Chromatographia (2011) 73:1057–1064
DOI 10.1007/s10337-011-1992-8
ORIGINAL
Temperature-Induced Ionic Liquids Dispersive Liquid–Liquid
Microextraction of Tetracycline Antibiotics in Environmental
Water Samples Assisted by Complexation
Dekun Hou • Yan Guan • Xiaowei Di
Received: 31 October 2010 / Revised: 8 January 2011 / Accepted: 25 February 2011 / Published online: 20 March 2011
Ó Springer-Verlag 2011
Abstract In this study, dispersive liquid–liquid mic-
roextraction (DLLME) combined with ultra high pressure
liquid chromatography (UHPLC)–tunable ultraviolet detec-
tion (TUV), was developed for pre-concentration and
determination of trace levels of tetracyclines, including
4-epitetracycline, 4-epichlortetracycline, doxycycline, chlo-
rtetracycline oxytetracycline, tetracycline, 4-epianhydro-
tetracycline and anhydrotetracycline, in aqueous samples.
La (III) was used as the chelating agent to form a hydro-
phobic complex compound with tetracyclines, followed by
extraction with ionic liquids. Some important parameters
that may affect extraction efficiencies were examined and
optimized. Under the optimum conditions, linearity of the
method was observed in the range of 0.1–200 lg L-1, with
correlation coefficients (r2) [0.992. The limits of detection
and quantification were 0.031–0.079 and 0.10–0.26 lg
L-1, respectively. The spiked recoveries of eight target
compounds in river water, fishpond water and hog leachate
were achieved in the range of 62.6–96.3, 58.9–94.5,
55.1–86.1%, respectively.
Keywords Column liquid chromatography  DLLME 
Ionic liquid  Tetracyclines
D. Hou (&)  Y. Guan  X. Di (&)
College of Chemistry and Chemical Engineering,
Inner Mongolia University, No.235, Daxue Xi Road,
Hohhot 010021, Inner Mongolia, China
e-mail: houdekun707_2@163.com
X. Di
e-mail: cexwdi@imu.edu.cn
D. Hou
Research Center of Eco-Environment Sciences,
Chinese Academy of Sciences, Beijing 100085, China
Introduction
In recent years, concerns have increasingly emerged with
regard to the occurrence of pharmaceuticals and personal
care products entering into the aquatic environment. One
important reason is their potential impact on environment
water quality if the sources supplying natural water to the
reservoirs are contaminated by these compounds [1, 2].
Tetracyclines (TCs) as a major group of pharmaceuticals
have long been use for treating and protecting the health of
animals and to promote growth and feed efficiency. How-
ever, the incorrect use or the dosages of veterinary drugs
results in the antibiotic residues being contained in animal
feces, and these residues are transported into the environ-
ment, TCs have been found widely disseminated in water
[1, 3–7] and sewage [5, 8–10], and have also occurred in
soils [11], these antibiotics in the environment may lead to
the development of antibiotic resistance in human patients
and may have direct toxic effect on people. Therefore, it is
important to develop a reliable identification and confir-
mation method to determine the presence of TCs in the
environment.
More recently, room temperature ionic liquids (RTILs),
which are ionic media resulting from the combination of
organic cations and various anions [12], have attracted
much attention taking into account their special features
such as a very low volatility, often good thermal stability,
electrical conductivity, good solvating properties, and
water or solvent solubility [13, 14], and they are applied
more and more as extraction solvents. Their low volatility
and unique solvating properties were considered as
attractive properties that would make them very useful for
use in separation techniques [15–21]. Classical liquid–
liquid extraction (LLE) based on ionic liquids has been
reported previously by Li et al. [22], but this method is
123
1058
tedious and consumes large amounts of ionic liquids, which
is expensive. Microextraction based on ILs was developed
by Liu et al. [23], and some novel microextraction tech-
niques like solid phase microextraction (SPME), stir-bar
sorptive extraction (SBSE) and single drop microextraction
(SDME) have been developed. The main limitations of
these methods include time-consuming extraction proce-
dures, lower enrichment factors, tedious operation, and the
large amounts of poisonous organic solvent used. In fact,
ILs has been recently proposed as extractants in analytical
chemistry [17], the use of RTILs can replace the use of
highly toxic chlorinated solvents, usually employed as
extractants, with a simple injection into HPLC systems
after dilution [24, 25].
Dispersive liquid–liquid microextraction (DLLME) is a
new type of solvent microextraction (SME) which was
developed by Assadi and coworkers in 2007 [26]. DLLME is
based on a ternary solvent system like homogeneous liquid–
liquid extraction and cloud point extraction, in which ana-
lytes in aquatic samples were extracted by a cloudy solution
formed by extraction solvents, then the extraction solvent
was transferred after centrifugation and target compounds in
the extraction solvent were determined by gas chromatog-
raphy (GC) or liquid chromatography (LC). Compared to
other microextraction techniques mentioned above,
DLLME is simplicity, rapidity, low cost, good recovery and
high enrichment factor, therefore it has been successfully
applied for the determination of trace organic and inorganic
compounds in water samples [27–31]. However, in previous
literature reported, most of the solvents used in dispersive
liquid–liquid microextraction are traditional solvents, which
are generally toxic, flammable and volatile [32, 33]. So,
dispersive liquid–liquid microextraction based on ILs is
preferred in this research. Besides, IL-DLLME seems to be a
much lower-cost method.
The objective of the present work was to evaluate the
possibility of using ionic liquids in DLLME. The analysis
method was based on ultra high pressure liquid chromatog-
raphy with tunable ultraviolet visible detection to analyze
residues of eight tetracycline antibiotics. The studied target
compounds were chosen from major use for human and
veterinary antibiotics which either have been found existent
in natural water in previous studies [1, 3, 4, 19, 34] or could
potentially be present in the environment, including 4-epi-
tetracycline (ETC), 4-epianhydrotetracycline (EATC),
oxytetracycline (OTC), 4-epichlortetracycline (ECTC),
tetracycline (TC), anhydrotetracycline (ATC), chlortetra-
cycline (CTC) and doxycycline (DC). DLLME behaviors of
these analytes were studied and recoveries of all target
compounds at different stages of sample processing were
evaluated. The tetracycline antibiotics contamination in
river, fishpond, and leaching water of a pig farm soil was
investigated using this method. The developed method using
123
D. Hou et al.
ILs as the extraction agent was successfully applied to ana-
lyze tetracycline residues of environment samples.
Experimental
Materials and Reagents
Antibiotic standards were obtained from the National
Institute for the Control of Pharmaceutical and Biological
Products (Beijing, China). Acetonitrile (HPLC grade) was
purchased from Tedia (Fairfield, OH, USA). Methanol
(HPLC grade) was bought from Fisher (Pittsburgh, PA,
USA). Lanthanum nitrate, trifluoroacetic acid, magnesium
chloride, sodium chloride and potassium chloride were of
analytical grade and were obtained from Beijing Chemical
Works (Beijing, China). [Bmim]PF6 was purchased from
Shanghai Chengjie Chemical Co. (Shanghai, China). Triton
x-100 and triton x-114 were purchased from Fluka (Buchs,
Switzerland). Deionized water was purified with a Milli-
pore Mill-Q plus System (Bedford, MA, USA).
Instrumentation
Tetracycline antibiotics were analyzed by an Acquity Ultra
Performance Liquid Chromatography system (Waters,
Milford, MA, USA), consisting of a binary solvent manager
and a sample manager coupled to a tunable UV detector.
UPLC analyses were performed on a bridged ethylene hybrid
(BEH) C18 analytical column (50 9 2.1 mm, 1.7 lm).
Empower software was used for chromatographic data
gathering and integration of chromatograms. A model
CR22GII High-speed Refrigerated Centrifuge (Hitachi,
Tokyo, Japan) was used for centrifugation and the super-
mixer (Melrose, Park, IL, USA) was used to disperse the
samples after the extracting solvents was injected into the
aqueous samples. A DIKEW-2-6 water bath was obtained
from the Beijing Zhongxing Company (Beijing, China).
Standard Solution
The individual standard solutions of eight tetracycline anti-
biotics were prepared accurately by weighing 10 mg of each
compound, dissolving in 10 ml of LC grade methanol (stock
solution).The stock solutions were stored in the dark at
-20 °C until analysis. The working blended standard solu-
tions containing eight analyte compounds were prepared daily
by mixing and diluting the stock solutions with methanol.
UPLC Conditions
The mobile phase consisted of methanol/acetonitrile
(50:60, v/v, A), and trifluoroacetic acid (0.05%, v/v, B) with
Temperature-Induced Ionic Liquids Dispersive Liquid–Liquid Microextraction
the gradient elution as follows: 0 min, 0% A/100% B with
a flow rate of 0.3 mL min-1, then 10% A/90% B with a
flow rate of 0.3 mL min-1 at 1.0 min (Waters curve type
6), 30% A/70% B with a flow rate of 0.3 mL min-1 at
5.0 min (Waters curve type 10), 35% A/65% B with a flow
rate of 0.3 mL min-1 at 6.5 min (Waters curve type 6),
finally, reconditioning the column with 0% A/100% B after
washing the column with 90% A at a rate of 0.3 mL min-1
for 1.5 min. The total run time for analysis was 10 min.
The peaks were detected at a wavelength of 380 nm. 20
and 30 °C were adopted as sample temperature and column
temperature, respectively. The injection volume was
5.0 lL.
Temperature-Induced Ionic Liquids Dispersive
Liquid–Liquid Microextraction Procedure
The 5.00-mL sample solution was placed in a 10-mL
plastic test tube with a conical bottom, and La (III)
(0.4 mmol L-1, 5.0 lL) and triton x-114 (1.0%, v/v,
0.5 mL) were added successively. The mixture solution
was blended and held for 40 min. Then, the ionic liquid
was added to the solution above, and the tube was put into
the 30 °C water bath to dissolve the ionic liquids com-
pletely, after shaking the solution to mix evenly, the tube
was transferred into the ice-water bath and a milky cloudy
mixture was formed. The milky cloudy mixture was cen-
trifuged for 10 min at 11,8009g, then, the dispersed fine
particles had sedimented to the bottom of the conical test
tube. The supernatant was discarded and the sedimented
phase was dissolved in methanol. Then 5.0 lL of this was
injected automatically for UPLC analysis.
Results and Discussion
To obtain the high enrichment factors and recoveries, the
parameters which may affect the partition of analytes
among the different phase were optimized using mixed
working solutions. A step-by-step optimization scheme
was designed for analysis including the type and amount
the extraction solvents, the effect of an anti-sticking agent
and complex reaction time, pH, temperature and extraction
time. In order to calculate the enrichment factor (EF), the
following equation was used:
Csed
EF ¼
C0
where the EF was defined as the ratio between the tetra-
cycline concentration in the sedimented phase (Csed) and
the initial concentration of analytes (C0) in the aqueous
sample.
1059
Csed was calculated from the calibration graph of a
tetracycline standard solution in the concentration range of
0.1–200 lg L-1.
Selection of Ionic Liquid
Ionic liquids are unique solvents that consist entirely of
ions, and they are widely applied in extraction. In our
experiment, there were some requirements that had to be
satisfied when selecting ionic liquids: they should have:
(a) low water solubility, (b) high extraction efficiency of
target compounds and (c) inexpensive and have no effect
on chromatographic behavior. Based on these consider-
ations, 1-butyl-3-methylimidazolium hexafluorophosphate
([Bmim]PF6; density, 1.36 g mL-1; water solubility,
1.88 g 100 mL-1; 25 °C),1-hexyl-3-methyl imidazolium
hexafluorophosphate ([Hmim]-PF6; density, 1.29 g mL-1;
water solubility, 0.75 g 100 mL-1; 25 °C), 1-octyl-3-
methylimidazolium hexafluorophosphate ([Omim]PF6;
density, 1.20 g mL-1; water solubility, 0.20 g 100 mL-1;
25 °C) were examined in this research [23]. For evaluating
these three ionic liquids, a series of sample solutions were
studied by contrasting the extraction recovery of target
compounds in 5.0 mL of the spiked water sample. The
operating conditions were: 0.100 g ILs, 0.4 lmol L-1
La3?, 1.0% (v/v) triton-x 114, 30 min complexing time,
50 °C heating temperature, 30 min cooling time and cen-
trifugation at 11,8009g for 10 min at 4 °C. After injection,
it was observed that the spiked recoveries of ETC and TC
were very low and almost neither of the two target peaks
were found in the chromatograms when using [Hmim]PF6
as extractant; while when [Omim]PF6 was used as the
extraction agent, the chromatographic peaks of CTC and
DC could not be completely separated, which made
quantification of CTC and DC inaccurate. As for
[Bmim]PF6, it had good chromatographic behavior and no
interruption for the target compounds in chromatograms,
but comparing [Hmim]PF6 and [Omim]PF6, the solubility
of [Bmim]PF6 was slightly high in the water sample, so the
amount of [Bmim]PF6 needed was also a little too high. For
the reasons mentioned above, [Bmim]PF6 was selected as
the extraction ionic liquid.
Effect of Anti-Sticking Agent and Amounts of ILs
A phenomenon was found in the experiment: some of the
IL-phase sticks on the wall of the centrifuge tube after
centrifugation, which may result in low recoveries of target
compounds. Baghdadi and coworkers [26] reported that a
non-ionic surfactant could decrease the interaction of IL
with the wall of the tube during the phase separations,
molecules of the surfactant surround the fine droplets of IL
123
1060
Triton x-100 Triton x-114
25000
Peak area
12500
0 ETC
ETC OTC TC ECTC
Target compounds
Fig. 1 Effect of triton x-100 and triton x-114 on the peak area of La–
TCs complex. Utilized conditions: Water volume: 5.0 mL,
0.4 lmol L-1 La3?, concentration of surfactant: 1.0% (v/v), temper-
ature: 50 °C, extraction time: 30 min, centrifugation time: 10 min,
concentration of spiked standard: 2.5 lg mL-1
ETC OTC
120 CTC DC
Extraction recovery (%)
60
0
0.0 0.2 0.4
The concentration of triton x-114 (%)
Fig. 2 Effect of Triton x-114 concentration on the extraction
recovery. Utilized conditions: Concentration of spiked standard:
2.5 lg mL-1, water volume: 5.0 mL, 0.4 lmol L-1 La3?, triton
x-114, temperature: 50 °C, extraction time: 30 min, centrifugation
time: 10 min
and the IL-phase do not stick on the wall of the centrifuge
tube. Based on above considerations, triton x-100 and tri-
ton x-114 were selected to study the effect of non-ionic
surfactant. According to Fig. 1, in the presence of non-
ionic surfactant, the target compounds peak area increased
obviously. Compared with triton x-100, in the case of using
triton x-114, the peak area increased. So triton x-114 was
chosen as the anti-sticking agent.
In our study, the concentration of triton x-114 with the
percentage of 0, 0.02, 0.10, 0.20, 0.60 and 1.0% (v/v), was
investigated. Figure 2 presents the curve of extraction
recovery of target compounds while using different con-
centrations of triton x-114, which reveals that the extrac-
tion recovery increased clearly after adding triton x-114
and is nearly constant in the concentration range of
0.1–1.0%. Therefore, a concentration of 0.1% was chosen
for the subsequent experiments.
The effect of the [Bmim]PF6 amount on extraction
performance was investigated by adding a certain amount
123
D. Hou et al.
no surfactant 30000 0.100g 0.125g 0.150g 0.175g
Peak area
15000
0
OTC TC ECTC CTC DC EATC ATC
CTC DC EATC ATC
Tetracyclines
Fig. 3 Effect of [Bmim]PF6 amount. Utilized conditions: Concentra-
tion of spiked standard: 2.5 lg mL-1, water volume: 5.0 mL,
0.4 lmol L-1 La3?, ILs: [Bmim]PF6, triton x-114 0.1% (v/v), temper-
ature: 50 °C, extraction time: 30 min, centrifugation time: 10 min
of [Bmim]PF6 to a purified aqueous sample under the
TC ECTC
EATC ATC
above-mentioned procedure, and then this was spiked with
a concentration of 2.5 lg mL-1 of eight target compounds.
In this experiment, 0.100, 0.125, 0.150 and 0.175 g of
[Bmim]PF6 were studied while keeping other conditions
mentioned above constant. Result shown in Fig. 3 indicate
that the peak area of the eight target compounds increased
gradually when the amount of [Bmim]PF6 increased from
0.100 to 0.150 g, this was because of the fact that the
[Bmim]PF6 had a certain solubility in the aqueous sample,
and the dissolved part of IL did not sediment down when
0.6 0.8 1.0
the temperature decreased and centrifuged at 11,800
9g [35]. When the amount of [Bmim]PF6 was over
0.150 g, the peak areas of the target compounds showed
almost no further increase. So, 0.150 g [Bmim]PF6 was
selected in the subsequent experiments.
Effect of Complex Reaction Time
It takes some time to form the La3?–TCs complex com-
pounds. So, it is very important to select an appropriate
time for obtaining a good complex reaction rate. In our
study, 10, 20, 30, 40, 60 and 120 min were investigated
while keeping the other conditions mentioned above con-
stant. Figure 4 shows the curve of recovery of eight target
compounds versus different complex reaction times. From
Fig. 4, we can clearly know that the recoveries increased
by increasing complex reaction time from 10 to 40 min.
Though the continuous increasing of time from 40 to
60 min, the recoveries have little variation, indicating
equilibrium had been reached. So, we selected 40 min as
the complex reaction time in our experiments.
Effect of pH and Temperature
In the procedure of complex compounds formation with
sufficient hydrophobicity to be extracted into the IL-phase,
pH is a critical parameter because the pH value of a
Temperature-Induced Ionic Liquids Dispersive Liquid–Liquid Microextraction 1061

ETC OTC TC ECTC CTC DC EATC ATC 100

100 ETC
Extracton recovery (%)

Extraction recovery (%)

OTC

TC

ECTC
50
50
CTC

DC
EAT C

ATC
0 0
10 20 30 40 60 120 30 40 50 60 70
Complexing time (min)
Heating temperature
Fig. 4 Effect of complexing time on the extraction recovery. Utilized Fig. 6 Effect of temperature on the recovery of TCs. Utilized
conditions: Concentration of spiked standard: 2.5 lg mL-1, water conditions: Concentration of spiked standard: 2.5 lg mL-1, water
volume: 5.0 mL, triton x-114 0.1% (v/v), 0.4 lmol L-1 La3?, ILs: volume: 5.0 mL, triton x-114: 0.1% (v/v), 0.4 lmol L-1 la3?, ILs:
0.150 g [Bmim]PF6, temperature: 50 °C, extraction time: 30 min, 0.150 g [Bmim]PF6, complexing time: 40 min, centrifugation time:
centrifugation time: 10 min 10 min

ETC OTC TC ECTC

100 CTC DC EATC ATC a stable chelate-metal complex only in neutral environ-
Extraction recovery (%)

ment. So, neutrality was selected as extraction condition in

our experiment.
As temperature is the driving force for the complete
50 dispersion of [Bmim]PF6 into the aqueous solution [37], it
plays an important role on the determining whether the
sensitivity of the developed method is satisfactory or not.
After adding [Bmim]PF6, they were heated in the range of
0
30–70 °C. As is shown in Fig. 6, in the range of 30–60 °C
3 5 7 9 10
the extraction recoveries have a little increase and kept
pH value
satisfactory results, the extraction efficiency showed an
Fig. 5 Effect of pH on the recovery of TCs. Utilized conditions: optimum at 60 °C. While from 60 to 70 °C, the extraction
Concentration of spiked standard: 2.5 lg mL-1, water volume: recoveries decreased, probably due to decompositions of
5.0 mL, triton x-114 0.1% (v/v), 0.4 lmol L-1 la3?, ILs: 0.150 g
[Bmim]PF6, temperature: 50 °C, complexing time: 40 min, centrifu-
the TCs or increased formation of 4-epimers [38]. So,
gation time: 10 min 60 °C was chosen for heating step.

Effect of Extraction Time

solution determines the existing state of analytes, as well
determines the extraction recovery of target compounds. In
this experiment, in order to examine the effect of pH,
diluted hydrochloric acid was used to regulate acidity and
sodium hydroxide to regulate alkalinity [25]. The effect of
pH was studied in the range of 3.0–10.0. The results
illustrated in Fig. 5 reveal that the extraction efficiency of
target compounds. It can be seen that when the pH values
were in the range of 3–7, the recovery of all tetracycline
antibiotics increased. Whereas, when the pH value
increased from 7 to 10, the recovery decreased gradually.
A better extraction efficiency for all the target compounds
was observed at pH 7.0, it happens due to the fact that, in
weak acid solution (pH 3.0–6.0), the chemical structures of
tetracyclines would be isomerized to 4-epimers, while in
alkaline medium tetracyclines were known to be easily
oxidized [36]. In addition, La3? and tetracyclines can form
In this study, the extraction time is defined as the interval
time between adding the IL and starting to centrifuge. We
investigated the extraction time influence from 5.0 to
120.0 min with the optimized other operation parameters
described above. The extraction time has little influence on
the extraction recoveries in the experiment. This could be
explained that the formation of cloudy solution causes the
infinitely large interface of IL-phase and aqueous phase,
which results in the rapid transfer of analytes from aqueous
phase to IL-phase and equilibrium was established quickly
so that the extraction time is very short [12]. Thus, cen-
trifugation procedure could be done immediately after
forming milky solution, which is an important element in
dispersive liquid–liquid microextraction for rapid deter-
mination of pollutants. In our study, 5 min was selected for
further discussion.

123
1062 D. Hou et al.

Evaluation of Method Performance ETC, OTC, EATC and ATC, 100 lg L-1 for TC, ECTC,
CTC and DC, respectively (n = 5). The enrichment factor
Main Method Parameters was found to be 43.

Important parameters such as linear range, correlation
coefficient and detection limits for these analytes with
DLLME-UHPLC method were tested using spiked water
samples under the optimum conditions. Calibration curve
was obtained on a concentration range of 0.1–200 lg L-1
for all eight tetracycline antibiotics at six different con-
centration levels. The experiment results were shown in
Table 1. As can be seen, linearity was observed in the
range of 0.1–200 lg L-1. The correlation coefficients (r2)
were higher than 0.99, Limits of detection (LOD) and
limits of quantification (LOQ) were calculated with signal-
to-noise ratios of 3 and 10, respectively. LOD ranged from
0.031 to 0.079 lg L-1 and LOQ ranged from 0.10 to
0.26 lg L-1. The relative standard deviations varied
between 5.4 and 11.6% with the levels of 200 lg L-1 for
Real Water Samples Analysis
The efficiency of the proposed method was evaluated by
determining the concentrations of eight tetracyclines in
river water, fishpond water and leaching water of a pig
farm soil. River water was sampled from Qinghe, Beijing,
China. Fishpond water was sampled from Miyun Fish
Farm, Beijing, China. Leaching water of the pig farm soil
was sampled from a specialized Gaobeidian pig farm,
Beijing, China. All samples were filtered through a
0.22 lm filter membrane and stored at -20 °C before
being used, extracted using DLLME method and analyzed
by UPLC–TUV. As a result, CTC was detected with a
concentration of 2.14 lg L-1 and ECTC was detected with
a concentration of 0.09 lg L-1 (below LOD) in hog

Table 1 Main method parameters of RTILs-DLLME

Compounds r2 Linear range (lg L-1) RSD (%) (n = 5) LOD (lg L-1) LOQ (lg L-1)

4-epi-Tetracycline 0.9989 0.1–200 9.5 0.046 0.15

Oxytetracycline 0.9964 0.1–200 11.6 0.079 0.26
Tetracycline 0.9957 0.1–200 10.2 0.031 0.10
4-epi-Chlortetracycline 0.9929 0.1–200 11.1 0.066 0.22
Chlortetracycline 0.9951 0.1–200 9.1 0.039 0.13
Doxycycline 0.9982 0.1–200 9.6 0.043 0.14
4-epi-Anhydrotetracycline 0.9918 0.1–200 8.2 0.039 0.13
Anhydrotetracycline 0.9970 0.1–200 5.4 0.043 0.14
3? -1
Extraction conditions: sample volume, 5.0 mL, concentration of La : 0.4 lmol L , triton x-114 (v/v): 0.1%, complexing time: 40 min,
temperature: 60 °C, extraction time: 5 min; centrifugation time: 10 min; 0.150 g [Bmim]PF6
RSD relative standard deviation, LOD limits of detection, LOQ limits of quantitation

Table 2 Relative recoveries and standard deviations of eight TCs from spiked river water, fishpond water and hog leachate samples
Antibiotics River water Fishpond water Hog leachate
Found (lg L ) Recovery (%) RSD (%) Found (lg L ) Recovery (%) RSD (%) Found (lg L-1) Recovery (%) RSD (%)
-1 a -1

ETC ND 89.2 2.4 ND 76.8 13.2 ND 77.9 5.4

OTC ND 72.1 12 ND 58.9 5.7 ND 55.1 8.2
TC ND 62.6 3.8 ND 67.4 4.9 ND 79.4 9.7
ECTC ND 72.3 10 ND 94.5 3.9 BL 82.9 16
CTC ND 76.9 10 ND 61.5 9.1 2.14 60.2 9.6
DC ND 74.6 8.1 ND 75 8 ND 70.4 6.9
EATC ND 69.9 6.8 ND 58.9 11 ND 62.8 9.1
ATC ND 96.3 8.5 ND 69.6 7.4 ND 86.1 5.8
-1
Extraction conditions: as in Table 1, added amounts of tetracyclines 1.0 lg L
ND not detected, BL below LOD
a
n=5

123
Temperature-Induced Ionic Liquids Dispersive Liquid–Liquid Microextraction 1063

Table 3 Accuracy and

Antibiotics Spiked River water Fishpond water Hog leachate
precision of the method for TCs
level
in spiked river water, fishpond Recovery RSD Recovery RSD Recovery RSD
(lg L-1)
water and hog leachate (n = 5) (%) (%) (%) (%) (%) (%)

4-epi-Tetracycline 5 90.1 4.9 78.8 10.6 74.9 7.6

20 91.7 6.3 84.2 7.2 85.3 3.9
Oxytetracycline 5 72.2 10.6 61.4 7.3 60.7 11.4
20 78.8 9.3 64.9 4.8 69.1 6.8
Tetracycline 5 64.1 6.1 69.9 15.8 78.3 10.1
20 73.4 8.7 74.8 5.1 88.1 8.8
4-epi-Chlortetracycline 5 76.6 12.4 93.1 8.1 84.1 13.8
20 84.4 9.0 99.2 4.2 92.5 7.7
Chlortetracycline 5 80.9 5.2 65.0 9.6 67.2 8.9
20 88.3 6.7 74.4 8.1 81.7 7.5
Doxycycline 5 76.7 3.1 76.1 12.5 75.1 8.3
20 87.1 5.6 82.3 7.3 79.4 7.9
4-epi-Anhydrotetracycline 5 77.1 9.4 64.7 10.1 65.3 16.4
20 84.2 7.2 71.7 6.1 80.4 12.3
Anhydrotetracycline 5 95.6 14.1 70.3 9.6 88.1 9.1
20 103.1 9.6 79.5 5.4 92.6 6.3

leachate, and no target compounds were found in the other
water samples (see in Table 2). The concentration of ECTC
detected in leaching water of the pig farm soil was very
low, this was probably because the confirmation of CTC
changed and transformed into ECTC. To evaluate the
matrix effect, 12.5 lL 0.4 mg L-1 ETC., OTC, EATC and
ATC, and 25 lL 0.2 mg L-1 TC, ECTC, CTC and DC
were spiked to the water samples. As shown in Table 2, the
spiked recoveries and relative standard deviation (RSD)
were in the range of 55.1–96.3 and 2.4–15.6%, respec-
tively. Table 3 shows the recoveries of all analytes at
spiked level 5 and 20 lg L-1 were in the range of
60.7–103.1% with the RSD% less than 16.4%, which
indicated that IL-DLLME with UPLC–TUV was feasible
for the determination trace level of tetracyclines mentioned
above in aqueous samples.
Conclusion
This paper describes a confirmed mode RTILs-DLLME–
UHPLC method for the analysis of trace amounts of eight
tetracycline antibiotics in aqueous samples, this method
provides an approach to monitor the tetracycline antibiotics
in environmental water. In addition, the proposed method
has many practical advantages such as small sample vol-
ume, short sample preparation time, as well as only small
amounts of toxic organic solvents are needed, also
including high sensitivity and a convenient extraction
procedure, all of which indicates that RTILs-DLLME–
UHPLC is an attractive technique for the preconcentration
and rapid determination of tetracycline antibiotics men-
tioned above in aqueous samples. This method is also
probably capable of being expanded for the extraction and
separation of other pollutants in the aqueous environment.
Acknowledgments We gratefully acknowledge the financial sup-
port of this work by the National Basic Research Program of China
(20877092).
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