Residue Depletion Of Ketoprofen In Milk Of Lactating Dairy Cows After Intramuscular Administration

In recent years,the safe use of veterinary drugs has attracted more and more attention the veterinary drug residues in the animal product food has caused global concern.Ketoprofen has many advantages,such as anti-inflammatory effect,high curative effect,little side effect,convenient use and so on.To establish the milk discard time of ketoprofen,ensure the quality of animal food safety and provide the basis for scientific rational drug use,the study presented a sensitive and rapid liquid chromatography tandem mass spectrometry（LC-MS/MS）method for the determination of ketoprofen in milk,and milk residue depletion profiles of ketoprofen in lactating dairy cows after intramuscular administration were determined.The determination of ketoprofen in milk was conducted using LC-MS/MS.Target analyte could be extracted by methanol and taken to dryness under a gentle flow of nitrogen at40℃.The residues were redissolved by 0.1%formic acid in acetonitrile（90/10,v/v）and filtered through a 0.22μm nylon filter into an autosampler vial for LC-MS/MS analysis.Chromatographic separation for ketoprofen was performed on an Agilent HPLC system with the column oven temperature maintained at 30℃via an Agilent EcliPse XDB-C18（4.6×15mm i.d.,0.5μm particle size）.The mobile phase of 0.1%formic acid aqueous solution（A）and acetonitrile（B）with a flow rate of 0.25 mL/min.Gradient elution program was performed as follows:0¹.0 min,90%A;1.0¹.5min,90¹0%A;1.5⁷.5min,10%A;7.5⁸.0min,10⁹0%A;8.0¹3.0 min,90%A.The injection volume was 5μL.The MS instrument was a triple quadrupole 6410B（Agilent Technologies）,fitted with an electrospray ionization（ESI）source.The ESI interface was operated in positive ionization mode.The triple quadrupole was used in multiple reaction monitoring（MRM）mode.The quantitative ion for ketoprofen and ketoprofen-D₃ were 255.1>209.2,258.1>212.1,respectively.At 5μg/kg²00μg/kg concentration range showed a good linear relationship with the concentration ratio of ketoprofen and ketoprofen-D3,correlation coefficient is greater than 0.99.In 5μg/kg,25μg/kg,50μg/kg and 100μg/kg four concentration,the average recovery rate was 100.53%¹03.05%,CV was 1.72%⁷.82%,coefficient of variation between groups was 1.13%¹0.48%,ketoprofen in milk detection limit was 1μg/kg,the limit of quantification was 5μg/kg.20 healthy Holstein dairy cows（10 high yield,10 low yield）were intramuscular injected of ketoprofen injection at a dose of 3 mg/kg.b.w.,once daily,for three days.At 2,15,26,39,50,64,74,87,98 h after the first injection,milk samples were collected and immediately frozen at-20°C until further analysis.The highest levels residue concentration was38.43±3.95μg/kg at 2h after the first injection,which was lower than the MRL（50μg/kg）.No milk level of ketoprofen was detected 15 h after the last injection.This study established a method for the determination of ketoprofen residues in milk by LC-MS/MS with good reproducibility,high sensitivity,specificity,simple and easy pretreatment.The analysis meets the requirements of veterinary drug residues.The exploring of ketoprofen residue elimination through injection of in healthy Holstein cows proved that the milk disposed for a period of 0 day.