UPLC-MS/MS法同时测定阿胶中20种氨基酸含量及其化学模式识别分析

Simultaneous Determination of 20 Amino Acids in Asini Corii Colla by UPLC-MS/MS and Its Chemical Pattern Recognition

目的建立阿胶中20种氨基酸的含量测定方法及不同产地阿胶的化学模式识别分析法。方法采用超高效液相色谱串联质谱(UPLC-MS/MS)法测定阿胶中氨基酸含量,分析柱为Waters BEHC_(18)柱(50 mm×2.1 mm,1.7μm),流动相为0.1%甲酸乙腈-0.1%甲酸水溶液(5∶95,V/V),流速为0.35 mL/min,柱温为45℃,进样体积为5μL;采用电喷雾离子源(ESI),正离子模式下多反应监测(MRM)。采用层次聚类分析(HCA)法、主成分分析(PCA)法和正交偏最小二乘-判别分析(OPLS-DA)法分析测定结果。结果20种成分在相应质量浓度范围内与峰面积线性关系良好,检测限为0.002~0.075μg/mL,定量限为0.004~0.134μg/mL;精密度、稳定性、重复性试验的RSD均不超过4.70%,平均加样回收率为93.20%~107.80%,RSD为0.80%~3.90%。HCA法及PCA法均可将18个产地的药材样品分为2类;OPLS-DA法筛选出指标性成分异亮氨酸、丙氨酸、谷氨酰胺、天冬氨酸、谷氨酸、脯氨酸、精氨酸。结论所建方法可用于阿胶中氨基酸类成分的含量测定,筛选的7种成分可作为区分不同厂家阿胶的潜在指标。

Objective To establish a method for the determination of 20 amino acids in Asini Corii Colla and a chemical pattern recognition method for Asini Corii Colla from different producing areas.Methods The content of amino acids in Asini Corii Colla was determined by the ultra-high-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)methed the analytical column was the Waters BEH C_(18)column(50 mm×2.1 mm,1.7μm),the mobile phase was 0.1%formic acid acetonitrile-0.1%formic acid aqueous solution(5∶95,V/V),the flow rate was 0.35 mL/min,the column temperature was 45℃,and the injection volume was 5μL.The electrospray ionization(ESI)with positive ion mode and multi-reaction monitoring(MRM)was adopted.The content determination was analyzed by the hierarchical clustering analysis(HCA),principal component analysis(PCA),and orthogonal partial least squares-discriminant analysis(OPLS-DA).Results Twenty components had a good linear relationship with the peak area within the corresponding mass concentration range.The limits of detection(LOD)were in the range of 0.002-0.075μg/mL,the limits of quantification(LOQ)were in the range of 0.004-0.13~4μg/mL.The RSDs of precision,stability and repeatability tests were not over 4.70%.The average recovery rates of 20 components were in the range of 93.20%-107.80%with RSDs in the range of 0.80%-3.90%.The medicinal materials from 18 producing areas were classified into two categories by HCA or PCA methods;seven indicator components including isoleucine,alanine,glutamine,aspartic acid,glutamic acid,proline,and arginine were screened out by the OPLS-DA method.Conclusion The established method can be used to determine the content of amino acids in Asini Corii Colla,and the screened seven components can be used as the potential indicators to distinguish Asini Corii Colla from different manufacturers.