Metabonomic Approach Based On Ultra-high Performance Liquid Chromatography-quadrupole Time-of-flight Mass Spectrometry For Urine Profiling Of Swine Treated With β₂-agonists

β2-agonists are used illegally for their ability to improve growth rate and reduce carcass fat when fed to farm animals. However, the residues of β2-agonists in animal tissues would be harmful to consumers’health. The illegal use of β2-agonists in livestock production has been banned in China and was previously detected by efficient methods based on mass spectrometry to control the residues of these drugs. Nevertheless, such methods still remain a challenging task for authorities because the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention of illegal use of growth-promoting agents. Metabonomics based on mass spectrometry has emerged as a promising technology in the quantitative and qualitative analysis of large-scale small-molecular metabolites in complex biological samples. The objective of this study were to build a metabonomic approach for urine profiling of swine treated with β2-agonists and develope a novel method for the determination of β2-agonists in swine urine.Firstly, Metabolomics analysis on Wistar rats treated clenbuterol was performed using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Wistar rats were randomly assigned to four group:the low dose group (2mg/kg-bw), the middle dose group (5mg/kg-bw), the high dose group (10mg/kg-bw), and control group. The urine was collected at different time points and was analyzed by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Data were processed by MarkerLynx XS software (Waters). The results showed the changes of metabolite in the rat urine of the low or middle dose group was not significant, and potential biomarker was found in the rat urine of the high dose group. In this study, untargeted metabonomics based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry has been developed for the analysis of rat urine treated with clenbuterol.Then, a metabonomics-based strategy for detecting the use of three β2-agonists via urine profiling was outlined. Twelve female and twelve male swine were divided into four groups randomly (n=6) and orally administered clenbuterol, salbutamol, ractopamine, and "cocktails" consisting of the above three drugs, respectively. The dosage of single treated group was10mg/kg. The dosage of cocktail-treated group was3mg/kg of clenbuterol,3mg/kg of salbutamol, and3mg/kg of ractopamine. After28days drug administration, urine was daily collected and the urine collected24h prior to treatment was used as control. Urine profiles of controls and swine treated with single clenbuterol, salbutamol, and ractopamine and mixture of low amounts of three above compounds were analyzed by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The metabolic differences between controls and β2-agonists-treated groups were compared using multivariate data analysis including a non-supervised approach of principal components analysis (PCA) and an orthogonal partial least-squares to latent-structures discriminant-analysis (OPLS-DA). The ions selection criteria were as follows:(1) variable importance plots (VIP) value>5;(2) P-value<0.05;(3) fold change>1.5;(4) intensity>1,000. The ions selected was then submitted for online database searching, such as with Chemspider, KEGG, MassBank, Human Metabolome Database (HMDB), and METLIN. Fourteen metabolites were identified related with the β2-agonists treatment, while two co-biomarkers,2-indolecarboxylic acid and fluorometholone acetate, either in single or "cocktails" of low-dose mixture of clenbuterol, salbutamol, and ractopamine, were identified with their standards by UHPLC-QTOF/MS analysis and could be considered as diagnostic markers for the detection of illegal use of β2-agonists. The results of depletion study demonstrated that it is practical to use the markers for monitoring of β2-agonists.Finally, the proposed method for the two biomarkers was optimized and Oasis MAX solid phase extraction cartridge was selected for two biomarkers, provieding technical support for monitoring residur of β2-agonists in urine. A total of52swine urine samples collected from different origins were detected urinary metabolites by a UPLC system coupled with a triple-quadrupole tandem mass spectrometer (MS/MS) in multiple reaction monitoring (MRM). Two potential positive urine samples detected by metabonomics method were validated by a Chinese national standard targeted method. Twenty negative urine samples collected from different origins were used as control samples. The urine samples were purified and the two proposed biomarkers were determined by the above mentioned UPLC-MS/MS method. Fold change of the two proposed biomarkers was calculated by comparing the response of treated samples versus the mean response of all the control samples. The threshold was built for detection the two markers for monitoring of β2-agonists by a UPLC system coupled with a triple-quadrupole tandem mass spectrometer (MS/MS). The metabolomic profiling would be developed into a promising diagnostic tool to overcome current limitations of targeted methods in controlling the abuse of new-emerging unknown β2-agonists.