超高效液相色谱-三重四极杆质谱法检测阿胶补血颗粒中的阿胶

目的:建立阿胶补血颗粒中阿胶的专属性检测方法。方法:采用胰蛋白酶对阿胶补血颗粒中阿胶进行酶解,利用超高效液相色谱-三重四极杆质谱(UPLC-QQQ MS)对阿胶的专属性特征肽段进行检测。色谱柱为Agilent SB-C₁₈(2.1 mm×100 mm,1.8 μm),流动相为0.1%甲酸溶液(A)-乙腈(B),梯度洗脱 (0~25 min,95%A→80%A;25~40 min,80%A→50%A),流速0.3 mL·min^-1,柱温40 ℃,进样量5 μL;离子化模式为ESI⁺,进行多反应监测,选择阿胶特征分子离子峰m/z 539.8(双电荷)→612.4,m/z 539.8(双电荷)→923.8作为检测离子对。结果:3批市售样品中均可检出阿胶的特征肽段。结论:所建立的方法经方法学验证,专属性强,可用于阿胶补血颗粒中阿胶的检测。

Objective:To establish an analysis method for specificity identification of donkey-hide gelatin in Ejiao Buxue granula.Methods:Samples were dissolved by water containing 1% NH₄HCO₃ and digested by trypsin.Ultra-high performance liquid chromatography(UPLC)coupled to triple quadruple mass spectrometry(QQQ MS)was used to detected the specific marker peptides in donkey-hide gelatins.Determination was carried out with the application of an Agilent SB-C₁₈(2.1 mm×100 mm,1.8 μm)column at temperature of 40 ℃.The mobile phase was composed of water solvent containing 0.1% formic acid(A)and acetonitrile(B)with gradient elution(0-25 min,95%A→80%A;25-40 min,80%A→50%A)at a flow rate of 0.3 mL·min^-1.The electrospray ionization(ESI)source was performed in multiple reaction monitoring(MRM)mode of the transitions of m/z 539.8(double charge)→612.4,m/z 539.8(double charge)→923.8.Results:The marker peptides can be detected in all samples by this method.Conclusion:The established method is specific,which is suitable for identification of donkey-hide gelatin in Ejiao Buxue granula.

[1] WS3-B-2535-97-2003 国家食品药品监督管理局药品标准[S]. 2003 WS3-B-2535-97-2003 China Food and Drug Administration National Drug Standard[S].2003
[2] 中国药典2010年版.一部[S].2010:175 ChP 2010.Vol Ⅱ[S].2010:175
[3] 汪冰,肖新月,程显隆,等.凝胶排阻色谱法研究阿胶中蛋白质及多肽相对分子质量分布规律[J].药物分析杂志,2009,29(11):1886 WANG B,XIAO XY,CHENG XL,et al.Study on relative molecular mass distribution of protein and peptide in donkey-hide glue by gel exclusion chromatography[J].Chin J Pharm Anal,2009,29(11):1886
[4] 程显隆,李文杰,魏锋,等.动物胶类药材的鉴别方法研究进展. 亚太传统医药,2011,7(3):167 CHENG XL,LI WJ,WEI F,et al.Advances in studies on identificated method of gelatins[J].Asia Pac Tradit Med,2011,7(3):167
[5] 程显隆,肖新月,邹秦文,等.柱前衍生化HPLC法同时测定阿胶中4 种主要氨基酸的含量[J].药物分析杂志,2008,28(12):1997 CHENG XL,XIAO XY,ZOU QW,et al.Pre-column derivatization HPLC simultaneous determination of 4 main amino acids in donkey-hide glue[J].Chin J Pharm Anal,2008,28(12):1997
[6] CHENG XL,WEI F,CHEN J,et al.Using the doubly charged selected ion coupled with MS/MS fragments monitoring(DCSI-MS/MS)mode for the identification of gelatin species[J].J Anal Methods Chem,2014:764397;doi:10.1155/2014/764397
[7] 程显隆,陈佳,李明华,等.特征肽段检测技术用于胶类药材专属性鉴别方法研究[J].中国药学杂志,2015,50(2):104 CHENG XL,CHEN J,LI MH,et al.Identification study of glue medicines[J].Chin Pharm J,2015,50(2):104
[8] 程显隆,李文杰,张小龙,等.UPLC-QTOF-MS结合主成份分析法用于龟甲胶、鹿角胶中添加牛皮源成分的检测研究[J].药物分析杂志,2012,32(6):931 CHENG XL,LI WJ,ZHANG XL,et al.Identification of bovine-hide gelatin from glue of tortoise shell and deer-horn gelatin by UPLC-QTOF-MS and principal component analysis[J].Chin J Pharm Anal,2012,32(6):931
[9] CHENG XL,WEI F,XIAO XY,et al.Identification of five gelatins by ultra performance liquid chromatography/time-of-flight mass spectrometry(UPLC/Q-TOF-MS)using principal component analysis[J].J Pharm Biomed Anal,2012,62(2):191
[10] 陈佳,程显隆,魏锋,等.超高效液相色谱-三重四极杆质谱法检测复方阿胶浆中阿胶[J].药物分析杂志,2015,35(2):328 CHEN J,CHENG XL,WEI F,et al.Qualitative RRLC-QQQ/MS analysis of dongkey-hide gelatin in Fufang Ejiao Jiang[J].Chin J Pharm Anal,2015,35(2):328
[11] 程显隆.胶类药材质量控制关键技术研究[D].北京:北京中医药大学,2014 CHENG XL.Study on the Key Technology of Quality Control of Gelatin Drugs [D].Beijing:Beijing University of Chinese Medicine,2014
[12] 周岩,赵永刚,张蓓蓓,等.超高效液相色谱-三重四极杆/复合线性离子阱质谱测定水样中三苯甲烷类及代谢产物[J].分析化学,2014,42(3):367 ZHOU Y,ZHAO YG,ZHANG BB,et al.Determination of tnphenylmethane dye residues in water by ultra performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry[J].Chin J Anal Chem,2014,42(3):367