液相色谱-串联质谱法同时测定饲用血液制品中18种β-受体激动剂

Simultaneous Determination of 18 β-agonists in Blood Products for Feeds by Liquid Chromatography Tandem Mass Spectrometry

【背景】饲用血液制品是一种非常规动物源性饲料原料,由家畜或家禽的血液凝成块后经高温蒸煮,压除汁液、晾晒、烘干后粉碎而成,主要用于畜牧养殖。但由于动物养殖过程中存在非法使用β-受体激动剂的现象,一些含有β-受体激动剂的血液制成饲用血液制品可能成为对人类健康潜在的危害源头。为了降低安全风险,研究饲用血液制品中β-受体激动剂的方法是十分必要的。β-受体激动剂检测方法主要有酶联免疫吸附分析(ELISA)法、高效液相色谱(HPLC)法、气质联用(GC-MS)法和液相色谱串联质谱(LC-MS/MS)法等。目前所有方法或标准大多针对商业成品饲料或动物源性食品,缺乏对饲用血液制品中β-受体激动剂的检测技术进行相关的研究。【目的】为了研究和监控饲用血液制品中β-受体激动剂的情况,研究建立了固相萃取结合液相色谱-串联质谱检测饲用血液制品中18种β-受体激动剂的方法。【方法】称取2 g(精确至0.01 g)血液制品样品于50 mL离心管中,准确加入20 mL乙酸铵提取液(pH=5.2)和50μLβ-葡萄糖苷酸酶/芳基硫酸酯酶,漩涡混合均匀,于37℃水解过夜(应大于16h),然后8000 r/min离心5 min,取5 mL上清液转移至另一离心管中,加入0.5mL高氯酸溶液,漩涡混合30 s,然后于8000r/min离心5min,上清液备用。PCX固相萃取小柱依次用3 mL甲醇,3 mL水活化。取上清液过柱,用3 mL水和3 mL甲醇淋洗,抽干,用3 mL氨水甲醇溶液洗脱,洗脱液于50℃氮气吹至近干,用1.0 mL 0.1%甲酸水+乙腈溶液(95+5)溶解,过0.22μm滤膜。进Waters TQ液相色谱串联质谱仪检测,使用ACQUITY UPLC BEH C18(100 mm,2.1 mm,1.7μm)色谱柱。乙腈和0.1%甲酸溶液做流动相,梯度洗脱。质谱电离方式采用电子喷雾离子源,正离子检测方式,多反应监测(HRM)。喷雾电压为3.5 kV;雾化气温度为480℃;源温度为150℃;雾化气流速为600L·h^-1;锥孔气流速为5 L·h^-1。脱溶剂气、锥孔气、碰撞气均为高纯氮气。【结果】18种β-受体激动剂在5-100μg·L^-1呈良好的线性关系,相关系数在0.99-0.999之间。在血粉、血浆蛋白粉、血球蛋白粉三种基质中在5、10和50μg·kg^-1三个添加水平上的平均回收率为65.1%-110%之间,相对标准偏差小于15%,批间变异系数小于20%。检出限为低于5ng·g^-1。【结论】从方法回收率、精密度结果及实际样品的检测结果来看,该方法适用于血液制品中β-受体激动剂的监测。

【Background】Blood product for feeds is a kind of unconventional animal-derived feed material.It is made through coagulating the blood of livestock or poultry,cooking at high temperature,pressing out juice,drying and grinding.However,due to the existence of illegal use of β-agonists,the use of blood product from blood containing β-agonists may become a potential source of harm to human health.In order to reduce the safety risk,it is necessary to study the methods of β-agonists in blood product for feeds.The detection methods of β-agonists include enzyme-linked immunosorbent assay(ELISA),high performance liquid chromatography(HPLC),gas chromatography-mass spectrometry(GC/MS)and liquid chromatography-tandem mass spectrometry(LC-MS/MS).At present,most of the methods or standards are aimed at commercial finished feed or animal-derived food,however there is a lack of relevant research on the detection technology of β-agonists in blood product for feeds.【Objective】In order to study and monitor the status ofβ-agonists in blood product for feeds,a method of LC-MS/MS combined with solid phase extraction(SPE)was developed for the determination of 18β-agonists in blood product for feeds.【Method】2 g(accurate to 0.01 g)blood product sample was weighed in 50 mL centrifugal tube,and then 20 mL ammonium acetate extract(pH=5.2)and 50 mL beta-glucuronidase/arylsulfatase were added accurately.The eddies were mixed evenly hydrolyzed overnight at 37(>16 hours),then centrifuged for 5 min at 8000 r/min,the supernatant was transferred to another centrifugal tube,and 0.5 mL 30%perchloric acid solution was added.After vortex mixing for 30 seconds and centrifugation for 5 minutes at 8000r/min,supernatant was reserved.PCX solid phase extraction column was activated with 3 mL methanol and 3 mL water in turn.The supernatant was load and washed by 3 mL water and 3 mL methanol,then drained,eluted by 3 mL 5%ammonia methanol solution.The eluent was blown to near dry by nitrogen at 50℃,dissolved by 1.0 mL 0.1%formic acid water+acetonitrile solution(95+5)and filtered through 0.22μm filter membrane,then detected by Waters TQ liquid chromatography tandem mass spectrometer.The column ACQUITY UPLC BEH C18(100 mm,2.1 mm,1.7μm)was used as analysis column;acetonitrile and 0.1%formic acid solution were used as mobile phase for gradient elution.The ionization modes of mass spectrometry were electron spray ion source,positive ion detection method and multi reaction monitoring(HRM),the spray voltage was 3.5 kV,the dissolvent temperature was 480℃,the source temperature was 150℃,flow rate of the dissolvent gas was 600L·h^-1,and flow rate of the cone gas was 5 L·h^-1.The dissolvent gas,cone gas and collision gas were all high purity nitrogen gas.【Result】18β-agonists showed a good linear relationship between 5 and 100μg·L^-1,with correlation coefficients ranging from 0.99 to 0.999.The average recovery of blood powder,plasma protein powder and globulin powder was 65.1%-110%at the levels of 5,10 and 50μg·kg^-1,and the relative standard deviation below 15%.The coefficient of variation between batches was less than 20%.The detection limit was less than 5 ng·g^-1.【Conclusion】The results of recovery,precision and actual samples showed that the method was suitable for monitoring β-agonists in blood products for feed.