Analytical

Methods
View Article Online
PAPER View Journal | View Issue
Published on 04 January 2013. Downloaded by University of California - Santa Cruz on 31/10/2014 12:47:32.

On-site solid phase extraction and HPLC determination

of chloramphenicol in surface water and sewage
Cite this: Anal. Methods, 2013, 5, 1150

Sheng Liu,*a Xian-Zheng Wu,†b Zi-Hui Gaocd and Fang Jiaod

Received 8th October 2012
Accepted 26th December 2012
DOI: 10.1039/c2ay26162f
www.rsc.org/methods
A mini-solid phase extractor (MSPE) was designed and applied in the on-site determination of trace levels
of chloramphenicol. The results showed that 1.5 g of the macroporous XAD-16 resin was suitable for
capturing chloramphenicol from 500 mL of solution. Methanol was used as the eluent to desorb
chloramphenicol from the resin. Chloramphenicol was determined by HPLC at 278 nm with a mobile
phase of 0.01 mol L
1 oxalic acid–acetonitrile (40 : 60 v/v), the retention time was around 5.07 min. The
detection scope of chloramphenicol was between 0.004 and 0.120 mg L
1. Using the MSPE,
chloramphenicol in surface water and hospital sewage was determined with recoveries of 96 to 108%
and RSDs of less than 6.8%. The MSPE achieved a rapid on-site preconcentration of chloramphenicol
and avoided the inconvenient transportation and storage of large volumes of water sample.

Introduction requirement for accurate, simpler, faster and improved analyt-

Antibiotics are widely used in animals for the treatment of
diseases and also as animal growth promoters. The use of anti-
biotics may lead to the presence of drug residues in animal-
derived foods and in water, the side effects of which would
threaten public health. Chloramphenicol, i.e. D(
)-threo-2-
dichloroacetamido-1-p-nitrophenyl-1,3-propanediol is a broad-
spectrum antibiotic, previously used in veterinary medicine.1
The adverse effects of this compound have led to its restricted use
in both human and veterinary medicine. Among other applica-
tions, this drug has been used for the treatment of childhood
meningitis. It is also a potent drug for typhoid fever. Chloram-
phenicol has been used in veterinary practice for the prevention
and treatment of many bacterial infections. However, toxic
effects in humans such as aplastic anemia have been described.
It has been banned for use in foodstuffs of animal origin in the
European Union.2,3 Chloramphenicol is still illegally used in
animal farming because of its easy access and low cost. Further,
in the EU, application of chloramphenicol to food production
has been prohibited since 1994. Moreover, the EU has dened a
minimum required performance limit of 0.3 mg kg
1 for chlor-
amphenicol in food of animal origin, because a safe level of
chloramphenicol dosage has yet to be identied. With growing
concerns over food safety and the need to increase sample-
throughput in analytical testing laboratories, there is a constant
a
Anhui Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Hefei
230001, China. E-mail: liusheng@yahoo.cn
b
Tongji Hospital, Shanghai 200065, China
ical methods. The complexity of matrices and the presence of
much potential interferences, require specic and selective
methods for analysis.
Various methods have been developed for determining
chloramphenicol, including immunoassays,4,5 microbiological
methods,6 sensors,7 molecularly imprinted polymer-based
methods8 and chromatographic methods i.e. GC–MS,9 LC–
MS10–12 and capillary zone electrophoresis.13 However, most of
these methods need expensive apparatus and reagents and they
are time-consuming. Many methods are focused on the deter-
mination of chloramphenicol in food but few on its determi-
nation in water. The residues of chloramphenicol have an
important inuence on the safety of seafood, so the determi-
nation of chloramphenicol in water is necessary, especially in
aquaculture water-bodies. It is therefore important to develop
accurate techniques to quantify chloramphenicol in water. Due
to the low concentration in environmental samples and the
complexity of matrices, it is usually difficult to detect chlor-
amphenicol directly. Two principal sample pretreatment
methods developed for the determination of chloramphenicol
are liquid–liquid extraction (LLE)14 and solid-phase extraction
(SPE).15 However, LLE usually requires some poisonous volatile
organic solvents. SPE is an important method with good puri-
cation and concentration effect. During the years, it has
evolved to meet such requirements because of several advan-
tages e.g. exibility, low cost, simplicity and ease of automation.
The traditional SPE device has some disadvantages. For
example, a vacuum pump is essential so that it is used only in
the laboratory. In addition, water must be kept at a very low ow

rate to pass through an SPE column lled with sorbent. A

c
College of Politics and Economics, Kyung Hee University, Seoul, 130-701, Korea
d
Shanghai Green Empire Environmental Protection Science & Technology Corporation,
portable SPE device consisting of the sorption, separation and
Shanghai 200092, China
elution functional units has been used to preconcentrate heavy
† Share the rst author.

1150 | Anal. Methods, 2013, 5, 1150–1154 This journal is ª The Royal Society of Chemistry 2013

Paper
metals ions from aqueous solution with satisfactory results.16 In
this work, the portable SPE device was further miniaturized, i.e.
a mini-solid phase extractor (MSPE) was created for on-site
preconcentration of organic compounds at trace level in water.
Chloramphenicol existing in three water samples was pre-
concentrated with the MSPE device and Amberlite XAD-16
macroporous resin as sorbent and was then determined
Published on 04 January 2013. Downloaded by University of California - Santa Cruz on 31/10/2014 12:47:32.
by HPLC.
Experimental
Instruments and materials
The MSPE device was designed (Fig. 1A), assembled (volume –
15  15  12 cm3, weight – only 1.5 kg, Model GE MSPE-02,
Shanghai Green Empire Environmental Protection S&T Co.,
China) (Fig. 1B) and employed for on-site preconcentration of
chloramphenicol. An HPLC instrument (Model L-2000, Hitachi,
Japan) was used with a chromatographic column (Model All-
sphere ODS-2 5 mm, Length 250 mm) for determination of
chloramphenicol.
A chloramphenicol (Aladdin Reagents Co., China) stock
solution was prepared with methanol (Chromatographic Grade,
Fig. 1 Design of the MSPE device. (A) The function units, and (B) cartoon illus-
tration of the device-working. (a) Magnetic stirrer, (b) sample flask, (c) flow-
guiding pipe, (d) SPE column, (e) wriggle pump, (f) mini-ultrasonic transducer, (g)
reservoir bottle, and (h) detector. (1) Water sample, (2) the XAD-16 resin, (3) the
resin adsorbing chloramphenicol, (4) raw eluent i.e. methanol, and (5) eluent
containing chloramphenicol.
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Analytical Methods
Aladdin Reagents), and further diluted for use. Amberlite
XAD-16 macroporous resin (Shanghai Mosu Science Equip-
ments, China) was used to capture chloramphenicol from
aqueous media. Methanol was used to elute chloramphenicol
from the XAD-16 resin. Oxalic acid and acetonitrile (Chro-
matographic Grade, Aladdin Reagents) were used as the mobile
phase. The other chemical reagents were GR grade.
Preparation of sorbent
Amberlite XAD-16 macroporous resin was selected as the
sorbent in this work. Prior to use, it was immersed in acetone
for 4 h, then distilled water was used to wash the resin to pH 7.
Then 2 mol L
1 HCl and 2 mol L
1 NaOH solutions were used to
immerse the resin for 2 h, respectively and distilled water was
used to wash the resin to pH 7.
Preconcentration and determination of chloramphenicol
According to Fig. 1, 1.5 g of the XAD-16 resin (2) was added into
a water sample (1) containing chloramphenicol. The liquid in
the ask (b) was stirred (a) for 20 min and then pumped (e)
through a ow-guiding pipe (c) with an empty column (d). The
resin with chloramphenicol (3) was separated from the liquid.
The resin (3) was washed with 5.0 mL of eluent (4) for 1 min with
an ultrasonic unit (f). The eluent (5) was pushed into a 5 mL
glass bottle (g) with an injector and chloramphenicol in the
eluent was determined by HPLC with detection at 278 nm (h)
the retention time was around 5.07 min . The mobile phase
consisted of both 0.01 mol L
1 oxalic acid and acetonitrile
(40 : 60 v/v) and the ow rate through the chromatographic
column was 1.0 mL min
1. The injection volume was 20 mL.
Application to on-site water monitoring
Chloramphenicol in three water samples (Taihu Lake, The
Beijing-Hangzhou Grand Canal and hospital sewage) was
captured and eluted using the MSPE device and determined by
HPLC according to the above procedures.
Results and discussion
Design of the MSPE device
An MSPE device was designed, which consisted of the sorption
of chloramphenicol, separation of sorbent and desorption of
chloramphenicol (Fig. 1). Being different from the traditional
SPE, it is favourable for on-site preconcentration of chloram-
phenicol in aqueous media.
Effect of sorbent
A known amount of XAD-16 resin from 0.5 to 3 g was added into
500 mL of solution containing 0.100 mg L
1 chloramphenicol.
Aer adsorption and elution, the recovery of chloramphenicol
was calculated and the results are shown in Fig. 2A. The
recovery of chloramphenicol increased to 91% from 31% with
an increase in the amount of XAD-16 resin. Thus, 1.5 g of XAD-
16 resin was added and the recovery approached a constant
maximum.
Anal. Methods, 2013, 5, 1150–1154 | 1151

Analytical Methods
Published on 04 January 2013. Downloaded by University of California - Santa Cruz on 31/10/2014 12:47:32.
Fig. 2 Effects of the experimental conditions on the recovery of 0.100 mg L
1 chloramphenicol. (A) Addition of XAD-16 resin, (B) the sorption time, (C) the solution
volume and (D) the elution time of XAD-16 resin.
Effect of the sorption time
1.5 g of XAD-16 resin was mixed into 500 mL of solution con-
taining 0.100 mg L
1 chloramphenicol. The effect of the sorp-
tion time from 5 to 30 min was investigated. From Fig. 2B it can
be seen that the recovery of chloramphenicol increased with an
increase in time in the rst 20 min and then approached a
constant value.
Effect of solution volume
The effect of the solution volume from 200 to 1000 mL on
adsorption of chloramphenicol was investigated. The recovery
of chloramphenicol decreased to 89.1% from 93.3% with an
increase in the water volume from 200 to 1000 mL (Fig. 2C).
Effect of the ultrasonic elution time
1.5 g of XAD-16 resin was mixed into 500 mL of the solution
containing 0.100 mg L
1 chloramphenicol. The effect of the
ultrasonic elution time from 0.5 to 3 min was investigated. The
recovery of chloramphenicol increased in the rst 1 min and
then decreased (Fig. 2D). The possible reason is that the long
1152 | Anal. Methods, 2013, 5, 1150–1154
View Article Online
Paper
ultrasonic elution time caused the decomposition of chloram-
phenicol. Only 1 min was selected in this work.
Calibration graph
According to the above optimized conditions, 1.5 g of XAD-16
resin was added to 500 mL of the standard series of chloram-
phenicol from 0 to 0.12 mg L
1. Aer preconcentration, chlor-
amphenicol was determined by HPLC, the retention time was
around 5.07 min (Fig. 3A) and the detection wavelength was
278 nm (Fig. 3C). The good linearity (Fig. 3B) indicated that the
recommended method is suitable for the preconcentration and
determination of chloramphenicol in aqueous media. The
detection limit (3s) of chloramphenicol was calculated to be
2 mg per 500 mL, i.e. 0.004 mg L
1.
Application to water monitoring
The MSPE device was used for on-site monitoring of Taihu Lake,
The Beijing-Hangzhou Grand Canal and hospital sewage.
According to the recommended method, chloramphenicol in
these water samples was determined as given in Table 1. The
This journal is ª The Royal Society of Chemistry 2013
 View Article Online

Paper Analytical Methods

Published on 04 January 2013. Downloaded by University of California - Santa Cruz on 31/10/2014 12:47:32.

Fig. 3 Separation and determination of chloramphenicol by HPLC. (A) Chromatograph curves of the Taihu Lake water eluent (1) and its chloramphenicol-adding
sample eluent (2), detected at 278 nm, (B) calibration graph, i.e. plots of the peak area around 5.07 min vs. chloramphenicol concentration and (C) absorption spectra of
the eluent (1 and 2) at 5.07 min.

recovery of chloramphenicol was between 96 and 108% and the Conclusions

RSDs were calculated to be 0.4% for the Taihu Lake sample and
6.8% for the hospital sewage sample.
From Table 1 it can be seen that over 0.1 mg
L
1chloramphenicol was detected in Taihu Lake. In addition,
the determination of chloramphenicol in Taihu Lake was found
to be 0.117 mg L
1 by a conventional GC–MS method,17 which
was in accordance with the recommended method. The
possible reasons are that lots of tourists oen gather around the
scenic region and waterfowls are stocked. In the hospital
sewage, more than 2 mg L
1 chloramphenicol was found. This
indicates that the hospital is a major emission source of
antibiotics.
In recent years, chloramphenicol has been detected frequently
in natural water18 owing to the abuse of antibiotics. The toxicity
and health risk of antibiotic drugs has begun to raise
concerns.19 Regarding the determination of ultra-trace amounts
of harmful substances in aqueous media, some new sample
preparation methods have increasingly been developed e.g.
magnetic separation,20 microextraction,21 membrane extrac-
tion22 and so on. Nonetheless, this work provided an easy-to-
use, light and handy on-site preconcentration device for
harmful substances in trace levels. In addition, experiments
show that the MSPE device is rapid, effective and avoids the

Table 1 Determination of chloramphenicol in water samples (n ¼ 3)

Chloramphenicol added Chloramphenicol Chloramphenicol

Sampled from into 500 mL of sample/mg found/mg Recovery (%) in sample/mg L
1

The B-H Grand Canal 0 <2 98.5–99 <0.004

40 39.4–39.6
Taihu Lake 0 63.0–63.2 99.8–105 0.126
60 123–126
Hospital sewagea 0 930–1057 96.0–108 2.05
1000 1985–2107
a
25 mL of the sewage was diluted to 500 mL with distilled water.

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 1150–1154 | 1153

Analytical Methods
inconvenient transportation and storage of a large volume of
water sample. Therefore, it is particularly useful for water
monitoring in mountains, tableland and other remote areas.
Notes and references
1 K. N. Woodward and D. H. Watson. Pesticide, Veterinary and
Published on 04 January 2013. Downloaded by University of California - Santa Cruz on 31/10/2014 12:47:32.
Other Residues in Food, Woodward Published Limited,
Cambridge, 2004, vol. 8.
2 R. Council, Off. J. Eur. Communities: Legis., 1994, 6.
3 R. L. Epstein, C. Henry, K. P. Holland and J. Dreas, J. AOAC
Int., 1994, 77, 570.
4 P. Jarujamrus, R. Chawengkirttikul, J. Shiowatana and
A. Siripinyanond, J. Anal. At. Spectrom., 2012, 27, 884.
5 G. Scortichini, L. Annunziata, M. N. Haouet, F. Benedetti,
I. Krusteva and R. Galarini, Anal. Chim. Acta, 2005, 535, 43.
6 C. J. Singer and S. E. Katz, J. AOAC Int., 1985, 68, 1037.
7 J. Ferguson, A. Baxter, P. Young, G. Kennedy, C. Elliott,
S. Weigel, R. Gatermann, H. Ashwin, S. Stead and
M. Sharman, Anal. Chim. Acta, 2005, 529, 109.
8 C. Zhou, T. Wang, J. Liu, C. Guo, Y. Peng, J. Bai, M. Liu,
J. Dong, N. Gao, B. Ning and Z. Gao, Analyst, 2012, 137, 4469.
9 A. Posyniak, J. Zmudzki and J. Niedzielska, Anal. Chim. Acta,
2003, 483, 307.
10 P. Mottier, V. Parisod, E. Gremaud, P. A. Guy and
R. H. Stadler, J. Chromatogr., A, 2003, 994, 75.
1154 | Anal. Methods, 2013, 5, 1150–1154
View Article Online
Paper
11 M. J. Bogusz, H. Hassan, E. Al-Enazi, Z. Ibrahim and
M. Al-Tufail, J. Chromatogr., B: Anal. Technol. Biomed.
Life Sci., 2004, 807, 343.
12 U. R. Tjaden, D. S. Stegehuis, B. J. E. M. Reeuwijk,
H. Lingeman and J. van der Greef, Analyst, 1988, 113, 171.
13 W. R. Jin, X. Y. Ye, D. Q. Yu and Q. Dong, J. Chromatogr.,
Biomed. Appl., 2000, 741, 155.
14 M. H. Akhtar, C. Danis, A. Sauve and C. Barray, J. Chromatogr.,
A, 1995, 696, 123.
15 H. Kubala-Drincic, D. Bazulic, J. Sapunar-Postruznik,
M. Grubelic and G. Stuhne, J. Agric. Food Chem., 2003, 51,
871.
16 F. Jiao and H. W. Gao, Environ. Monit. Assess., 2013, 185, 39.
17 L. A. van Ginkel, H. J. van Rossum, P. W. Zoontjes, H. van
Blitterswijk, G. Ellen, E. van der Hee, A. P. J. M. de Jong
and G. Zomer, Anal. Chim. Acta, 1990, 237, 61.
18 H. Liu, G. Zhang, C. Q. Liu, L. Li and M. Xiang, J. Environ.
Monit., 2009, 11, 1199.
19 C. Song, N. Y. Gao and H. W. Gao, Mol. BioSyst., 2010, 6,
1901.
20 Y. Cui, J. Q. Liu, Z. J. Hu, X. W. Xu and H. W. Gao, Anal.
Methods, 2012, 4, 3095.
21 M. Cruz-Vera, R. Lucena, S. Cárdenas and M. Valcárcel, Anal.
Methods, 2011, 3, 1719.
22 M. Kaykhaii, A. Sarafraz-Yazdi, M. Chamsaz and J. Pawliszyn,
Analyst, 2002, 127, 912.
This journal is ª The Royal Society of Chemistry 2013