SHORT COMMUNICATION
Simultaneous Determination of Zoalene and Its Metabolite
in Chicken Muscle and Liver by SPE and UPLC–MS-MS
Yin-Liang Wu
•
Yong Xu
•
Ting Yang
•
Wei-Guo Huang-Fu
Received: 27 May 2011 / Revised: 21 August 2011 / Accepted: 22 August 2011 / Published online: 10 September 2011
Ó Springer-Verlag 2011
Abstract This paper presents an analytical method for
the simultaneous determination of zoalene and its meta-
bolite 3-amino-5-nitro-o-toluamide (3-ANOT) in chicken
muscle and liver by solid phase extraction and UPLC–MS-
MS operated in the positive and negative ionization
switching mode. Samples were extracted with phosphate
buffer solution and purified with OASIS
TM
HLB cartridge
after pH adjustment. The determination was carried out
using UPLC–MS-MS on a Waters Acquity BEH C
18
column with 0.1% formic acid in water/acetonitrile as
mobile phase with gradient elution. The linearity of the
analytical response across the studied range of concentra-
tions (2.0–1,000 lgL
-1
) was excellent, obtaining corre-
lation coefficients higher than 0.999. Matrix effects had
been investigated for zoalene and 3-ANOT. Recovery
studies were carried out on spiked chicken muscle and liver
blank samples, at four concentration levels (50, 1,500,
3,000, and 4,500 lgkg
-1
for chicken muscle and 50,
3,000, 6,000, and 9,000 lgkg
-1
for chicken liver) per-
forming six replicates at each level. Mean recoveries of
77.9–94.2% with CVs of 3.2–8.7% were obtained. The
method demonstrated to be suitable for the simultaneous
determination of zoalene and 3-ANOT in chicken tissues.
Keywords Zoalene Á 3-ANOT Á Chicken tissues Á
Column liquid chromatography–electrospray tandem mass
spectrometry Á Solid phase extraction
Introduction
Zoalene (3,5-dinitro-o-toluamide) is a poultry feed addi-
tive, widely used to prevent coccidiosis infections due to its
advantages, such as high effect, low toxicity, stability, and
less drug resistance. Despite its relatively low toxicity, the
maximum residue limits (MRLs) of 3,000 lgkg
-1
in
chicken muscle, 6,000 lgkg
-1
in liver and kidney, and
2,000 lgkg
-1
in fat for zoalene and its metabolite (3-
amino-5-nitro-o-toluamide, 3-ANOT) have been estab-
lished by many countries to protect consumers [1, 2].
Therefore, specific and sensitive methods for the identifi-
cation and quantification of zoalene and 3-ANOT in
chicken tissues are required.
To determine zoalene and its metabolite in biological
samples, only few methods using thin layer chromatogra-
phy [3], spectrophotometry [4, 5], and LC [6–9] have been
developed. Moreover, most of these methods cannot be
used for the simultaneous determination of zoalene and
3-ANOT. Among LC methods, only the method developed
by Parks and Doerr [6] can be used for simultaneous
determination of zoalene and 3-ANOT. It is well known
that LC–MS-MS has more widespread application in resi-
due analysis due to its shorter chromatographic run time
and higher sensitivity in the past 10 years. Recently, a LC–
MS-MS method operated in negative ionization mode had
been developed for determination of zoalene residue in
foodstuffs by Liu et al. [10]. It is regrettable that the
method can not be used for determination of 3-ANOT
residues in foodstuffs. Up to now, a method for the
simultaneous determination of zoalene and 3-ANOT in
chicken tissues by LC–MS-MS has not been reported yet.
Therefore, the purpose of this study was to develop a
simple and sensitive LC–MS-MS method for simultaneous
determination of zoalene and 3-ANOT in chicken tissues.
Y.-L. Wu (&) Á T. Yang Á W.-G. Huang-Fu
The Ningbo Academy of Agricultural Sciences,
Ningbo 315040, People’s Republic of China
e-mail: wupaddyfield@tom.com
Y. Xu
Institute for the Control of Agro-chemicals of Zhejiang Province,
Hangzhou 310020, People’s Republic of China
123
Chromatographia (2011) 74:833–838
DOI 10.1007/s10337-011-2142-z