Analytica Chimica Acta 707 (2011) 92–99

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Analytica Chimica Acta

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Ionic liquid-based microwave-assisted dispersive liquid–liquid microextraction

and derivatization of sulfonamides in river water, honey, milk, and animal plasma
Xu Xu, Rui Su, Xin Zhao, Zhuang Liu, Yupu Zhang, Dan Li, Xueyuan Li, Hanqi Zhang, Ziming Wang ∗
College of Chemistry, Jilin University, Changchun 130012, PR China

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

Article history: The ionic liquid-based microwave-assisted dispersive liquid–liquid microextraction (IL-based MADLLME)
Received 4 July 2011 and derivatization was applied for the pretreatment of six sulfonamides (SAs) prior to the determina-
Received in revised form tion by high-performance liquid chromatography (HPLC). By adding methanol (disperser), fluorescamine
13 September 2011
solution (derivatization reagent) and ionic liquid (extraction solvent) into sample, extraction, derivati-
Accepted 14 September 2011
zation, and preconcentration were continuously performed. Several experimental parameters, such as
Available online 19 September 2011
the type and volume of extraction solvent, the type and volume of disperser, amount of derivatization
reagent, microwave power, microwave irradiation time, pH of sample solution, and ionic strength were
Keywords:
Ionic liquid-based microwave-assisted
investigated and optimized. When the microwave power was 240 W, the analytes could be derivatized
dispersive liquid–liquid microextraction and extracted simultaneously within 90 s. The proposed method was applied to the analysis of river
In situ derivatization water, honey, milk, and pig plasma samples, and the recoveries of analytes obtained were in the range of
Sulfonamides 95.0–110.8, 95.4–106.3, 95.0–108.3, and 95.7–107.7, respectively. The relative standard deviations varied
High-performance liquid between 1.5% and 7.3% (n = 5). The results showed that the proposed method was a rapid, convenient and
chromatography-fluorescence detection feasible method for the determination of SAs in liquid samples.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction with ultraviolet (UV) [13–15], fluorescence detection (FD) [16–19],

Sulfonamides (SAs) are commonly used in animal husbandry
owing to their broad-spectrum activities, low cost, and effective-
ness as growth promoters [1–3]. SAs are the priority pollutants to
be monitored in animal derived food products and in a wide variety
of matrices, because their undesirable residues can be incorporated
into waters, soils, animal tissues, honey, milk and the likes [4,5]. The
presence of SA residues is of great public concern because some of
SAs could be carcinogenic, promote occurrence of the antibiotic-
resistant bacteria, and cause allergic reactions in humans [6,7]. In
the European Union (EU), the total level of SA residues should not
exceed 100 ␮g kg−1 [8]. This maximum residue level (MRL) is valid
for target tissues (muscle, fat, liver, and kidney) and for milk from
all food-producing species [9]. Therefore, a reliable, rapid, and high
sensitive method is required for the determination of SA residues
in different kinds of samples.
Various methods have been developed for the determination
of SA residues, such as gas chromatography–mass spectrome-
try (GC–MS) [10,11], capillary electrophoresis-ultraviolet detection
(CE-UV) [12], and high-performance liquid chromatography(HPLC)
∗ Corresponding author. Tel.: +86 431 85168399; fax: +86 431 85112355.
E-mail addresses: analchem@jlu.edu.cn, xux10@mails.jlu.edu.cn (Z. Wang).
and MS [20]. HPLC methods are widely applied owing to their
high selectivity, sensitivity and simple sample treatment by using
different detection systems. SAs have to be derivatized prior to
separation and determination by HPLC-FD [16–19]. However, the
derivatization represents a major expenditure of time and effort. In
order to avoid the problem mentioned above, a fast in situ deriva-
tization method for SA determination by HPLC-FD is necessary.
Due to the very low concentrations of SA residues and the com-
plexity of the matrices, the analyte enrichment and sample cleanup
are of great importance for the determination of SAs. Many sample
preparation methods, such as liquid–liquid extraction (LLE) [21,22]
and solid-phase extraction (SPE) [16,17] were applied for SAs. How-
ever, in LLE, large volume of organic solvents was required, and in
SPE, SPE cartridges were usually expensive. These methods were
very time-consuming. In recent years, microextraction has been
applied in the analytical chemistry. Solid-phase based microex-
tractions, such as solid-phase microextraction (SPME) and stir bar
sorptive extraction (SBSE), provide a simple and solventless sam-
ple preparation and have been applied for extracting SAs from food
samples [13,14]. However, SPME and SBSE are time-consuming
and the coated fibers or bars are generally expensive and easily
destroyed. Compared with the solid-phase based microextrac-
tions, liquid phase microextraction (LPME) has the benefits of both
cost-effectiveness and convenience, and has been applied for the
extraction of SAs in environment water [15]. However, LPME still

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.09.018
 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99 93

has some drawbacks, such as instability of the microdrop, opera- Table 1

Structures of SAs.
tional difficulty and bubble formation during the extraction.
Recently, a novel microextraction method termed as disper- General structure Compound –R
sive liquid–liquid microextraction (DLLME) based on the use of
CH3
an appropriate extraction solvent and disperser has been devel- O H N
oped [23]. This method has many advantages, including simplicity H 2N S N R Sulfamethazine (SMZ)
of operation, rapidity, low cost, high recovery and enrichment O N
CH3
factor, and obtains success in many applications [24–27]. How-
ever, the commonly used high-density extraction solvents, such
OCH 3
as chlorobenzene, chloroform, and carbon tetrachloride were typ- Sulfamethoxypyridazine (SMP)
N N
ically highly toxic and environment-unfriendly. Ionic liquids (ILs)
can be used as green extraction solvents instead of organic solvents N
Sulfamethoxydiazine (SMD) OCH3
due to their unique physicochemical properties, such as negligible N
vapor pressure, miscibility with water and organic solvents, good
solubility for organic and inorganic compounds, and high thermal CH3
stability [28,29]. The ILs have been used in DLLME as extraction Sulfamethoxazole (SMX)
solvents with good results [30–36]. However, to the best of our N O
knowledge, IL-based DLLME has not been applied for the extraction OCH3
of SAs. In addition, considering the central molecules of ILs are the
combination of the organic cations and various anions [37], they are Sulfadimethoxine (SDM) N
suitable for dissipation of microwave energy. Based on these, the N
OCH3
studies using ILs as solvents in the microwave-assisted extraction
(MAE) of organic compounds have been reported. [38–40]. Com-
N N
pared with conventional extraction methods, IL-based MAE could
improve extraction efficiency and save time. Sulfaphenazole (SPP)
In the present study, ionic liquid-based microwave-assisted
dispersive liquid–liquid microextraction (IL-based MADLLME) and
derivatization followed by HPLC-FD was first applied for the deter-
mination of six sulfonamides in some liquid samples. The aim of
this work is to simplify the analytical step, reduce the consump- 2.2. Instruments
tion of toxic solvents and improve the sensitivity. The effects of
various experimental parameters were studied and optimized. The The IL-based MADLLME was performed on a household
developed method was successfully applied to the analysis of real microwave oven (SANYO, China) modified in the laboratory with
samples. a maximum microwave output power of 600 W. The microwave
energy can be continuously transmitted to the reactor, and the
microwave output power can be controlled with a continuously
2. Experimental
regulable transformer.
A Shimadzu LC-20A HPLC system (Shimadzu, Kyoto, Japan)
2.1. Chemicals and reagents
equipped with a RF-10AXL fluorescence detector was used. Chro-
matographic separation of target analytes was performed on a
The standards of sulfamethazine (SMZ), sulfamethoxypyri-
Zorbax SB-C18 column (150 mm × 4.6 mm I.D., 3.5 ␮m) (Agilent,
dazine (SMP), sulfamethoxydiazine (SMD), sulfamethoxazole
Palo Alto, CA, USA).
(SMX), sulfadimethoxine (SDM), and sulfaphenazole (SPP) were
The fluorescence spectra of SAs were measured on a Shi-
obtained from National Institute for the Control of Pharmaceutical
madzu RF-5301PC fluorescence spectrophotometer (Kyoto, Japan).
and Biological Products (Beijing, China). The chemical structures
A DELTA-320 acidity meter (Mettler-Toledo Instruments Co., Ltd,
of the compounds are shown in Table 1. The mixed stock solu-
Shanghai, China) was used for pH measurement. An Allegra 64R
tion containing the analytes was prepared monthly by dissolving
High-Speed Refrigerated Tabletop Centrifuge (Beckman Coulter,
appropriate amount of SAs in methanol and stored in amber
Inc., USA) was used for sample treatment.
glass bottles at 4 ◦ C. The mixed working solutions were obtained
daily by appropriately diluting the stock solution with acetoni-
trile or pure water. Fluorescamine was purchased from Acros 2.3. Sample preparation
Company (New Jersey, USA). The derivatization reagent was pre-
pared by dissolving fluorescamine in acetone (0.2%, w/v), and 2.3.1. River water
the reagent would be stable for at least 3 months. Chromato- River water samples were taken from Yitong river (Changchun,
graphic grade methanol and acetonitrile were purchased from China). The spiked river water sample was prepared by spiking the
Fisher Corporation (Pittsburgh, PA, USA). Analytical grade anhy- mixed working solutions into 10 mL of river water. Then the spiked
drous sodium acetate, glacial acetic acid, and sodium chloride were river water sample was adjusted with sodium acetate buffer to a
purchased from Beijing Chemical Co. (Beijing, China). Pure water pH of 3.5. The resulting solution was referred to as sample solution,
was obtained with a Milli-Q water system (Millipore, Billerica, MA, filtered through 0.45 ␮m filters and then stored at 4 ◦ C.
USA). All the solvents and solutions were passed through a 0.45 ␮m
nylon filter (Jinteng Instrument Co., Tianjin, China) before they 2.3.2. Honey
were used. 1-Butyl-3-methylimidazolium hexafluorophosphate Honey samples were purchased from a local supermarket. 1 g
([C4 MIM][PF6 ]), 1-hexyl-3-methylimidazolium hexafluorophos- of honey sample was diluted to 10 mL with sodium acetate buffer
phate ([C6 MIM][PF6 ]), and 1-octyl-3-methylimidazolium hexaflu- (pH 3.5) and then spiked with the mixed working solution of SAs.
orophosphate ([C8 MIM][PF6 ]) were purchased from Chengjie The resulting solution was referred to as sample solution, filtered
Chemical Co., Ltd. (Shanghai, China). through 0.45 ␮m filters, and then stored at 4 ◦ C.
94 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99
2.3.3. Milk
Whole milk samples were purchased in a local supermarket.
The fat content of the milk was 3.5 g/100 mL. 10 mL of homogenized
milk was accurately transferred to a 15 mL polypropylene tube. The
spiked sample was prepared by spiking the mixed working solu-
tion into the milk sample. Then, 1.0 mL trichloroacetic acid (15% in
water) was added, and the solution was shaken and centrifuged at
15,000 rpm for 10 min [41]. The supernatant was collected, diluted
to 10 mL with sodium acetate buffer (pH 3.5). The resulting solution
was referred to as sample solution, filtered through 0.45 ␮m filters
to remove the denatured proteins and then stored at 4 ◦ C.
2.3.4. Animal plasma
Fresh pig blood samples were obtained from local markets. The
sample was centrifuged for 15 min at 3500 rpm first, and the super-
natant was collected and stored at −20 ◦ C. 10 mL of the thaw plasma
was spiked with the mixed working solution of SAs and then mixed
with methanol at 1:2 ratio to remove protein and other substances.
The solution was afterwards centrifuged for 15 min at 15,000 rpm.
The supernatant was collected and evaporated to dryness under
vacuum at 40 ◦ C. The residue was dissolved in 10 mL of sodium
acetate buffer (pH 3.5). The resulting solution was referred to as
sample solution, filtered through 0.45 ␮m filters and stored at 4 ◦ C.
2.4. IL-based MADLLME and derivatization
The sample solution obtained above and 0.3 g NaCl were placed
in a 15 mL of centrifugal tube. 0.75 mL methanol (disperser), 200 ␮L
of fluorescamine solution (derivatization reagent) and 100 ␮L IL
(extraction solvent) were injected rapidly into the sample solu-
tion using a syringe. A cloudy solution was formed in the tube,
and then the tube was immediately placed in the microwave oven
and irradiated under the microwave power of 240 W for 90 s. The
IL was dispersed into the sample solution, and the derivatives of
the analytes were transferred to the IL phase. Then the solution
was centrifuged at 15,000 rpm for 10 min at 0 ◦ C. The upper aque-
ous phase was removed, and the IL phase was dissolved in 100 ␮L
acetonitrile. The resulting solution was referred to as analytical
solution, filtered through a 0.22 ␮m PTFE filter membrane (Jinteng
Instrument Co., Tianjin, China) and stored for HPLC analysis.
2.5. Chromatographic conditions
The mobile phases A and B were 10 mmol L−1 sodium acetate
buffer (pH 3.5) and acetonitrile, respectively. The gradient con-
dition was as follows: 0–5 min, 35–40% B; 5–10 min, 40% B;
11–15 min, 40–50% B; 15–20 min, 50–55% B; 20–25 min; 55–35%
B; 25–30 min; 35% B. The flow rate of the mobile phase was kept
at 0.7 mL min−1 . The injection volume of analytical solution was
20 ␮L, and temperature of the column was controlled at 35 ◦ C. The
excitation and emission wavelengths for the determination of SA
derivatives were 401 and 493 nm, respectively. The fluorescence
intensities of the derivatives could remain constant for about 2 h.
3. Results and discussion
3.1. Optimization of IL-based MADLLME and derivatization
In order to optimize the simultaneous IL-based MADLLME
and derivatization, several experimental parameters affecting the
extraction and derivatization efficiency were studied, includ-
ing type and volume of extraction solvent, type and volume of
disperser, microwave power and irradiation time, volume of fluo-
rescamine solution, pH of sample solution as well as ionic strength.
As shown in Table 1, six SAs used widely in veterinary medicine
Fig. 1. Effect of type of IL. Spiked concentration, 0.5 ␮g L−1 ; extraction solvent vol-
ume, 100 ␮L; disperser volume, 0.75 mL; microwave power, 240 W; microwave
irradiation time, 90 s; derivatization reagent volume, 200 ␮L; pH, 3.5.
were selected as representatives because only –R group is dif-
ferent (Table 1) and the chemical properties should be similar. A
pre-optimization for the experimental parameters was performed
and based on the preselected optimum parameters, the effects
of experimental parameters were investigated again. When one
parameter changed, the other parameters were fixed at the prese-
lected optimal values. The spiked samples were prepared by spiking
the standard solution in the pure water and used in the experi-
ments. All the experiments were performed in triplicate and the
concentration of SAs in the spiked samples was 0.5 ␮g L−1 .
3.1.1. Type of extraction solvent
Selection of an appropriate extraction solvent is important
for IL-based MADLLME. In this study, three kinds of hydropho-
bic imidazolium-ILs, including [C4 MIM][PF6 ], [C6 MIM][PF6 ], and
[C8 MIM][PF6 ], were investigated. When [C4 MIM][PF6 ] was used
and the aqueous phase was separated from the IL phase, no
IL phase appeared at the bottom of the tube after centrifuga-
tion. The main reason may be due to the higher solubility of
[C4 MIM][PF6 ] in water (1.88 g/100 mL) than those of [C6 MIM][PF6 ]
(0.75 g/100 mL) and [C8 MIM][PF6 ] (0.2 g/100 mL), and the lower
viscosity of [C4 MIM][PF6 ] (450 mPa s) than those of [C6 MIM][PF6 ]
(585 mPa s) and [C8 MIM][PF6 ] (710 mPa s) at 25 ◦ C [42]. As shown
in Fig. 1, the peak areas obtained with [C6 MIM][PF6 ] are larger
than those obtained with [C8 MIM][PF6 ]. Thus, [C6 MIM][PF6 ] was
selected as extraction solvent in the subsequent experiments.
3.1.2. Type of disperser
The disperser should be miscible between IL phase and aque-
ous phase. In this experiment, acetonitrile, methanol, acetone and
ethanol were selected as disperser. The effect of the dispersers on
the extraction efficiency of SA derivatives was studied, and the
results are shown in Fig. 2. It can be seen that the highest extrac-
tion efficiency is obtained when methanol is used as the disperser.
Therefore, methanol was chosen as the disperser in the subsequent
experiments.
3.1.3. Effect of the volume of IL
In order to evaluate the influence of extraction solvent vol-
ume on extraction efficiency of SA derivatives, different volumes of
[C6 MIM][PF6 ] (80, 90, 100, 110, and 120 ␮L) were used as extrac-
tion solvents. As shown in Fig. 3, the peak areas of the SA derivatives
increase with the increase of the volume of [C6 MIM][PF6 ] from 80 to
100 ␮L, and then decrease with the further increase of the volume
of [C6 MIM][PF6 ] because the increase of the IL phase volume can
 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99
Fig. 2. Effect of type of disperser. Spiked concentration, 0.5 ␮g L−1 ; extraction sol-
vent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser volume, 0.75 mL; microwave power,
240 W; microwave irradiation time, 90 s; derivatization reagent volume, 200 ␮L; pH,
3.5.
Fig. 3. Effect of the volume of IL. Spiked concentration, 0.5 ␮g L−1 ; disperser
(methanol) volume, 0.75 mL; microwave power, 240 W; microwave irradiation time,
90 s; derivatization reagent volume, 200 ␮L; pH, 3.5.
result in the dilution of the analytical solution. Therefore, 100 ␮L of
[C6 MIM][PF6 ] was selected in the following studies.
3.1.4. Effect of the volume of disperser
The effect of disperser volume on the extraction efficiency was
studied. As shown in Fig. 4, the peak areas of the SA derivatives
Fig. 4. Effect of the volume of disperser. Spiked concentration, 0.5 ␮g L−1 ; extrac-
tion solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; microwave power, 240 W; microwave
irradiation time, 90 s; derivatization reagent volume, 200 ␮L; pH, 3.5.
95
Fig. 5. Effect of microwave power. Spiked concentration, 0.5 ␮g L−1 ; extraction
solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser (methanol) volume, 0.75 mL;
microwave irradiation time, 90 s; derivatization reagent volume, 200 ␮L; pH, 3.5.
are largest when the volume of methanol is 0.75 mL. The volume
of disperser can directly affect the solubility of IL in aqueous phase
and the volume of IL phase. When the volume of methanol was
excessively small, cloudy suspension could not be formed well.
However, when the volume of methanol was excessively large, both
the volume of IL phase and extraction efficiency of target analytes
decrease due to the increase of the solubility of IL in the aque-
ous phase. Therefore, 0.75 mL methanol was selected in the further
experiments.
3.1.5. Effect of microwave power and irradiation time
The temperature can affect the mass transfer rates of analytes
and the contact area between IL and aqueous solution. Additionally,
temperature also can affect the rate of the in situ derivatization
reaction. The microwave power and irradiation time will affect the
extraction efficiency because the temperature of sample solution is
strongly related to the microwave irradiation power and time. The
effect of microwave power was studied in the range of 60–300 W
when the irradiation time was 90 s. As shown in Fig. 5, the peak
areas of SA derivatives increase from 60 to 240 W and decrease
quickly thereafter. Excessively high microwave power can cause
the decrease of IL phase volume, which resulted in the decrease of
extraction efficiency. Therefore, 240 W of microwave power was
selected for further experiments.
The effect of microwave irradiation time was also studied. As
shown in Fig. 6, the peak areas of the SA derivatives increase grad-
ually with the increase of extraction time from 30 to 90 s and then
Fig. 6. Effect of microwave irradiation time. Spiked concentration, 0.5 ␮g L−1 ;
extraction solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser (methanol) volume,
0.75 mL; microwave power, 240 W; derivatization reagent volume, 200 ␮L; pH, 3.5.
96 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99
Fig. 7. Effect of derivatization reagent volume. Spiked concentration, 0.5 ␮g L−1 ;
extraction solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser (methanol) volume,
0.75 mL; microwave power, 240 W; microwave irradiation time, 90 s; pH, 3.5.
decrease when the time exceeds 90 s. The increase of extraction
time was beneficial to the decrease of the viscosity of ILs, the accel-
eration in the rate of derivatization reaction and the mass transfer
of analytes. However, the increase of extraction time can result in
the increase of solubility of [C6 MIM][PF6 ] in the sample solution,
which resulted in the decrease of extraction efficiency. Based on
the experimental results, the irradiation time selected was 90 s.
3.1.6. Effect of derivatization reagent volume
Fluorescamine and its hydrolytic products are nonfluorescent.
This property eliminates extensive clean-up and chromatographic
separation of the fluorescent derivatives from the excess reagent.
To investigate the effect of derivatization reagent volume on extrac-
tion efficiency for in situ derivatization of SAs, different volumes of
derivatization reagent ranging from 50 to 300 ␮L were used. As
shown in Fig. 7, the peak areas of SAs derivatives increase with the
increase of the volume of derivatization reagent from 50 to 200 ␮L,
and then slightly decrease, which is probably due to quenching
effect produced by fluorescamine hydrolytic products [43]. There-
fore, 200 ␮L of derivatization reagent was selected in the following
experiments to ensure quantitative derivatization of SAs.
3.1.7. Effect of pH of sample solution
The pH of sample solution plays an important role in the extrac-
tion of organic compounds because the pH value of the solution
determines the present state of analytes. Additionally, the deriva-
tization reaction of SAs with fluorescamine needs an acidic medium
[16–18]. On the other hand, when the pH value was too low, the
emulsive phenomenon did not occur, and the volume of IL phase
was sharply reduced. In this experiment, effect of pH on the extrac-
tion performance was investigated, and the results are shown in
Fig. 8. It can be seen that the peak areas of SA derivatives are highest
when pH is 3.5, which is in agreement with the results previously
reported for SA derivatization [18]. Hence, pH 3.5 was chosen in the
following experiments.
3.1.8. Effect of salt concentration
Generally, NaCl is added into sample solution to increase the
ionic strength, which can improve the partition of analytes between
aqueous phase and organic phase. However, the effect may be dif-
ferent when ILs are used as the extraction solvent. The experimental
results shown in Fig. 9 indicate that the peak areas of SA derivatives
increase slightly with the increase of NaCl concentration from 0 to
3%, and then sharply decrease when NaCl concentration exceeds
3%. The addition of NaCl could reduce the solubility of the ana-
lytes in sample solution and improve the extraction efficiency. On
Fig. 8. Effect of pH of sample solution. Spiked concentration, 0.5 ␮g L−1 ; extrac-
tion solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser (methanol) volume, 0.75 mL;
microwave power, 240 W; microwave irradiation time, 90 s; derivatization reagent
volume, 200 ␮L.
the other hand, when the concentration of NaCl was excessively
high, the ion exchange between IL and chloride occurs, which could
make [C6 MIM][Cl] soluble in water, leading to the decrease of the
amount of IL phase and the poor extraction performance [44]. On
basis of these results, concentration of NaCl was chosen as 3% in all
subsequent experiments.
3.2. Method evaluation
3.2.1. Analytical performances
Under the optimal experimental conditions, a series of experi-
ments were performed for obtaining linear ranges, precision, and
the limits of detection (LODs) and quantification (LOQs). The work-
ing curves were constructed by plotting the peak areas measured
versus the concentrations of analytes in the samples. All the experi-
ments were performed in triplicate. The linear regression equations
and correlation coefficients are listed in Table 2. The correlation
coefficients (r2 ) ranging from 0.9989 to 0.9998 are obtained for
all the analytes. The working curves are applied to evaluating the
method LODs and LOQs. The LODs and LOQs were calculated by the
following equations:
s s
LODs = 3 ; LOQs = 10
k k
where s is the standard deviation of blank signal, and was obtained
by analyzing the blank sample 11 times. k is the slope of the
Fig. 9. Effect of the NaCl concentration. Spiked concentration, 0.5 ␮g L−1 ; extrac-
tion solvent ([C6 MIM][PF6 ]) volume, 100 ␮L; disperser (methanol) volume, 0.75 mL;
microwave power, 240 W; microwave irradiation time, 90 s; derivatization reagent
volume, 200 ␮L; pH, 3.5.
 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99 97

Table 2
Analytical performance.

Sample Analyte Liner rangea Regression equationb A = (a ± SDa ) + (b ± SDb )ca Correlation coefficient (r2 ) EF LODsa LOQsa

Water SMZ 0.10–5.00 A = (5.189 ± 0.622) × 10 + (1.946 ± 0.005) × 10 c

3 5
0.9998 28 0.015 0.051
SMP 0.10–5.00 A = (9.029 ± 1.082) × 103 + (1.716 ± 0.005) × 105 c 0.9995 30 0.018 0.061
SMD 0.05–5.00 A = (8.593 ± 1.020) × 103 + (3.388 ± 0.008) × 105 c 0.9996 35 0.014 0.048
SMX 0.05–5.00 A = (9.894 ± 1.092) × 103 + (3.854 ± 0.009) × 105 c 0.9997 37 0.014 0.045
SDM 0.05–5.00 A = (1.020 ± 0.094) × 104 + (3.637 ± 0.008) × 105 c 0.9997 44 0.012 0.041
SPP 0.05–5.00 A = (1.053 ± 0.085) × 104 + (3.771 ± 0.007) × 105 c 0.9995 41 0.011 0.036

Honey SMZ 1.00–50.00 A = (2.962 ± 0.904) × 103 + (1.837 ± 0.007) × 104 c 0.9994 26 0.233 0.778
SMP 1.00–50.00 A = (2.959 ± 0.916) × 103 + (1.615 ± 0.007) × 104 c 0.9993 28 0.269 0.898
SMD 1.00–50.00 A = (3.483 ± 1.147) × 103 + (3.133 ± 0.009) × 104 c 0.9998 32 0.174 0.579
SMX 1.00–50.00 A = (3.481 ± 1.111) × 103 + (3.483 ± 0.009) × 104 c 0.9994 33 0.151 0.505
SDM 1.00–50.00 A = (4.276 ± 0.981) × 103 + (3.312 ± 0.008) × 104 c 0.9993 40 0.141 0.469
SPP 1.00–50.00 A = (5.481 ± 0.885) × 103 + (3.492 ± 0.007) × 104 c 0.9990 37 0.120 0.401

Milk SMZ 0.10–5.00 A = (3.529 ± 1.005) × 103 + (1.795 ± 0.008) × 105 c 0.9994 26 0.027 0.089
SMP 0.10–5.00 A = (3.845 ± 1.023) × 103 + (1.571 ± 0.008) × 105 c 0.9998 28 0.031 0.095
SMD 0.10–5.00 A = (8.155 ± 1.482) × 103 + (3.112 ± 0.012) × 105 c 0.9997 32 0.023 0.075
SMX 0.10–5.00 A = (6.553 ± 1.473) × 103 + (3.557 ± 0.011) × 105 c 0.9991 34 0.020 0.066
SDM 0.10–5.00 A = (6.472 ± 1.227) × 103 + (3.308 ± 0.010) × 105 c 0.9989 40 0.018 0.059
SPP 0.10–5.00 A = (5.164 ± 1.318) × 103 + (3.458 ± 0.010) × 105 c 0.9993 38 0.018 0.060

Pig plasma SMZ 0.10–5.00 A = (5.431 ± 0.958) × 103 + (1.651 ± 0.008) × 105 c 0.9997 24 0.028 0.092
SMP 0.10–5.00 A = (3.928 ± 0.996) × 103 + (1.451 ± 0.008) × 105 c 0.9995 25 0.033 0.104
SMD 0.10–5.00 A = (9.275 ± 1.556) × 103 + (2.878 ± 0.013) × 105 c 0.9991 30 0.026 0.086
SMX 0.10–5.00 A = (1.090 ± 0.151) × 104 + (3.176 ± 0.012) × 105 c 0.9994 31 0.023 0.075
SDM 0.10–5.00 A = (9.924 ± 1.291) × 103 + (3.120 ± 0.010) × 105 c 0.9990 38 0.020 0.065
SPP 0.10–5.00 A = (9.679 ± 1.314) × 103 + (3.221 ± 0.011) × 105 c 0.9992 35 0.018 0.062
a
The units of concentrations are ␮g L−1 for river water, milk, pig plasma and ␮g kg−1 for honey.
b
A, peak area of SA; c, SA concentration in ␮g L−1 for river water, milk, pig plasma and ␮g kg−1 for honey; a, intercept; b, slope; SDa , SDb , standard deviations of intercept
and slope, respectively.

working curve. The method LODs and LOQs for all types of matri-
ces are shown in Table 2, and the results indicate that the proposed
method should be a feasible method in the determination of trace
SAs in various matrices.
Enrichment factor (EF) was calculated by the following equa-
tions:
ca
EF =
cs
where ca and cs are the concentrations of the analyte in analytical
and sample solutions, respectively. In order to calculate the EF for
the analytes, five replicates were conducted for the sample solu-
tions containing 5 ␮g L−1 of the analytes. It was found that under
optimal conditions, EFs in the range of 24–44 were obtained.
3.2.2. Analysis of samples
To evaluate the applicability of the proposed method, some real
samples, including environmental water (river water), food (honey
and milk), and biological fluid (pig plasma) were analyzed. The typ-
ical chromatograms of the blank and spiked samples are shown in
Fig. 10. As can be seen, the SAs in the samples are not detectable,

Fig. 10. Typical chromatograms for spiked (A) and blank (B) samples. (a) River water, (b) honey, (c) milk, and (d) pig plasma. The concentrations of analytes are 0.1 ␮g L−1 in
spiked river water, milk, and pig plasma samples and 1 ␮g kg−1 in spiked honey sample. 1: SMZ; 2: SMP; 3: SMD; 4: SMX; 5: SDM; 6: SPP.
98 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99

Table 3

RSD (%)
The intra- and inter-day precision and recoveries of the assay.

3.62.3

5.53.9

3.14.2

5.23.5
6.44.1
Sample Analytes Spiked Intra-day Inter-day
concentrationsa

Recovery (%)
Recovery RSD Recovery RSD

103.6105.7

101.3104.9

99.8101.5
106.0104.2

102.298.6
(%) (%) (%) (%)

River water SMZ 0.10 98.8 3.6 98.5 6.0

SPP
1.00 101.0 4.3 99.8 5.6
SMP 0.10 100.5 1.9 100.1 5.5

RSD (%)
1.00 96.4 2.7 98.0 4.0

1.83.1

3.64.1

3.96.4

5.23.3
3.01.9
SMD 0.10 104.5 3.5 106.3 3.8
1.00 110.8 4.6 108.2 5.2
SMX 0.10 97.0 4.1 95.9 5.1

Recovery (%)
1.00 96.1 5.3 95.0 4.0

104.4102.1

104.1105.9

98.4100.1
99.597.6
99.097.3
SDM 0.10 101.8 1.8 104.4 3.1
1.00 98.1 2.6 99.5 5.3

SDM
SPP 0.10 103.6 3.4 105.7 4.2
1.00 105.7 3.1 104.8 6.5

Honey SMZ 1.00 100.2 1.5 99.2 6.4

RSD (%)

1.92.6

3.13.6

2.95.1
4.53.0

3.63.0
10.00 106.0 2.1 104.8 3.9
SMP 1.00 95.9 2.6 97.1 5.4
10.00 96.4 3.4 98.4 4.3
SMD 1.00 104.7 2.6 106.3 3.0

Recovery (%)

101.8103.7

104.3101.2
105.3102.0

106.1102.0
10.00 104.1 3.1 103.8 5.8

96.899.4
SMX 1.00 101.8 1.5 99.8 3.4
10.00 104.0 2.5 106.1 5.6

SMX
SDM 1.00 97.3 3.0 95.4 4.7
10.00 96.7 4.6 98.0 7.3
SPP 1.00 100.5 1.9 103.1 4.5

RSD (%)

3.44.2

4.52.9

6.22.5
4.63.0

2.63.0
10.00 102.9 3.1 104.0 5.1

Milk SMZ 0.10 105.1 1.8 103.8 7.0

1.00 106.4 2.9 104.1 4.3

Recovery (%)
SMP 0.10 101.5 2.5 99.8 6.1

98.6102.8
104.2106.0

102.999.1

97.195.6

103.199.7
1.00 103.0 3.4 105.6 6.8
SMD 0.10 96.7 2.5 95.0 5.2

SMD
1.00 97.3 4.0 99.4 4.6
SMX 0.10 99.6 2.3 102.1 4.2
1.00 104.2 3.6 106.9 6.5

RSD (%)
SDM 0.10 103.9 1.9 101.1 4.8

2.65.8

3.63.1

2.82.1

4.56.4
7.86.4
1.00 107.9 2.7 106.3 6.7
SPP 0.10 105.2 1.5 107.0 4.4
1.00 106.8 3.3 108.3 5.4
Recovery (%)

The units of concentrations are ␮g L−1 for river water, milk, pig plasma and ␮g kg−1 for honey.
102.9103.4

102.6103.9

105.2105.9
104.1106.5
Pig plasma SMZ 0.10 96.1 2.4 98.5 5.8
95.798.3
1.00 101.0 3.8 99.6 7.2
SMP 0.10 104.5 3.6 105.8 6.3
SMP

1.00 107.7 2.9 106.1 6.9

SMD 0.10 103.1 1.9 102.6 3.8
1.00 98.6 3.0 100.1 5.3
RSD (%)

3.92.9

5.62.3

3.23.5

3.63.4
5.24.0
SMX 0.10 104.2 2.1 102.8 5.8
1.00 101.8 4.1 103.4 4.9
SDM 0.10 95.7 3.5 97.0 5.9
1.00 98.4 2.0 96.4 7.3
Recovery (%)

104.6101.1

103.4105.7

SPP 0.10 100.9 3.4 97.1 5.5

100.298.1

98.599.1
97.399.0

1.00 103.6 1.7 105.4 4.0

SMZ

a
The units of concentrations are ␮g L−1 for river water, milk, pig plasma and
␮g kg−1 for honey.
concentrationsa of

and no significant interference peaks are found at the retention

positions of SAs. To evaluate precision and accuracy of the proposed
the analyte

1.0010.00

method, the spiked samples were analyzed. The intra-day precision

0.101.00

0.101.00

0.101.00
0.101.00
Spiked

was determined by analyzing the samples five times in one day. The
inter-day precision was achieved by analyzing the samples once a
The stability of SAs in the sample.

day in five consecutive days. The analytical results are shown in

Freeze–thaw

Table 3. The relative standard deviations (RSDs) and recoveries are

1-Month

1-Month

1-Month

1-Month

in the range of 1.5–7.3% and 95.0–110.8%, respectively. It can be

Storage

considered that the present method provides acceptable recoveries

and precision for the determination of SAs in real samples.
The stability of SAs in the samples was studied. In this study,
River water

Pig plasma

spiked samples stored for one month were analyzed. The spiked
samples were all stored at −4 ◦ C except that the spiked pig plasma
Sample

Honey
Table 4

Milk

sample was stored at −20 ◦ C. The results from Table 4 indicate that
a

SAs do not show significant degradation for at least one month.

 X. Xu et al. / Analytica Chimica Acta 707 (2011) 92–99 99

Table 5
Comparison of this method with other extraction methods for determination of SAs.

Method Matrix Extraction time (min) Recovery (%) Enrichment factor LODsa LOQsa Ref.

SPE-LC-FD Serum 120 91.2–119.0 – 0.25–0.30 0.75–1.00 [17]

SAE-HPLC-FD Honey 60 90.9–99.6 – 0.6–0.9 – [18]
SPME-HPLC-UV Milk 28 11.5–96.5 – 1.7–22.0 5.5–75.0 [13]
SBSE-HPLC-DAD Milk 100 54.8–126.0 – 1.3–7.9 4.29–26.30 [14]
HF-LPME-HPLC-UV Environment water 480 82.2–103.2 58–135 0.1–0.4 – [15]
This method River water 15 95.0–110.8 28–44 0.011–0.018 0.036–0.061
Honey 15 95.4–106.3 26–40 0.120–0.269 0.401–0.898
–
Milk 15 95.0–108.3 26–40 0.018–0.031 0.059–0.095
Plasma 15 95.7–107.7 24–38 0.018–0.033 0.062–0.104
a
The units of concentrations are ␮g L−1 for river water, milk, serum, plasma and ␮g kg−1 for honey.

Freeze–thaw stability was especially evaluated for the analytes in
plasma samples. The sample was submitted to three freeze–thaw
cycles. The each cycle consisted of removing sample from freezer,
thawing at room temperature, keeping at room temperature and
refreezing at −20 ◦ C. The results shown in Table 4 indicate that SAs
in pig plasma have an acceptable stability after three freeze–thaw
cycles.
3.2.3. Comparison of IL-based MADLLME and derivatization with
other methods
Some other methods reported in literature, such as solid-phase
extraction liquid chromatography with fluorescence detection
(SPE-LC-FD) [17], sugaring-out extraction HPLC with fluorescence
detection (SAE-HPLC-FD) [18], solid-phase microextraction HPLC
with ultraviolet detection (SPME-HPLC-UV) [13], stir bar sorp-
tive extraction HPLC with ultraviolet detection (SBSE-HPLC-UV)
[14], and hollow fiber supported liquid phase microextraction
HPLC with diode array detection (HF-LPME-HPLC-DAD) [15], were
compared with the proposed method, and the results are pre-
sented in Table 5. Compared with the reported methods, when the
proposed method is applied, the LODs are lower and the extrac-
tion time is shorter. Those reported methods normally involved
multi-step operation, which would make the extraction proce-
dure complex and time-consuming. The proposed method was
timesaving and convenient because IL-based microwave irradia-
tion could obviously improve extraction efficiency and two-step
operation, including preparation of sample solution, and extrac-
tion, derivatization and preconcentration of the analytes, was
involved.
4. Conclusion
In this work, a new sample pretreatment method, IL-based
MADLLME and derivatization combined with HPLC-FD, was suc-
cessfully applied to the determination of trace SAs in various
matrices such as river water, honey, milk, and pig plasma. When
IL was used as the extraction solvent not only the consumption of
organic solvent can be reduced but also the extraction time can be
shortened. Compared with other pretreatment methods reported,
the proposed method can be performed in two steps and has some
advantages in extraction time, extraction efficiency and organic
solvent consumption.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (No. 20905030) and the China Postdoctoral Science
Foundation (No. 20090461039).
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