马皮特征肽的发现及其在阿胶中马皮源成分检测中的应用  

Identification of the horse skin marker peptide for test of horse-hide gelatin in donkey-hide gelatin

作　　者：巩丽萍 杭宝建 迟连利[2] 李雪[2] 徐丽华 魏峰[3] 程显隆[3] 马双成[3] 石峰 

机构地区：[1]山东省食品药品检测研究院,济南250101 [2]山东大学,济南250100 [3]中国食品药品检定研究院,北京100050

出　　处：《药物分析杂志》2018年第2期364-369,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：国家重大新药创制中药安全检测技术及标准平台(No.2014ZX09304307)--中药质量安全检测和风险控制技术平台(No.2014ZX0930437-002)

摘　　要：目的:发现马皮特征肽,建立阿胶中马皮源成分专属性的检测方法。方法:采用序列比对技术发现理论马皮胶原蛋白特征肽,利用胰蛋白酶酶切马皮胶样品,通过纳升液相-高分辨质谱确证了马皮特征肽GASGPAGVR。利用超高效液相色谱-三重四极杆质谱(UPLC-QQQ/MS)对马皮特征肽进行检测。色谱柱为ACQUITY UPLC BEH C_(18)(2.1 mm×150 mm,1.7μm),流动相为0.1%甲酸溶液(A)-乙腈(B),梯度洗脱(0~3 min,96%A;3~8 min,96%A→92%A;8~10 min,92%A→50%A;10~12 min,50%A;12~12.1 min,50%A→96%A;12.1~15 min,96%A),流速0.3 mL·min^(-1),柱温40℃,进样量5μL;离子化模式为ESI+,进行多反应监测,选择马皮特征肽特征分子离子峰m/z 386.25→499.25,m/z 386.25→556.51作为检测离子对。结果:在马皮胶样品中检出了马皮特征肽,在阿胶、牛皮胶、猪皮胶、羊皮胶样品中均未检出马皮特征肽。该方法的检出限度为1%马皮胶的阿胶样品。对10批阿胶样品进行检测,在2批样品中检出了马皮特征肽。结论:所建立的方法经验证,专属性强,可用于阿胶中马皮源成分的检测。Objective：To find marker peptide of horse hide and establish a specific method for identification of horse-hide gelatin in donkey-hide gelatin.Methods：Theoretical species-specific peptide of horse skin was predicted by sequence comparison as GASGPAGVR,which was verified by Nano LC-Orbitrap MS followed by sample digestion with trypsin.Ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry（UPLC-QQQ/MS）was used to detect horse-hide marker peptide.Determination was carried out on an ACQUITY UPLC BEH C_（18）（2.1 mm×150 mm,1.7 μm）column at temperature of 40 ℃.The mobile phase was composed of 0.1% formic acid（A）and acetonitrile（B）with gradient elution（0-3 min,96%A;3-8 min,96%A → 92%A;8-（-1）0 min,92%A → 50%A;10^-12 min,50%A;12^-12.1 min,50%A → 96%A;12.1^-15 min,96%A）at a flow rate of 0.3 m L·min^-1.The electrospray ionization（ESI）source was performed in multiple reaction monitoring（MRM）mode with the transitions of m/z 386.25 → 499.25 and m/z 386.25 → 556.51.Results：Marker peptide was found in horse hide gelatin,but not in donkey hide gelatin,cow hide gelatin,pig hide gelatin nor sheep hide gelatin.The detection limit is 1% horse hide gelatin in donkey hide gelatin.Marker peptide was detected in 2 out of 10 donkey-hide gelatin samples.Conclusion：The established method is proved to be specific,which is suitable for the identification of horse-hide gelatin in donkey-hide gelatin.

关 键 词：序列比对 液质联用技术 阿胶鉴别 马皮胶 马皮特征肽 

分 类 号：R917[医药卫生—药物分析学]