Analytical MethodsSimultaneous determination of mequindox, quinocetone, and their major metabolites in chicken and pork by UPLC–MS/MS
Introduction
Quinoxaline-1,4-dioxides (QdNOs) are synthetic antibacterial agents that are used in animal husbandry because of their high antimicrobial activity (Carta, Corona, & Loriga, 2005). Over the past few decades, some QdNO derivatives have been widely used as feed additives in swine and chickens for their growth-promoting effects (Wu et al., 2009). Mequindox (MEQ) and quinocetone (QCT) are new well known members of this class of compounds (Ihsan et al., 2010), and they can significantly improve growth and prevent bacterial disease in swine. However, reports of these compounds causing toxicity, including photoallergy, mutagenicity and carcinogenicity have appeared (Chen et al., 2009, Ihsan et al., 2013, Jin et al., 2009, Yang et al., 2013). Consequently, close attention should be paid to the safety of QdNOs used in animal husbandry.
To the best of our knowledge, the majority of pharmacokinetic studies concerning veterinary drugs have focused on their metabolites. To investigate the safety of MEQ and QCT, in vivo and in vitro metabolism studies of both compounds were conducted and numerous metabolites of MEQ and QCT were identified. These metabolites may also be associated with precursor toxicity (Liu et al., 2010, Liu et al., 2010, Shen et al., 2010). Certain metabolites of the QdNOs, especially their desoxy and reduction compounds, may cause severe toxicity (Huang et al., 2005, Zhang and Huang, 2005). In the metabolism studies cited above, there were totally 11 major metabolites identified which covered the vast majority of MEQ and QCT metabolites. To adequately monitor these drugs for food safety, it is necessary to develop a sensitive and accurate analytical method for the simultaneous determination of both the precursors as well as their metabolites.
Recently, many analytical techniques have been used to analyze for the QdNO precursor compounds, such as high-performance liquid chromatography coupled with detection by ultraviolet light (HPLC–UV) (Huang et al., 2008, Zhang et al., 2012) and by mass spectrometry (LC–MS/MS) (Wu et al., 2009). However, few methods have been developed to analyze the metabolites of the QdNOs (Ding et al., 2012, Huang et al., 2005, Li et al., 2012, Li et al., 2012, Zeng et al., 2012). These developed methods mainly focused on only a single precursor drug. There are currently no detection methods capable of analyzing for multi-precursor drugs along with their metabolites.
In this work, an analytical method for the 2 precursor drugs, MEQ and QCT, and their 11 metabolites was developed (structures are shown in Fig. 1). A novel method for simultaneous determination of these 13 compounds in chicken and pork tissues is presented, which uses SPE C18 cartridges for purification and UPLC–MS/MS for detection. Based on the literature, this study is the first to simultaneously monitor MEQ and QCT as well as their major metabolites. Currently, the residue marker metabolite(s) for MEQ and QCT have not been determined. Therefore, prior to the investigations of residue marker metabolites for these compounds, the method of detection presented here will allow the control of MEQ and QCT along with their major metabolites in food animal products and facilitate further pharmacokinetic and residue studies of similar QdNO veterinary drugs.
Section snippets
Reagents and materials
HPLC grade methanol and acetonitrile were purchased from Dima Technology Inc. (Muskegon, MI, USA). Formic acid (HPLC grade) was obtained from Fisher Scientific Inc. (Pittsburgh, PA, USA). Water was purified using a Milli-Q Synthesis system from Millipore (Bedford, MA, USA). Bond Elut C18 cartridges (500 mg, 6 cc) were purchased from Agilent Technologies (Santa Rosa, CA, USA). Ethyl acetate, metaphosphoric acid, Dimethyl sulfoxide (DMSO) and other reagents were purchased from Sinopharm Chemical
Optimisation of extraction procedure
The extraction procedure is one of the most crucial steps in the sample preparation process that can impact obtaining good recovery results. Based on the structure of each analyte, the physicochemical properties including polarity, solubility, and the pKa values would be different, which makes it difficult to extract all the analytes simultaneously. Furthermore, the best extraction for some of the compounds from animal tissues requires acidolysis, alkaline hydrolysis, or enzymolysis (Zhang,
Conclusion
This research describes the development and validation of an UPLC–MS/MS method for the simultaneous determination of 13 compounds (MEQ, QCT and their 11 metabolites) in chicken and pork samples. The developed method was validated and the data demonstrated good accuracy and precision and achieved low LODs. To our knowledge, most metabolites were quantified here for the first time. In the application of this method to real samples, 16.7% of the samples were determined with trace amounts of MQCA.
Acknowledgement
This work was financially supported by the National Basic Research Program of China (973 program; Grant No. 2009CB118801).
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2021, Electrochimica ActaCitation Excerpt :Quinoxaline-1,4-dioxides (QdNOs), a class of synthetic antibacterial veterinary drugs with strong antibacterial activity, are widely used as feed additives in terrestrial animal husbandry, including poultry and domestic animals [1-3].
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Yanshen Li and Kaili Liu contributed equally.