Analytica Chimica Acta 638 (2009) 139–145

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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Dispersive liquid–liquid microextraction combined with high performance liquid

chromatography–fluorescence detection for the determination of carbendazim
and thiabendazole in environmental samples
Qiuhua Wu, Yunpeng Li, Chun Wang, Zhimei Liu, Xiaohuan Zang, Xin Zhou, Zhi Wang ∗
Key Laboratory of Bioinorganic Chemistry, College of Science, Agricultural University of Hebei, Baoding 071001, China

a r t i c l e i n f o a b s t r a c t

Article history: A rapid and sensitive method for the determination of carbendazim (methyl benzimidazole-2-
Received 16 October 2008 ylcarbamate, MBC) and thiabendazole (TBZ) in water and soil samples was developed by using dispersive
Received in revised form 12 February 2009 liquid–liquid microextraction (DLLME) coupled with high performance liquid chromatography with flu-
Accepted 12 February 2009
orescence detection. The water samples were directly used for the DLLME extraction. For soil samples,
Available online 21 February 2009
the target analytes were first extracted by 0.1 mol L−1 HCl. Then, the pH of the extract was adjusted to 7.0
with 2 mol L−1 NaOH before the DLLME extraction. In the DLLME extraction method, chloroform (CHCl3 )
Keywords:
was used as extraction solvent and tetrahydrofuran (THF) as dispersive solvent. Under the optimum con-
Dispersive liquid–liquid microextraction
Benzimidazole fungicides
ditions, the enrichment factors for MBC and TBZ were ranged between 149 and 210, and the extraction
High performance liquid chromatography recoveries were between 50.8 and 70.9%, respectively. The linearity of the method was obtained in the
Water samples range of 5–800 ng mL−1 for water sample analysis, and 10–1000 ng g−1 for soil samples, respectively. The
Soil samples correlation coefficients (r) ranged from 0.9987 to 0.9997. The limits of detection were 0.5–1.0 ng mL−1 for
water samples, and 1.0–1.6 ng g−1 for soil samples. The relative standard deviations (RSDs) varied from
3.5 to 6.8% (n = 5). The recoveries of the method for MBC and TBZ from water samples at spiking levels of 5
and 20 ng mL−1 were 84.0–94.0% and 86.0–92.5%, respectively. The recoveries for soil samples at spiking
levels of 10 and 100 ng g−1 varied between 82.0 and 93.4%.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction organic solvents. SPE techniques typically require reduced amounts

Sample preparation is one of the most important and crucial
procedures in a whole analytical process. It is often also the bot-
tleneck for rapidly obtaining the desired results, especially for the
determination of trace analytes in a complex matrix sample. The
objective of the sample preparation is not only to isolate the target
analytes from the samples, thus reducing or even eliminating the
interferences originally present in the sample, but also simultane-
ously to concentrate the analytes to facilitate their determinations
at low levels. A variety of methods for the separation and precon-
centration of the analytes from sample matrix have been developed,
such as liquid–liquid extraction (LLE) [1], solid-phase extraction
(SPE) [2], solid-phase microextraction (SPME) [3] and liquid-phase
microextraction (LPME) [4–9].
LLE and SPE are the most commonly used techniques for
the preconcentration and cleanup of the selected analytes. How-
ever, LLE suffers from the disadvantages of being time-consuming,
expensive, and requiring large volumes of both samples and toxic
∗ Corresponding author. Tel.: +86 312 7521513; fax: +86 312 7521513.
E-mail address: wangzhi@hebau.edu.cn (Z. Wang).
of organic solvents relative to LLE, but SPE can still be tedious,
time-consuming, relatively expensive, and sometimes suffers from
analytes breakthrough when large sample volumes are analyzed.
SPME, a more recent procedure, is a simple, organic solvent-free
and efficient extraction technique [3]. However, SPME also suffers
from some problems such as sample carry-over, relatively high cost
and fiber fragility.
Recently, LPME has emerged as an attractive alternative for sam-
ple preparations because of its simplicity, effectiveness, low cost,
minimum use of solvents and excellent sample cleanup ability.
Different configurations of this technique have recently emerged,
including static LPME, dynamic LPME, single-drop LPME and hollow
fiber-based liquid-phase microextraction (HF-LPME) [4–9]. How-
ever, several disadvantages, such as the instability of liquid drop in
single-drop LPME, air bubbles formation in HF-LPME, long analysis
time and relatively low precisions, are often encountered for such
techniques.
Very recently, a novel microextraction technique, named disper-
sive liquid–liquid microextraction (DLLME), based on dispersion
of tiny droplets of the extraction solvent within the aqueous
solution has been developed by Assadi and co-workers [10]. DLLME
is a miniaturized LLE that uses microliter volumes of the extraction

0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.02.017
140 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145
solvent. For DLLME, water-immiscible extraction solvent dissolved
in a water-miscible dispersive solvent was rapidly injected into
an aqueous solution by syringe. A cloudy solution containing fine
droplets of extraction solvent dispersed entirely in the aqueous
phase was formed. The analytes in the sample were extracted into
the fine droplets, which were further separated by centrifugation
and the enriched analytes in the sedimented phase were deter-
mined by either chromatographic or spectrometric methods. The
advantages of the DLLME method are rapidity, low cost, simplicity
of operation and high enrichment factor. DLLME has been applied
for the analysis of a variety of trace organic pollutants and metal ions
in the environmental samples [11–17]. But until now, the reported
applications of DLLME have been mainly focused on simple water
samples. Therefore, the exploration of the potential applications of
the DLLME technique in more complex matrix samples, such as soil
and food, is very desirable.
Because of the widespread use of agricultural pesticides for
different applications, the pesticide residues may present a main
source of pollution, which poses risks to plant, animal and human
health. Benzimidazole fungicides are widely used pesticides in
agriculture for pre- and post-harvest treatment for the control
of a wide range of pathogens. They are either applied directly
to the soil, or sprayed over crop fields. Most of these com-
pounds persist in the environment after their application, with
some even remaining for many years [18]. Carbendazim (methyl
benzimidazole-2-ylcarbamate, MBC) and thiabendazole (TBZ) are
the widely used benzimidazole fungicides. The thermal insta-
bility of MBC and TBZ does not permit their analysis directly
by gas chromatography unless they are derived into thermally
stable derivatives. The most frequently used methods for the
determination of these benzimidazol fungicides are fluorescence
spectroscopy [19] and high performance liquid chromatography
(HPLC) with UV, fluorescence or mass spectrometric detection
[1–2]. The different pretreatment methods, such as LLE [1], SPE [2],
SPME [20], microwave-assistant extraction [21], supercritical fluid
extraction [22], and cloud point extraction [23] have been used for
the preconcentration and cleanup procedures of these fungicides
from different samples.
In continuation to our previous endeavors in the exploration of
novel sample pretreatment techniques [24–28], herein, a DLLME
method in combination with HPLC-fluorescence detection was
developed for the determination of MBC and TBZ in water and soil
samples. To the best of our knowledge, this may be the first report
about the application of the DLLME method for the determination
of these fungicides. The effects of various experimental parameters,
such as the kind and volume of extraction and disperser solvent,
extraction time and salt effect have been studied. The applicabil-
ity of the presented method for the analysis of real water and soil
samples has also been investigated.
2. Experimental
2.1. Reagents and materials
MBC (99%) and TBZ (99%) were purchased from the Eighth
Chemical Factory of Baoding (Baoding, Hebei, China). Chloroform
(CHCl3 ), carbon tetrachloride (CCl4 ) and chlorobenzene (C6 H5 Cl)
were purchased from Beijing Chemical Reagents Company (Bei-
jing, China). Acetone, tetrahydrofuran (THF), acetonitrile, ethanol
and HPLC-grade methanol were from Sinopharm Chemical Reagent
Co. Ltd (Tianjin, China). Sodium chloride (NaCl), sodium hydrox-
ide (NaOH) and hydrochloric acid (HCl) were from Tianjin Fuchen
Chemical Reagent Factory (Tianjin, China). All the reagents were
analytical reagent grade unless otherwise stated. Double-distilled
water was produced by a SZ-93 automatic double-distiller (Shang-
hai Yarong Biochemistry Instrumental Factory, Shanghai, China),
which was used for preparation of aqueous solutions.
Rain water, well water and lake water samples were collected
from Baoding (Baoding, China). Soil samples were collected from
the plough layer of the field at Ximachi and Wumazhuang (Baod-
ing, China), which were dried at room temperature, pulverized and
passed through 250-␮m sieve. All the solvents and water samples
were filtered through a 0.45-␮m membrane to eliminate particulate
matter before analysis.
A mixture stock solution containing each of MBC and TBZ at
1.0 mg mL−1 was prepared in methanol. A series of standard solu-
tions were prepared by mixing an appropriate amount of the stock
solution with double-distilled water in a 10-mL volumetric flask.
All the standard solutions were stored at 4 ◦ C in the dark.
2.2. Instrument
The HPLC system, assembled from modular components
(Waters, Milford, MA, USA), consisted of an in-line degasser, a 600E
pump, and a fluorescence detector. A Millennium32 workstation
(Waters) was utilized to control the system and for the acquisi-
tion and analysis of the data. The injection loop volume is 20.0 ␮L.
A Centurysil C18 column (4.6 i.d. × 250 mm, 5.0 ␮m) from Dalian
Jiangshen Separation Science Company (Dalian, China) was used
for separations. The mobile phase was a mixture of methanol–water
(60:40, v/v) and the flow rate was 1.0 mL min−1 . For detection, the
fluorescence excitation and emission wavelengths were set at 280
and 315 nm, respectively.
The pH of the solution was measured with a PHS-3C digital pH
meter (Hangzhou Dongxing Instrument Factory, Hangzhou, Zhe-
jiang, China).
2.3. Sample preparation before DLLME
Water samples were filtered through 0.45 ␮m filter prior to
extraction by DLLME.
The extraction of MBC and TBZ from soil samples before DLLME
was carried out according to the following procedures reported in
the documents [29,30]. Soil samples were air-dried at room tem-
perature, pulverized and passed through 250-␮m sieve. 20.0 g of
the soil sample was accurately weighed and put into a 100 mL
centrifuge tube, to which 40.0 mL 0.1 mol L−1 HCl was added. The
resultant sample mixture was first vigorously shaken on a vibrator
for 30 min and then filtrated under reduced pressure. The pH of the
filtrate was adjusted to 7.0 by 2 mol L−1 NaOH. A 5.0 mL aliquot of
the above sample solution was used for DLLME.
2.4. DLLME procedures
For the DLLME, a 5.00 mL aliquot of water sample was placed in
a 10 mL screw cap glass tube with conic bottom and 0.5 g NaCl was
added into the solution. A mixture of 0.75 mL of THF (as disperser
solvent) and 80.0 ␮L CHCl3 (as extraction solvent) was injected
into the sample solution by 1.00 mL syringe, and then the mixture
was vortexed for 10 s. A cloudy solution that consists of very fine
droplets of CHCl3 dispersed into aqueous sample was formed, and
the analytes were extracted into the fine droplets. After centrifu-
gation at 3500 rpm for 5 min, the CHCl3 phase was sedimented at
the bottom of the centrifuge tube. The sedimented phase was com-
pletely transferred to another test tube with conical bottom using
100-␮L HPLC syringe and blown to dryness with a mild nitrogen
stream. The residue was dissolved in 15 ␮L methanol and 10.0 ␮L
was injected into the HPLC system for analysis.
 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145
Fig. 1. Effect of different extraction solvents on the extraction recovery of MBC and
TBZ. Extraction conditions: sample volume, 5.0 mL; dispersive solvent, 1.0 mL ace-
tone; extraction solvent volume, 70 ␮L; concentration of fungicides, 100 ng mL−1 for
MBC and TBZ.
2.5. Calculation of enrichment factor and extraction recovery
In order to evaluate the effect of different experimental param-
eters such as the type and volume of the extraction and disperser
solvents, salt addition, and the extraction time on the performance
of DLLME, the terms of the enrichment factor and extraction recov-
ery were introduced and used according to the Eqs. (1) and (2) as
follows [10–12]:
Csed
EF =
C0
where EF, Csed and C0 are the enrichment factor, the analyte con-
centration in the sediment and the initial analyte concentration in
the aqueous samples, respectively.
Csed Vsed
R% = × 100
C0 Vaq
where R%, Vsed and Vaq are the extraction recovery, the volume of
the sediment phase and the volume of the aqueous sample, respec-
tively.
3. Results and discussion
In this experiment, 5.0 mL double-distilled water spiked with
100 ng mL−1 each of MBC and TBZ was used to study the extraction
performance under different experimental conditions. All experi-
ments were performed in triplicate and the means of the results
were used for optimization.
3.1. Selection of extraction solvent
The selection of an appropriate extraction solvent is critical
for the DLLME process. The extraction solvent has to meet some
requirements: it should have a higher density than water, a low sol-
ubility in water, and high extraction efficiency of the compounds
of interest; it also should have good chromatographic behavior
and no interference with the analyte peaks when directly injected
into a chromatographic system for analysis. Based on these crite-
ria, five extraction solvents including CCl4 , CHCl3 , dichloroethane
(C2 H4 Cl2 ), dichloromethane (CH2 Cl2 ), and C6 H5 Cl were investi-
gated for the extraction of the two target analytes. In the case
of C2 H4 Cl2 and CH2 Cl2 as extraction solvent, a two-phase system
could not be observed with any of the dispersive solvents studied
in this work. Fig. 1 shows the effect of the other three remaining
141
Fig. 2. Effect of different dispersive solvents on the extraction recovery of MBC
and TBZ. Extraction conditions: sample volume, 5.0 mL; dispersive solvent volume,
1.0 mL; extraction solvent, 70 ␮L CHCl3 ; concentration of fungicides, 100 ng mL−1 for
MBC and TBZ.
extraction solvents selected on the DLLME with the use of acetone
as disperser solvent. As can be seen in Fig. 1, among the three sol-
vents investigated, CHCl3 gives the highest extraction efficiency.
Therefore CHCl3 was selected as the extraction solvent.
3.2. Selection of disperser solvent
For DLLME method, the disperser solvent should be miscible
with both water and the extraction solvent, and should have a
good chromatographic behavior when directly injected for chro-
matographic analysis. In this study, acetone, methanol, ethanol,
acetonitrile and (THF) were investigated as disperser solvent. A
(1)
series of sample water solutions were studied by using 1.0 mL of
each above-mentioned disperser solvent and 70 ␮L of CHCl3 as
extraction solvent. The effect of different disperser solvents on the
extraction recovery of MBC and TBZ are shown in Fig. 2. As can
be seen from Fig. 2, the best extraction recoveries were achieved
(2) when THF was used as a disperser solvent. Consequently, THF was
selected.
3.3. Effect of extraction solvent volume
In order to study the effect of the volume of the extraction sol-
vent on the extraction efficiency, different volumes of CHCl3 (20.0,
40.0, 60.0, 80.0 and 100.0 ␮L) and a constant volume of the dis-
persive solvent THF (1.0 mL) were investigated. Fig. 3 illustrates
the variations of the extraction recovery versus the volume of the
extraction solvent. According to Fig. 3, the extraction recovery was
increased with the increase of the volume of CHCl3 when it was less
than 80.0 ␮L. However, a reduction of the extraction recovery for
TBZ was observed after the volume of CHCl3 exceeded 80.0 ␮L. This
phenomenon may be attributed to the amount of CHCl3 exceeding
the target dispersed amount. Some excess CHCl3 was adsorbed on
to the wall of the tube, and part of the analytes migrating into these
CHCl3 could not be centrifuged into the bottom of the tube [31].
Consequently, 80.0 ␮L of CHCl3 was chosen for the experiment.
3.4. Effect of disperser solvent volume
The influence of the volume of the disperser solvent THF was
investigated by changing its volume from 0.3 to 0.5, 0.75, 1.0 and
1.2 mL, respectively. The results are shown in Fig. 4. According to the
Fig. 4, the extraction efficiency is increased first and then decreased
by increasing the volume of THF for the both fungicides investi-
gated. The reason for this could be that at a low volume of THF, a
cloudy state could not be formed well, therefore, resulting in a low
142 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145
Fig. 3. Effect of the volume of extraction solvent (CHCl3 ) on the extraction recovery
of MBC and TBZ. Extraction conditions: sample volume, 5.0 mL; dispersive solvent,
1.0 mL THF; extraction solvent, CHCl3 ; concentration of fungicides, 100 ng mL−1 for
MBC and TBZ.
Fig. 4. Effect of the volume of dispersive solvent (THF) on the extraction recovery
of MBC and TBZ. Extraction conditions: sample volume, 5.0 mL; extraction solvent,
80.0 ␮L CHCl3 ; concentration of fungicides, 100 ng mL−1 for MBC and TBZ.
recovery. At a higher volume of THF, the solubility of the fungicides
in water was increased, leading to the decreased extraction effi-
ciency because of a decrease in distribution coefficient. Based on
the experimental results in Fig. 4, 0.75 mL of THF was chosen.
3.5. Effect of extraction time
Extraction time is one of the most important factors in DLLME
as in most extraction procedures. The extraction time is defined as
the time interval between the addition of the mixture of disper-
sive solvent (THF) and extraction solvent (CHCl3 ) to the sample and
the start of centrifugation. The effect of extraction time was stud-
ied over the time range between vortexing for 10 s and shaking for
20 min. The results indicated that the extraction time almost had
Table 1
Analytical performance data for MBC and TBZ in water sample by the DLLME method.
Fungicides Water sample
LRa (ng mL−1 ) r LOD (ng mL−1 )
MBC 5–800 0.9994
TBZ 5–800 0.9997
a
LR: linear range.
Fig. 5. Effect of salt addition on the extraction recovery of MBC and TBZ. Extraction
conditions: sample volume, 5.0 mL; extraction solvent, 80.0 ␮L CHCl3 ; dispersive
solvent, 0.75 mL THF; concentration of fungicides, 100 ng mL−1 for MBC and TBZ.
no impact on the extraction recoveries. It is revealed that after the
formation of a cloudy state of the solution, the surface area between
extraction solvent and aqueous sample phase is infinitely large. In
such a case, the equilibrium state can be achieved quickly and there-
fore the extraction time required can be very short [14–17]. The
short extraction time is one of the remarkable advantages of the
DLLME technique. In this method, the extraction was selected as
vortexing for 10 s.
3.6. Salt addition
To evaluate the possibility of salting out effect, the extraction
efficiency was studied with the sodium chloride concentration over
the range from 0 to 15% (w/v). As is shown in Fig. 5, there is an
increase in extraction efficiency with the increase of the salt con-
centration up to 10%. However, at the salt concentration of 15%,
the extraction solvent phase could not be sedimented at the bot-
tom of the centrifuge tube, but went to the upper layer in the tube.
Based on such an observation, 10% (w/v) of NaCl was added in all
the subsequent experiments.
Under the optimized experimental conditions, the enrichment
factors of this method for MBC and TBZ were 149 and 210, and
the absolute extraction recoveries (or extraction efficiency), which
were calculated according to Eq. (2), were 50.8 and 70.9%, respec-
tively.
3.7. Calibration curve, reproducibility, limits of detection, and
limits of quantification
3.7.1. Water samples
A series of working solution containing each of MBC and TBZ
at six concentration levels of 5.0, 20.0, 100.0, 200.0, 400 and
800.0 ng mL−1 were obtained for the establishment of the calibra-
tion curve. For each level, five replicate extractions were performed.
The characteristic calibration data listed in Table 1 were obtained
under optimized conditions. The linear response was observed in
LOQ (ng mL−1 ) RSD (%) (n = 5)
0.5 1.0 3.5
1.0 2.0 5.2
 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145 143

Table 2
Analytical performance data for MBC and TBZ in soil sample by the DLLME method.

Fungicides Soil sample

LRa (ng g−1 ) r LOD (ng g−1 ) LOQ (ng g−1 ) RSD (%) (n = 5)

MBC 10–1000 0.9987 1.0 2.0 5.3

TBZ 10–1000 0.9994 1.6 3.2 6.8
a
LR: linear range.

Table 3
Determination of MBC and TBZ residues and recoveries in lake, rain and well waters.

Fungicides Spiked (ng mL−1 ) Lake water (n = 5) Rain water (n = 5) Well water (n = 5)

Found (ng mL−1 ) Rc (%) RSD (%) Found (ng mL−1 ) Rc (%) RSD (%) Found (ng mL−1 ) Rc (%) RSD (%)

MBC 0 nda nda nda

5 4.2 ± 0.3b 84.0 6.3 4.5 ± 0.2b 90.0 4.7 4.7 ± 0.2b 94.0 3.5
20 17.2 ± 0.9 86.0 5.2 17.7 ± 0.6 88.5 3.6 18.5 ± 0.6 92.5 3.3

TBZ 0 nda nda nda

5 4.3 ± 0.3 86.0 5.8 4.4 ± 0.2 88.0 4.3 4.7 ± 0.2 94.0 3.6
20 18.1 ± 0.9 90.5 4.9 18.2 ± 0.6 91.0 3.5 18.3 ± 0.6 91.5 3.1
a
nd: not detected.
b
Mean value ± SD.
c
R: recovery of the method.

the range of 5–800 ng mL−1 of MBC and TBZ in water samples.
The correlation coefficients (r) ranged from 0.9994 to 0.9997. The
limits of detection (LOD, S/N = 3) for MBC and TBZ were 0.5 and
1.0 ng mL−1 , respectively. The limits of quantification (LOQ, S/N = 6)
for MBC and TBZ were 1.0 and 2.0 ng mL−1 , respectively. The repro-
ducibility study was carried out by performing five parallel replicate
extractions and analysis at the concentration of 20 ng mL−1 for each
of MBC and TBZ under the optimal conditions. The resultant repro-
ducibilities expressed as relative standard deviations (RSDs) were
3.5% and 5.2% for MBC and TBZ, respectively. These results (see
Table 1) show that the proposed method has a high sensitivity and
reproducibility.
3.7.2. Soil samples
20.0 g of air-dried soil sample, which was free of MBC and TBZ,
was accurately weighed and put into a 100 mL centrifuge tube. An
appropriate amount of mixture standard solution of MBC and TBZ
and 10 mL ethanol were added into it. The mixtures were air-dried
at room temperature to obtain spiked soil samples. A series of work-
ing samples containing each of MBC and TBZ at five concentration
levels of 10.0, 40.0, 200.0, 400.0 and 1000.0 ng g−1 were obtained
for the establishment of the calibration curve. The samples were
then prepared and extracted with the DLLME procedures estab-
lished above as described previously in the sections of 2.3 and 2.4.
For each level, five replicate extractions were performed. The lin-
ear range, correlation coefficients (r), the LODs (S/N = 3), the LOQs
(S/N = 6), and RSDs are summarized in Table 2. The signal was lin-
ear over the concentration range from 10 to 1000 ng g−1 for both
MBC and TBZ in soil, with the correlation coefficients (r) of 0.9987
and 0.9994, the LODs of 1.0 and 1.6 ng g−1 , and LOQs of 2.0 and
3.2 ng g−1 , respectively.
3.8. Recoveries of the method and samples analysis
3.8.1. Water samples analysis
To evaluate the accuracy and applicability of the proposed
method, the extraction and determination of MBC and TBZ in differ-
ent water samples, i.e., rain, well and lake waters, were performed.
The water samples were spiked with the standards of the two
fungicides at the concentration of 5 and 20 ng mL−1 , respectively.
For each concentration level, five replicate experiments with the
whole analysis process were made and the results are given in
Table 3. The recoveries of the method (expressed as the mean per-
centage between the amounts found and the ones added) for the
two fungicides in lake, rain and well waters were in the range
84.0–90.5%, 88.0–91.0% and 91.5–94.0%, respectively. Fig. 6 shows

Fig. 6. The typical chromatograms of (a) sample blank and (b) the spiked concentration of MBC (1) and TBZ (2) at each of 5 ng mL−1 in (A) well water and (B) rain water.
144 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145

Table 4
Determination of MBC and TBZ residues and recoveries in soil samples.

Fungicides Spiked (ng g−1 ) Ximachi (n = 5) Wumazhuang (n = 5)

−1
Found (ng g ) c
R (%) RSD (%) Found (ng g−1 ) Rc (%) RSD (%)

MBC 0 nd a
3.1 ± 0.2 b
5.4
10 8.2 ± 0.6b 82.0 7.3 11.6 ± 0.6 85.0 5.1
100 88.2 ± 4.9 88.2 5.6 96.5 ± 4.5 93.4 4.7

TBZ 0 nda 3.4 ± 0.2 6.1

10 8.4 ± 0.5 84.0 6.2 12.1 ± 0.7 87.0 5.8
100 90.3 ± 3.8 90.3 4.2 94.2 ± 4.1 90.8 4.4
a
nd: not detected.
b
Mean value ± SD.
c
R: recovery of the method.

Fig. 7. Chromatograms of (a) sample blank and (b) the spiked concentration of MBC (1) and TBZ (2) at each of 10 ng g−1 in soil samples. (A) Ximachi soil; (B) Wumazhuang
soil.

the typical chromatograms of the extracted MBC and TBZ from well
and rain waters before and after spiking with 5 ng mL−1 of the two
fungicides.
3.8.2. Soil sample analysis
The developed DLLME-HPLC method was applied to the assay of
MBC and TBZ in real soil samples (collected from local Ximachi and
Wumazhuang) under the optimum conditions established above.
As a result, no residues of MBC and TBZ were found in Ximachi soil.
For Wumazhuang soil, MBC and TBZ were found to be at 3.1 and
3.4 ng g−1 , respectively, The recoveries of MBC and TBZ for the soil
samples were studied in the same way as for water samples except
for spiking the soil samples with MBC and TBZ at the concentration
of 10 and 100 ng g−1 , respectively. The results are summarized in
Table 4. As can be seen from Table 4, the recoveries were ranged
from 82.0 to 93.4% with RSDs less than 7.3%, indicating a good per-
formance of the DLLME method for the determination of MBC and
TBZ in soil samples. The typical chromatograms for the soil samples
are shown in Fig. 7.
tion solvent and can provide a much higher EF (about 150–200 in
this study). For the determination of MBC and TBZ in water sam-
ples with SPME [20], 40 min of the extraction time was required
and the RSDs ranged between 6.6 and 9.0% which was higher than
those reported by us here with the DLLME method (3.5–5.2%). The
lower RSDs are probably because of a quicker achievement of the
equilibrium in DLLME. The LODs for MBC and TBZ in water samples
by the SPME–HPLC method [20] was 1.3 and 0.03 ng mL−1 , respec-
tively. The LOD for TBZ by the SPME method is lower than that by the
DLLME method, which indicates SPME has a higher sensitivity for
TBZ than the DLLME method. The detection limits for MBC and TBZ
by SPE–HPLC method [32] was about 50 ng mL−1 , which is higher
than that with our newly developed DLLME method. Meanwhile,
the SPE method [32] is very time consuming. Another additional
advantage of the DLLME is that it does not require special instru-
mentation. Therefore, DLLME is indeed simple, rapid, easy to use
and environmentally friendly.
4. Conclusions

3.9. Comparison of the DLLME with other sample preparation
techniques
DLLME has the advantages of short extraction time, high EF
and recovery, and lower solvent consumption. The main competing
method (traditional LLE) has lower enrichment factor and higher
solvent consumption. LLE usually uses about 10 mL or more of the
extraction solvent and the EF for analytes is about 10 or less in most
cases, whereas DLLME only consumes microliter level of the extrac-
A simple, rapid, and sensitive DLLME extraction technique cou-
pled with HPLC-fluorescence detection has been developed for the
determination of MBC and TBZ in water and soil samples. The
method has been proven to produce good reproducibilities, high
enrichment factors and good recoveries within a short analysis time
for both water and soil samples. Compared with other extraction
methods such as SPME and SPE, the DLLME can offer advantages
of fastness, simplicity, ease of operation and a low consumption of
organic solvent.
 Q. Wu et al. / Analytica Chimica Acta 638 (2009) 139–145
Acknowledgments
Financial support from the Natural Science Foundations of Hebei
(B2008000210) and the Scientific Research Foundation of Agricul-
tural University of Hebei are gratefully acknowledged.
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