杆菌肽在动物可食性组织和禽蛋中残留的HPLC-MS/MS检测方法

Determination of Bacitracin Residues in Animal Edible Tissues and Poultry Eggs by HPLC-MS/MS

关键词:  杆菌肽  高效液相色谱串联质谱法  残留检测  动物可食性组织  禽蛋

Key words:  bacitracin  HPLC-MS/MS  residue detection  animal edible tissue  poultry egg

摘要:

试验旨在建立动物(猪、鸡、鸭)可食性组织(肌肉、肝脏、肾脏、脂肪)和禽蛋(鸡蛋、鸭蛋)中杆菌肽的高效液相色谱串联质谱(HPLC-MS/MS)检测方法。使用0.1%甲酸水溶液提取样品中的杆菌肽,三氯乙酸乙腈沉淀蛋白,正己烷除脂,混合阳离子型固相萃取柱净化后,进HPLC-MS/MS检测。待测物使用C18色谱柱分离,以0.1%甲酸水溶液和乙腈为流动相进行梯度洗脱(乙腈：0~0.5 min 15%,5.0~5.5 min 85%,5.6~6.5 min 15%)；质谱采用电喷雾离子源(ESI),正离子扫描,多反应监测(MRM)模式进行检测,外标法定量。结果显示：在50~1000 ng/g的添加范围内,各组织中杆菌肽添加浓度与目标峰面积的线性关系良好,相关系数均大于0.999。猪、鸡、鸭的可食性组织及禽蛋在50、100、500、750 ng/g的添加浓度下,平均回收率分别为71.3%~80.1%、71.6%~80.7%、71.5%~84.7%和66.2%~80.9%,批内、批间变异系数均小于15%。试验建立的动物可食性组织和禽蛋中杆菌肽残留的HPLC-MS/MS检测方法满足动物可食性组织中杆菌肽残留限量的检测要求。

Abstract:

This study was aimed to establish a HPLC-MS/MS method for the determination of bacitracin in animal (pig, chicken and duck) edible tissues (muscle, liver, kidney, fat) and poultry eggs (eggs, duck eggs). Using 0.1% formic acid aqueous solution extractes bacitracin in sample, then precipitated protein by trichloroacetic acid acetonitrile, defatted by N-hexane, purified by mixed cationic SPE column and detected by HPLC-MS/MS. Chromatographic separation was performed on a C18 column. Mobile phases, which comprised of a mixture of (A)0.1% formic acid in water and (B)acetonitrile, were delivered under a gradient rlution program (0-0.5 min: 85% A, 15% B; 5.0-5.5 min: 15% A, 85% B; 5.6-6.5: 85% A, 15%B). Electrospray ionization(ESI), positive ion scanning and multipule reaction monitoring mode(MRM) were used for mass spectrometry. External srandard method was used for quantification. The result shows that within the range of 50-1000 ng/g of bacitracin, the linear relationship between the added concentration in each tissue and the target peak area was good, and the regression coefficient were all greater than 0.999. At the added concentration of 50, 100, 500, 750 ng/g, the mean recovery rate of edible tissues of pigs, chickens and ducks and poultry eggs were 71.3%-80.1%, 71.6%-80.7%, 71.5%-84.7% and 66.2%-80.9%. Within-run and between-run coefficient of variation(CV) all less than 15%. The HPLC-MS/MS method for the determination of bacitracin residues in animal edible tissues and poultry eggs was established in this study, which met the requirements of the determination of bacitracin residue limit in animal edible tissues.