| Our study selected44representative drugs from the prohibited list of the FEI (International Equestrian Federation), the fragmentation pathways of the drugs were investigated and the regularies of cleavage and the characteristics ions under electro-spray ion source have been summarized and established the analytical method using high performance liquid chromatography-tandem mass spectrometry for the residues of these drugs in complex matrix of horse feed.1.Grinded samples were first infiltrated and deproteinized with methanol, then purified by solid-phase extraction (SPE) using a MCX cartridge after extracted with0.02mol/L NaOH. Detection was performed with HPLC-MS/MS using10mmol/L ammonium acetate solution and acetonitrile as mobile phase under negative electrospray ionization (-ESI) in multiple reaction monitoring (MRM) mode in five different time segment except for coumarin with positive mode to achieve simultaneously quantitative analysis of five anticoagulants and the experimental data collection and processing was done by agilent workstations MassHunter software (Agilent MassHunter is the software for system of data collection and data processing software). The experimental results show that this method is of high sensitivity and reproducibility, the limits of detection were10ng/g for dicoumarin and warfarin;4ng/g for acenocoumarin and coumarin;15ng/g for penidione while limits of quantitation were as low as10～40ng/g and the intra-day and inter-day relative standard deviations(RSDs) were less than8.63and10.20%respectively; the recoveries for blank samples fortified at three levels ranged from60.3～99.2%with the relative standard deviations within12.4%.2.Grinded samples were first deproteinated and extracted with10mL1mmol/L HClO4and then purified by a mix-mode solid-phase extraction (SPE). and the extracts were analysed by HPLC-MS/MS with0.4%formic acid solution and acetonitrile as mobile phase in positive electro-spray ionization (+ESI) mode with multiple reaction monitoring (MRM); The results validated that this method with good reproducibility and sensitivity and the limits of detection and quantification of the drugs were in the range of0.20～0.75ng/g and0.50～1.5ng/g, respectively.The recoveries of all analytes spiked in blank samples at three levels were between77.4%～97.8%, the intra-day and inter-day relative standard deviations(RSDs) were less than6.2%and7.1%.3.Grinded samples were firstly deproteinated and extracted with1mmol/L HClO4for the basic drugs and then acidified acetonitrile with20min sonication. The purification was accomplish with mix-mode solid-phase extraction (SPE) by multi-step loading of the extracts in weak acid and organic solutions and multi-elution with dichloromethane/isopropanol/concentrated aqueous ammonia (5/4/1, v/v/v) and acetonitrile. The extracts were separated on a reverse phase with water and acetonitrile (both containing0.4%formic acid) and analysed by HPLC-MS/MS in positive electro-spray ionization (+ESI) mode with multiple reaction monitoring (MRM) for qualitative analysis and confirmation of the analytes and quantification used matrix-matched internal standard calibration curves. The method was validated in fortified blank samples at three levels and the results showed that the overall recoveries were within the range of57,6～115.6%with the relative standard deviations(RSDs) in the ranges of1.6～20.4%; the limits of detection and the limits of quantification were0.2～25.0μug/kg and1.0～40.0μg/kg, and more than67%of the drugs studied can be detected below2.5μg/kg. |
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