/mL (isoproturon), 0.1–50 mg/mL (aziprotryne),
1.2–100 mg/mL (procymidone), the value of r for almost all
pesticides was 1.000, except r-value for aziprotryne was equal
to 0.9999). Each solution was injected in triplicate under the
same chromatographic conditions. The calibration plots were
linear for all investigated pesticides (Table 2).
2.6 Validation of the HPLC method
Limits of detection (LOD) and quantification (LOQ) were
calculated by use of the formulas LOD 5 3.3(SD/S) and
LOQ 5 10(SD/S), respectively, where SD is the standard
deviation of the response and S is the slope of the calibration
plot (Table 2).
Recovery and precision (Table 3) were determined by the
standard addition method in which 2 g M. officinalis L.
(Labiatae) samples were spiked with seven pesticides: hexazi-
non, methabenzthiazuron, isoproturon, aziprotryne, procy-
midone, flufenoxuron, and cypermethrin at a concentration
level equal to 10 mg/g in plant material. Four samples were
combined and the procedure described above was applied.
3 Results and discussion
In the preliminary part of experiments, the eluates from
fortified samples were injected on C18 column and analyzed
by HPLC-DAD. The chromatogram obtained from a sample
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 27–36
of M. officinalis L. (Labiatae) fortified by seven pesticides is
shown in Fig. 1. The analytes were identified on the
basis of their retention times and by comparison between
the UV spectrum of the reference compound in the library
and the UV spectrum of the detected peak in the sample
(Fig. 2, left column). A match equal to or higher than 990
was fixed to confirm identification between both spectra for
all of the pesticides determined. Aziprotryne, flufenoxuron,
and a-cypermethrin were obtained as pure peaks
(Fig. 2, right column), but for other analytes, the peaks
were impure.
If the peaks of analyte are pure, then the surface area
under the compared spectra of standard and analyte is green
(on black-and-white print it is light-grey). If the peak of the
analyte is contaminated, the surface area would be red
(dark-grey on black-and-white print). The right column of
Fig. 2 (and Fig. 7) represents the purity of peaks of three
pesticides: aziprotryne, flufenoxuron, and cypermethrin.
Since these peaks are pure, the calculated surface areas of
the compared peaks are light-grey (green in original).
The matrix contains 1024 photodiodes which corre-
sponds to nominal difference of 0.9 nm in the UV range. In
the visual and near infrared range, the difference is some-
what greater. To correct this optical nonlinearity and
transform the discrete diode distances into a linear scale, a
linear interpolation algorithm is applied which utilizes a
calibration table of wavelengths and real wavelength values
obtained from emission lines of a deuterium lamp. The
Figure 3. Correlation hRF (100  RF) versus hRF (100  RF) for 2-D-
HPTLC system: RP, methanol–water (80:20, v/v) on octadecyl
silica adsorbent wettable with water (RP-18W) and NP, diox-
ane–n-heptane (20:80, v/v) on silica gel. 1, Isoproturon; 2,
aziprotryne; 3, hexazinon; 4, flufenoxuron; 5, methabenzthiazur-
on; 6, procymidone; 7, a-cypermethrin.
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J. Sep. Sci. 2011, 34, 27–36 Liquid Chromatography 33

A determination of peaks purity was carried out using an

0.6
interpolation algorithm which takes into account a calibra-
tion table of wavelengths from the emission lines of the
0.5
deuterium lamp at 486 and 656 nm.
0.4
The average recoveries from the spiked samples with
pesticide at a concentration level of 10 mg/g in plant mate-
Abs

0.3 rial, and the SD values, were calculated at optimal wave-

lengths for all analytes and are summarized in Table 3. The
0.2 average recoveries from the spiked samples for more polar
pesticides from investigated analytes were good in the range
0.1
from 36.0 to 57.0%. These recovery values are equal to 57,
48.5, 40.3, and 36.0% for isoproturon, aziprotryne, hexazi-
0.0
200.0 220.0 240.0 260.0 280.0 300.0 320.0 340.0 360.0 380.0 400.0 none, and procymidone, respectively (except average recov-
Spectrum from CS500.3D at 9.4000 [mm] nm ery for methabenzthiazuron which is equal to 16.0%). But
for very nonpolar analytes, with the highest log P-values
B 1.0
(4.83 and 5.53 for flufenoxuron and a-cypermethrin,
0.9
respectively), the recovery values were very low (4.171.5
0.8 and 11.072.0% for flufenoxuron and a-cypermethrin,
0.7 respectively).
In Table 3, the average recovery values of the analytes
Int. WP39

0.6

0.5 are listed after their concentration by SPE, subsequently

0.4
analyzed by HPLC-DAD after their isolation by 2-D high-
performance planar chromatography (2-D-HPTLC). In spite
0.3
of very good correlations for pairs of spectra of pesticide
0.2
standard and analyte and their satisfactory alignment, most
0.1
of the peaks were contaminated. However, the recoveries of
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
most analytes were relatively satisfactory (in the range of
Least Squares fit for W39 (2) vs. WP39 (1) Int. W39 16–57%, except for flufenoxuron and a-cypermethrin) which
indicates good choice of eluents applied for extraction of
Figure 4. (A) Comparisons of UV spectra of isoproturon found in
M. officinalis L. (Labiatae) extract with UV spectra of pesticides pesticides from medicinal plants and for the elution of
standards from library. (B) Correlation curves of peak purity of concentrated analytes from SPE sorbents, except in the case
spectra of isoproturon found in extract of M. officinalis L. of most nonpolar analytes, flufenoxuron and a-cyperme-
(Labiatae) and that of pesticide standard (library) after 2-D- thrin, for which the retention times on RP 18 gel were
HPTLC (step B) on Multi-K CS5 plates. Pearson’s r is equal to
longest and log P-values greatest.
0.9997.

Figure 5. The heart-cut bands of the analytes from the stationary phase of Multi-K CS5 were analyzed by HPLC-DAD: chromatograms
obtained by HPLC-DAD after SPE and 2-D-HPTLC from M. officinalis L. (Labiatae).

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
34 T. Tuzimski J. Sep. Sci. 2011, 34, 27–36

Relatively nonsatisfactory purity of peaks of the

analytes before their separation by 2-D-HPTLC is caused
by very complex matrix which contains a great number
of components some of which coincide with the peaks
of the analytes. The contaminations of the peaks could
probably be eliminated by the use of an additional column
(precolumn).
Applications of NP and RP systems during a single
experiment enable separation not only of analytes in a
multicomponent sample, but also separation of analytes
from impurities and other components of the matrix. In the
next experiments, the eluates were applied on bilayer Multi
K CS5 plates. On the basis of correlations of RF values in NP
and RP systems (Fig. 3), it was possible to separate seven
analytes from the components of the matrix. The Multi-K
CS5 plates were scanned in the wavelength range of
200–400 nm. Identification of analytes was confirmed by
comparison of UV spectra of the components of plants
extract with those of standards of analytes; the spectra were
acquired by use of the DAD densitometer (Fig. 4A). The
purities of the peaks were also determined. The peak–purity
index is a numerical index of the quality of the coincidence
of the data sets. A peak–purity index has values in the range
from 0 (compared spectra are different) to 1 (compared
spectra are absolutely (ideally) identical). Example of the
least-squares fit values (obtained by cross-correlation) of
spectra from a fortified sample of M. officinalis L. and
spectra from pesticide standards were also calculated and
the purity index (Pearson’s r) for compared spectra was
always between 0.9911 and 0.9997. Figure 4B shows the
example of both compared and correlated spectra – analyte
and standard pesticide – the peak purity was 0.9997.
Heart-cut bands of analytes from the stationary phase
(after 2-D-TLC experiments) were also analyzed by HPLC-
DAD on C18 column. The HPLC chromatograms obtained
from extract of M. officinalis L. (Labiatae) after 2-D-TLC show
the purities of peaks of analytes separation from compo-
nents of matrix (Fig. 5). Identification of the analytes was
accomplished on the basis of retention times of the analytes
(Fig. 5) and by comparing the UV spectrum of the reference
compound in the library with the UV spectrum of the peak
detected in the sample (Fig. 6). The purities of the peaks
were also determined (Fig. 7). Average recoveries at optimal
wavelengths from the spiked samples, and the SD, were Figure 6. Comparisons of the UV spectra of standards (library)
acceptable: 10.071.0, 10.071.0, 16.372.0, 9.072.0, and and spectra found in M. officinalis L. (Labiatae).
17.571.5% for hexazinon, methabenzthiazuron, azipro-
tryne, procymidone, and isoproturon, respectively. For very
nonpolar analytes (flufenoxuron and a-cypermethrin), quantified by scanning the HPTLC plates with DAD
recoveries values were very low. densitometer and comparing the spot areas of analytes with
Table 3 lists the average values of recoveries of analytes those of calibration curves of standards.
obtained after extraction of the spots from the adsorbent The proposed procedure was proved correct for spiked
surface and their separation by 2-D-HPTLC. The recovery samples with seven pesticides at concentration levels equal
values analyzed by HPLC-DAD after separation by 2-D- to 10 mg/g in plant material after 1, 5, and 11 days. The
HPTLC are lower. This is due to the partial losses during method characterized good reproducibility.
scraping the adsorbent from the layer, its filtration, and The method was validated for precision, repeatability,
dissolution followed by introduction on the HPLC column. and accuracy. The LODs and the LOQs for investigated
The recoveries would probably be greater if they were pesticides at five wavelengths were published in earlier

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J. Sep. Sci. 2011, 34, 27–36 Liquid Chromatography 35

Moreover, it does not necessitate the use of additional

columns in HPLC experiments to purify the matrix from the
ballast substances.

4 Concluding remarks

The described methods enable monitoring of popular

pesticides of different classes, widely used in Poland and
other countries worldwide. The present methodology proved
to be a reproducible and a suitable alternative to the
chromatographic techniques with the necessity of applying
additional purifying and absorbents of the matrix in SPE,
used to screen different classes of pesticides in herbs
samples (except very nonpolar analytes). TLC coupled with a
scanner with a DAD (TLC-DAD) and HPLC-DAD method
can be successfully applied for more credible, repeatable,
and correct identification of the analytes and their quanti-
tative analysis in the environmental samples. Application of
both chromatographic techniques with different systems,
e.g. NP and RP (in both steps of 2-D-TLC-DAD on the
bilayer Multi-K CS5 plates) and RP (HPLC-DAD) can be
useful for correct identification of pesticides in complicated
mixtures and separation of analytes from the components of
the matrix. Thanks to 2-D-TLC experiments, it is possible to
obtain pure peaks of almost all pesticides investigated and
acceptable values of recoveries of analytes (except for very
nonpolar pesticides, e.g. pyrethroids, benzoylphenylurea
chitin synthesis inhibitors).

The investigations were financially supported by a grant

(No. N N204 167136, 2009-2011) of the Ministry of Science
and Higher Education, Poland.

The author has declared no conflict of interest.

5 References

Figure 7. Purities of peaks found in M. officinalis L. (Labiatae).
articles [10–14]. The number of examined samples of
M. officinalis L. (Labiatae) was equal to 150.
The suggested procedure is efficient and simple. It
allows to analyze the quantities of pesticides in medical
herbs without the necessity of applying additional purifying
and absorbents (silica or Florisil) of the matrix in SPE.
[1] Huang, Z., Li, Y., Chen, B., Yao, S., J. Chromatogr. B
2007, 853, 154–162.
[2] Nguyen, T. D., Lee, K. J., Lee, M. H., Lee, G. H., Micro-
chem. J. 2010, 95, 43–49.
[3] Hwang, B.-H., Lee, M.-R., J. Chromatogr. A 2000, 898,
245–256.
[4] Carvalho, P. H. V., Barreto, A. S., Rodrigues, M. O.,
Menezes Prata, V., Alves, P. B., Mesquita, M. E., Júnior,
S. A., Navickiene, S., J. Sep. Sci. 2009, 32, 2132–2138.
[5] Lino, C. M., da Silveira, I. N., J. Chromatogr. A 1997, 769,
275–283.
[6] Ozbey, A., Uygun, U., Food Chem. 2007, 104, 237–241.
[7] Hajou, R., Afifi, F. U., Battah, A., Pharm. Biol. 2005, 43,
554–562.
[8] Park, S., Lee, S. J., Kim, H. G., Jeong, W. Y., Shim, J.-H.,
El-Aty, A. M. A., Jeong, S. W., Kim, S. T., Shin, S. C.,
J. Sep. Sci. 2010, 33, 493–501.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
36 T. Tuzimski
[9] Koesukwiwat, U., Lehotay, S. J., Miao, S., Leepipatpi-
boon, N., J. Chromatogr. A 2010, 1217, 6692–6703.
[10] Dallüge, J., Rijn, M., Beens, J., Vreuls, R. J. J., Brink-
man, U. A. Th., J. Chromatogr. A 2002, 965, 207–217.
[11] Tuzimski, T., J. AOAC Int. 2008, 91, 1203–1209.
[12] Tuzimski, T., J. Sep. Sci. 2008, 31, 3537–3542.
[13] Tuzimski, T., Sobczyński, J., J. Liq. Chromatogr. Relat.
Technol. 2009, 32, 1241–1258.
[14] Tuzimski, T., J. Planar Chromatogr. – Modern TLC 2009,
22, 235–240.
[15] Tuzimski, T., J. AOAC Int. 2010, 93, in press.
[16] Tuzimski, T., J. Sep. Sci. 2010; 33, 1954–1958.
[17] Tuzimski, T., J. Planar Chromatogr. – Modern TLC 2010,
23, 184–189.
[18] Marcé, R. M., Multidimensional chromatography in
environmental analysis, in: Mondello, L., Lewis, A. C.,
Bartle, K. D. (Eds.), Multidimensional Chromatography,
Chapter 13, Wiley, Chichester 2002, pp. 335–377.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 27–36
[19] Tuzimski, T., Use of planar chromatography in pesticide
residue analysis, in: Nollet, L. M. L., Rathore, H. S.
(Eds.), Handbook of Pesticides. Methods of Pesticide
Residue Analysis, Chapter 9, CRC Press, Taylor and
Francis Group, Boca Raton 2009, pp. 187–264.
[20] Tuzimski, T., Progress in separation of multicomponent
mixtures by combination of different modes of multi-
dimensional planar chromatography (MDPC) in: 8th
Balaton Symposium on High Performance Separation
Methods and 15th International Symposium on
Separation Sciences, 2009, September 2–4, Siófok,
Hungary p. 20.
[21] Tuzimski, T., Basic principles of planar chromatography
and its potential for hyphenated techniques, in:
Srivastava, M. M. (Ed.), High-Performance Thin-Layer
Chromatography (HPTLC), Chapter 14, Springer,
Heidelberg 2011, pp. 247–310.
[22] Dzido, T. H., Soczewiński, E., J. Chromatogr. 1990, 561,
461–466.
www.jss-journal.com