超高效液相色谱-串联质谱法测定牛奶中重组牛生长激素N-末端肽链.docVIP

超高效液相色谱-串联质谱法测定牛奶中重组牛生长激素N-末端肽链 刘海山，汤杭燕，吕春华，陈笑梅 （浙江省检验检疫科学技术研究院，杭州 310016） 摘要：建立了高效液相色谱-串联质谱测定牛奶中重组牛生长激素(rBST)N-末端肽链的方法。用乙腈沉淀样品中的蛋白质，胰蛋白酶水解，离心净化后，在ACQUITY UPLC BEH C18（50 mm× μm）色谱柱上分离0.1～2 mg?L-1范围内呈线性关系，测定下限为0.2 mg?kg-1(S/N10)。以空白牛奶样品为基体，进行加标回收试验，rBST N-末端肽链的回收率在78.1%～ 关键词：超高效液相色谱-串联质谱；重组牛生长激素；牛奶 Determination of N- terminal Peptide of Recombinant Bovine Somatotropin in Milk by Ultra Performance Liquid Chromatography-tandem Mass Spectrometry LIU Hai-shan, TANG Hang-yan, Lü Chun-Hua, CHEN Xiao-mei (Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou 310016, China) Abstract:A sensitive and selective LC-MS/MS method was presented for the determination of N- terminal peptide of recombinant bovine somatotropin(rBST) in milk. The proteins in sample were precipitated with addition of acetonitrile and then digested with trypsin overnight at 37℃. Separation was performed on an ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) with gradient elution by acetonitrile and 0.05 % formic acid. The analyte was ionized by ESI+ and detected on multiple reaction monitor(MRM) mode. Linear relationship between the peak area and the mass concentration of N- terminal peptide of rBST was obtained in the range of 0.1-2 mg?L-1, with quantitation limit(S/N10) of 0.2 mg?kg-1. Using blank sample of milk as matrix, test for recovery was made by standard addition method, values of recovery found were in the range of 78.1%-96.9% with RSD’S(n=6) less than 10%. Keywords: UPLC-MS/MS; Recombinant bovine somatotropin; Milk 牛生长激素是由脑垂体前叶嗜酸性细胞分泌的一种具有调节生长和催乳功能的蛋白质早在上世纪30年代，牛生长激素就被注射到母牛体内以增加牛奶产量。上世纪80年代研制出基因合成的重组牛生长激素rBST)，它可以使奶牛的产奶量增加10％以上。重组牛生长激素牛生长激素1993年美国FDA允许在奶牛中使用重组牛生长激素以提高牛奶产量 母牛被注射了rBST之后，所产牛奶中的IGF-1)含量会大幅增加IGF-1可能增加人体患癌症的风险。有研究表明，IGF-1可以进入血液并影响其他激素的活动水平。妇女产前和哺乳期尤其容易受其影响，它可以干扰乳房细胞正常生长，从而增加罹患乳腺癌的风险。rBST的另一个间接影响在于增加了抗生素的使用量，因为rBST在增加奶牛产奶量的同时，也导致了牛乳腺炎发病的几率。为了治疗奶牛的乳腺疾病，需要注射大量的抗生素。治疗乳腺炎所需要的抗生素会残留在牛奶当中，最终被摄入人体。这些残留的抗生素容易引起人体过敏反应、造成环境平衡紊乱或菌群失调，并在药物治疗中产生耐药性。基于这些健康考虑，加拿大、欧盟、