Study On The Metabolism Of β-agonists In Swine Using High Resolution Mass Spectrometry

β-Adrenergic agonists (P-AAs), used as bronchodilators in human and veterinary medicines, are also illegally used at high dose as growth promoters in food animal production. In view of the potential risk for consumers’ health, the use of β-AAs for growth-promoting purposes has been banned in many countries. In China, fourteen β-AAs have been has been prohibited from being used in animal feed and drinking water by the Ministry of Agriculture of China. However, no information about the metabolism and residue kinetics of banned β-AAs in livestock was available, except for clebuterol and ractopamine. In this study, in vivo metabolism of clenbuterol, salbutamol, clorprenaline and phenylethanolamine A were sutudied in swine, which will provide valuable data for establishing analytical method β-AAs residues, and contribute to more effective surveillance of PEAA abuse in livestocks.In this study, high resolution MS/MS spectras of nine β-AAs were obtained using ESI-Q-TOF MS, and element compositions and possible strutures of fragment ions were studied. Besides, fragmentaton positions and rearrangements were confirmed using deuterated analogues. Loss of H2O from P-OH group and elimination of N-alkyl were proposed as two primary fragmentation pathways of various β-AAs, corresponding to characteristics fragment ions of [M-H2O+H]+and [M+H-H2O-Alkyl]+. Informations povided in this study will be of help for method development and metabolite identicatin based on LC-MS/MS methods.After a single dose of10mg/kg b.w clenbuterol (CLE), eight metabolites were screened and identificated in swine urine collected0-24h after administration using ultra performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with MDF, EIC and IPF processing methods. The detected metabolites included4-N-hydroxylamine-CLE(4-N-OH-CLE),4-nitro-CLE(4-NO2-CLE) and several glucuronide acid conjugates(GLU-CLE and GLU-OH-CLE). In urine collected0-24h after administration, relatieve percentages of parent CLE and4-N-OH-CLE were69.9%and19.1%, respectively, suggesting that4-N-oxidization was the major metabolic pathway of CLE in swine.After a single dose of10mg/kg b.w salbutamol (SAL), ten metabolites were screened and identificated in swine urine collected0-24h after administration using UPLC-Q-TOF MS with MDF and EIC processing methods. The detected SAL metabolites included hydroxylated-SAL(OH-SAL), benzoic acid-SAL(COOH-SAL), methylated-SAL (CH3-OH-SAL) and several glucuronide acid conjugates (GLU-SAL and GLU-OH-SAL). Among those detcted metabolites, COOH-SAL and CH3-OH-SAL were found for the first time in animals. In urine collected0-24h after administration, relatieve percentages of parent SAL, GLU-SAL and COOH-SAL were44.3%,37.8%and16.6%, respectively, suggesting that glucuronidation and oxidation of benzyl alcohol were two major metabolic pathways of CLE in swine.After a single dose of10mg/kg b.w clorprenaline (CLO), nine metabolites were screened and identificated in swine urine collected0-24h after administration using UPLC-Q-TOF MS with MDF, IPF and EIC processing methods. The detected SAL metabolites included hydroxylated-CLO(OH-CLO), glucuronide acid conjugates (GLU-CLO and GLU-OH-CLO) and sulphate conjugates (SO3-CLO). Among those detcted metabolites, COOH-SAL and CH3-OH-SAL were found for the first time in animals. In urine collected0-24h after administration,the relatieve percentages of GLU-OH-CLO and GLU-CLO were53%and17.6%, respectively, suggesting that hydroxylation and glucuronidation of CLO were major metabolic pathways of CLO in swine.After a single dose of10mg/kg b.w phenylethanolamine A (PEAA), twenty-two metabolites were screened and identificated in swine urine, feces, muscle, liver, kindey and lung using UPLC-Q-TOF MS with MMDF and EIC processing methods. The primary metabolites of PEAA included demethylated-PEAA (DM-PEAA), hydroxylated-CLO (OH-PEAA), nitro reduction-PEAA (NH2-PEAA) and glucuronide acid conjugates (GLU-PEAA). Besides, glucuronide acid conjugates, sulphate conjugates and acetylatied products of its primary metabolites were also found in urine and various tissues. In urine, liver and kindey, phase II metabolites were more abundant, and GLU-DM-PEAA was the major metabolite PEAA. In feces, muscle and lung, DM-PEAA was the dominant metabolites. The result suggested that O-demethylation and glucuronidation were major metabolic pathways of PEAA in swine.In this study, quantitative analysis method for PEAA and DM-PEAA was developed using UPLC-Q-TOF MS in urine and various tissues, which were carefully validated in terms of specificity, sensitivity, linear ranges, accuracy and precision. With the validated UPLC-Q-TOF MS method, concentrations of PEAA and DM-PEAA in urine and various tissues after administration were determined without and with enzyme hydrolysis. The result showed that lung had the highest concentrations of PEAA and DM-PEAA of all tissues, indicating the potential accumutation effect on PEAA. In muscle and lung, the free PEAA and DM-PEAA were dominant forms. On the contrary, the conjugates were the primary forms of PEAA and DM-PEAA. Besides, DM-PEAA and its conjugates showed higher residual concentrations in swine, and were eliminated more slowly than than those of parent PEAA in urine. According to the results, DM-PEAA might be more appropriate potential marker in swine urine and tissues for monitoring the illicit use of PEAA.