超高效液相色谱-串联质谱法测定猪肉、鸡蛋、牛奶中9种食源性兴奋剂类药物残留

β-受体激动剂、β-阻断剂、蛋白同化制剂属于兴奋剂类药物,在动物饲养和屠宰过程中的违禁使用成为食源性兴奋剂类药物残留的来源,危害人类健康。目前β-受体激动剂、蛋白同化制剂的检测较多,β-阻断剂检测报道较少,动物源食品中β-阻断剂检测尚无标准方法。该文建立了超高效液相色谱-串联质谱测定猪肉、鸡蛋、牛奶中β-受体激动剂、β-阻断剂、蛋白同化制剂三大类共9种食源性兴奋剂类药物残留的方法。样品中加入乙酸铵缓冲液(pH 5.2)和β-葡萄糖醛酸酶/芳基硫酸酯酶,在37 ℃条件下酶解16 h,酶解后的试样冷却至室温,NaOH溶液调节pH 9.5后用乙腈提取,提取液盐析分层,经增强型脂质去除净化管(EMR-Lipid)净化,无水硫酸镁除水,氮吹浓缩至近干,残渣用1 mL乙腈-0.1%甲酸水溶液(1∶9, v/v)溶解。以甲醇-0.1%甲酸水溶液作为流动相进行梯度洗脱,采用ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)分离,电喷雾正离子(ESI⁺)电离,多反应监测(MRM)模式下测定,基质匹配标准曲线内标法定量。实验优化了前处理过程中提取溶剂和pH对提取效率的影响,讨论了仪器分析过程中色谱柱、流动相、定容液等影响因素。结果表明:9种兴奋剂在0.5~20 μg/L浓度范围内线性关系良好,相关系数(r²)均大于0.99,方法检出限为0.3~0.6 μg/kg,定量限为1.0~2.0 μg/kg,在1、2、5倍定量限添加水平下,平均回收率为65.2%~117.0%,相对标准偏差(RSD)为1.3%~14.4%。应用建立的方法对市场上购入的猪肉、鸡蛋、牛奶三类动物源性食品进行测定,9种兴奋剂类药物残留均未检出。该方法快速、灵敏、准确、稳定,适用于猪肉、鸡蛋、牛奶等样品中9种食源性兴奋剂类药物残留的检测。

Determination of nine food-borne stimulant drug residues in pork,egg,and milk by ultra-performance liquid chromatography-tandem mass spectrometry

β-Agonists,β-blockers,and protein assimilators are classified as stimulant drugs.Their illegal use during animal feeding and slaughtering leads to food-borne stimulant drug residues,which are harmful to human health.At present,methods for the detection ofβ-agonists and protein assimilators are prevalent,but those for the detection ofβ-blockers are rare.There is no national standard for the detection ofβ-blockers in food products of animal origin.A method based on ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was developed for the determination of nine food-borne stimulant drug residues,includingβ-agonists,β-blockers,and protein assimilators,in pork,egg,and milk.The optimal extraction conditions for this method were as follows:The samples were hydrolyzed withβ-glucuronidase/aryl sulfate esterase in pH 5.2 ammonium acetate buffer.Enzymatic hydrolysis was carried out in a constant-temperature(37℃)water bath oscillator for 16 h.The enzymolyzed samples were cooled to room temperature and then extracted with acetonitrile,which was adjusted to pH 9.5 with NaOH solution.After extraction and homogeneous mixing,the extract was added to a salt package for salting out stratification.The clear supernatant was cleaned up using an enhanced lipid removal tube(EMR-lipid),which was pre-activated by water.Then,anhydrous magnesium sulfate was added to ensure dehydration of the extract and concentrated to near dryness under nitrogen flow.The residue was dissolved in 1 mL acetonitrile-0.1%formic acid aqueous solution(1∶9,v/v).Separation was performed on an ACQUITY UPLC HSS T3 column(100 mm×2.1 mm,1.8μm)with gradient elution using methanol-0.1%formic acid aqueous solution as the mobile phase.The analytes were detected in the multiple reaction monitoring(MRM)mode after being ionized by an electrospray positive ion(ESI⁺).Quantitative analysis was performed by the internal standard method using matrix-matched calibration curves.The effects of the extraction solvent and pH on the extraction efficiency during pretreatment were optimized.The influence factors of different types of chromatographic column,mobile phase and dissolved solution in the process of instrumental analysis were discussed in detail.Under the optimal conditions,the method showed good linearity in the range of 0.5 to 20μg/L,with correlation coefficients(r²)greater than 0.99.The limits of detection(LODs)and limits of quantification(LOQs)were in the range of 0.3-0.6μg/kg and 1.0-2.0μg/kg,respectively.The average recoveries of all the compounds ranged from 65.2%to 117.0%with relative standard deviations(RSDs)in range of 1.3%-14.4%at spiked levels of 1,2,and 5 times the LOQs.The established method was used to determine the quality of animal-origin foods such as pork,eggs,and milk purchased from the market.The nine stimulant drug residues were not detected in these food samples.The analytical method is rapid,sensitive,accurate,and stable.It can be used for the determination of the nine food-borne stimulant drugs residue in pork,egg,and milk.