RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 3487–3496 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3236 Multi-class confirmatory method for analyzing trace levels of tetracyline and quinolone antibiotics in pig tissues by ultra-performance liquid chromatography coupled with tandem mass spectrometry Bing Shao1,4*, Xiaofei Jia1, Yongning Wu2, Jianying Hu3, Xiaoming Tu1 and Jing Zhang1 1Beijing Center for Disease Control and Prevention, Beijing 100013, China 2Institute of Nutrition and Food Safety, China Center for Disease Control and Prevention, Beijing 100085, China 3College of Environmental Science, Peking University, Beijing 100871, China 4School of Public Health and Family Medicine, Capital Medical University, Beijing 100089, China Received 8 June 2007; Revised 26 August 2007; Accepted 27 August 2007 An ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/ MS) method was developed to screen and confirm multi-class veterinary drug residues in pig tissues including pig kidney, liver and meat. Twenty-one drugs of two different classes including seven tetracyclines and four types of quinolones (quinoline, naphthyridine, pyridopyrimidine and cino- line) were determined simultaneously in a single run. The homogenized sample tissues were extracted with EDTA–McIlvaine buffer solution and further purified using a polymer-based Oasis TM HLB solid-phase extraction (SPE) cartridge. An ACQUITY UPLC BEH C18 column was used to separate the analytes followed by tandem mass spectrometry using an electrospray ionization source. MS data acquisition was performed in the positive ion multiple reaction monitoring mode, selecting two ion transitions for each target compound. Recovery studies were performed at different fortification levels. The overall average recoveries from pig muscle, kidney, and liver fortified with quinolones and tetracyclines at three levels ranged from 80.2 to 117.8% based on the use of matrix-fortified calibration with the coefficients of variation ranging from 2.1 to 17.8% (n ¼ 6). The limits of quantitation (LOQs) of quinolones and tetracyclines in different tissues ranged from 0.03–4.50 mg/kg and 0.16–10.00 mg/kg, respectively. The effects of the extraction solvent, SPE cartridge, elution solvent and sample matrix on the analyte recovery as well as the effects of the mobile phase composition and column temperature on the chromatographic behavior were also studied. Copyright # 2007 John Wiley & Sons, Ltd. In modern animal husbandry, animals are reared in into the environment per year in China.8 To protect restricted accommodations, which inevitably increase the populations from health risks, food safety regulatory incidence and spread of diseases. Hence, there has been an agencies in many countries, including China, have estab- increasing need for therapeutic agents to safeguard animal lished maximum residue limits (MRLs) for the residues of welfare and the economic benefits of animal husbandry. tetracylines and quinolones9–11 in foods of animal origin and Tetracycline and quinolone antibiotics are the two classes of restricted their use in treating both animals and humans. veterinary drugs that are widely used for the prevention or Previous analytical methods for screening and confirming treatment of bacterial infections in various animal species. tetracyclines and quinolones in foods of animal origin However, abuse from failure to adhere to prescribed dosages include liquid chromatography coupled with UV12–14 or and withdrawal periods may present potential health risks1–3 fluorescence detection15-18 and the liquid chromatography/ and a threat to the eco-environment.4–6 It is estimated that the mass spectrometry (LC/MS)19–22 technique, which were veterinary usage of quinolones is about 500 tons per year in developed only for surveillance at the MRL levels. According China.7 Although there are no accurate data on the veterinary to the European Commission Decision 2002/657/EC,23 the usage of tetracyclines, it is estimated that one hoggery with reported method validations were also at 0.5, 1 and 2 times 10 000 swine may discharge 300–500 kg chlorotetracycline the MRL levels, which were often higher than the true levels. However, the effects of long-term exposure to low levels of *Correspondence to: B. Shao, Beijing Center for Disease Control residues below the MRLs are still unknown. On the other and Prevention, Beijing 100013, China. 23 E-mail: [email protected] hand, the European Commission Decision 2002/657/EC Contract/grant sponsor: Beijing Natural Science Foundation; contract/grant number: 7041004. Contract/grant sponsor: National Natural Science Foundation; contract/grant number: 20607004. Copyright # 2007 John Wiley & Sons, Ltd. 3488 B. Shao et al. states that, ‘‘Methods based only on chromatographic methanol and quinolones were first dissolved in 50 mL analysis without the use of molecular spectrometric detec- formic acid and then diluted to 10 mL with methanol. tion are not suitable for use as confirmatory methods’’. Different volumes of each stock standard solution (200 mLof Therefore, analytical methods using mass spectrometric pipemdilic acid, norfloxacin, pefloxacin, sarafloxacin, 250 mL technology for screening and confirming trace levels of of ciprofloxacin, 100 mL of other quinolones, 600 mL antibiotic residues in foods of animal origin are necessary for of chlortetracycline, doxycylines, 900 mL of minocycline and protecting the long-term health of consumers. 300 mL of other tertracyclines) were measured precisely and After several years of research, especially with the put in the same 10-mL black volumetric flask according to application of ultra-performance liquid chromatography their LC/MS/MS response, diluted with methanol to 10 mL, (UPLC), column materials, and the acquisition and transition and mixed to homogeneity as middle stock solution. The six speed of mass spectrometry, the analytical throughput has levels of fortification solutions were prepared by serially been remarkably enhanced, making it possible to monitor diluting the middle stock solution 1, 2, 5, 10, 20 and 40 times more than 50 analytes in a single run.24 The major benefit with methanol. These solutions were stored at À188C in the from the use of UPLC and the 1.7 mm particles column is the dark. increased column efficiency that results in narrow peaks and an improved separation. Presently, the multi-residue Reagents methods have become the prevailing techniques for the All solvents used in sample preparation and chromato- analysis of veterinary drug residues in foods of animal graphic separations were HPLC grade. Methanol (MeOH) origins, such as sulfonamides,25 tetracyclines,19,20 quino- and acetonitrile (ACN) were supplied by Fisher Scientific lones,21,22 and hormones.26 To the best of our knowledge, (Fair Lawn, NJ, USA). Formic acid (HCOOH, 99%) was from most of these methods aim at one class of drugs for each Acros Organics (Morris Plains, NJ, USA). Ultra-pure water method. Compared to single-class residue detection was obtained using a Milli-Q Ultrapure system (Millipore, methods, the multi-class methods are relatively scarce Bedford, MA, USA). Ethylenediaminetetraacetic acid dis- because of the difficulties in simultaneous extraction and odium salt (Na2EDTAÁH2O), disodium hydrogen phosphate purification of compounds with different physicochemical (Na2HPO4Á12H2O) and citric acid (C6H8O7ÁH2O) were all properties. Nevertheless, a few multi-residue and multi-class from Beijing Chemical Co. (Beijing, China). methods have been successfully developed for analysis of veterinary drug residues in foods of animal origin.27–30 Li Sample preparation et al. established a multi-class method to monitor veterinary The frozen samples were naturally thawed at room drug residues in shrimp.27 Heller et al. used hydrophilic temperature. Fifty pig tissues were minced and mixed. solid-phase extraction (SPE) cleanup followed by LC/MS/ Aliquots of 5 g sample were weighed and transferred into a MS to detect a variety of polar drug residues in eggs at levels 50-mL polypropylene centrifuge tube. Then 20 mL higher than 10 mg/kg.28 Granelli et al. used LC/MS/MS to EDTA-McIlvaine buffer31 was added, and the specimens screen nineteen analytes from five classes of antibiotics.29 and solution were homogenized for 2 min using a Pro 300A Yamada et al. developed a method to detect 130 analytes of homogenizer (Proscientific, Inc., Monroe, CT, USA). The most classes of antibiotics and growth promoters except mixture was centrifuged at 10 000 rpm for 10 min at 08C. The tetracycline antibiotics.30 supernatant was decanted into a conical flask. The residues In this study, a comprehensive sample preparation were vortexed and extracted with 20 mL EDTA-McIlvaine procedure and UPLC/MS/MS method was developed to buffer solution for 5 min and then with another 10 mL simultaneously monitor the wide range polarity of 21 drugs EDTA-McIlvaine buffer. The supernatants were pooled and of two different classes including seven tetracyclines and subjected to SPE. four types of quinolones (quinoline, naphthyridine, pyrido- An Oasis HLB SPE cartridge (200 mg, 6 mL; Waters Corp., pyrimidine, and cinoline) in meat, liver, and kidney tissues. Milford, MA, USA) was sequentially preconditioned with Compounds with different polarities were extracted, con- 6 mL methanol, 6 mL water and 6 mL EDTA-McIlvaine centrated, and purified using the same method. Mobile phase buffer solution. The extract was applied