柱前衍生化RP-HPLC同时测定不同产区锁阳中17种游离氨基酸含量及其多元统计分析

目的:建立柱前衍生化反相高效液相色谱法(RP-HPLC)同时测定不同产区锁阳中17种游离氨基酸含量的方法并进行多元统计分析。方法:采用柱前衍生化RP-HPLC检测,以异硫氰酸苯酯(PITC)为衍生化试剂,采用Wonda Sil C18-WR色谱柱(4.6 mm×150 mm,5μm),流动相A为0.05 mol·L^-1乙酸钠缓冲液(pH 6.5),流动相B为乙腈-甲醇-水(3∶1∶1),梯度洗脱,流速0.8 mL·min^-1,检测波长254 nm,柱温35℃。建立主成分分析(PCA)及系统聚类分析(HCA)模型进行多元统计分析并进行质量评价。结果:从锁阳中共分离鉴定了17种氨基酸,含7种人体必需氨基酸。17种氨基酸在各自质量浓度范围内呈良好线性关系,r在0.9990~0.9999;平均回收率在98.03%~103.89%,RSD均<3.5%。PCA和HCA结果基本一致,均可将12个市级产区明显区分为6个区域;PCA可根据不同市级或省级产区对锁阳进行聚类,而HCA可根据省级产区对锁阳进行归类,说明不同市级、省级产区锁阳的氨基酸含量具有产区差异性,且含量分布表现出明显的地域聚集性特点;PCA表明甘肃、内蒙古所产锁阳氨基酸含量较高,质量较佳。结论:所建方法可用于不同产区锁阳中17种游离氨基酸的含量测定,为其综合质量评价提供参考。

Pre-column Derivatization RP-HPLC for Simultaneous Determination of 17 Amino Acids in Cynomorii Herba from Different Producing Areas and Its Multivariate Statistical Analysis

Objective:To establish a pre-column derivatization reverse-phase high-performance liquid chromatography(RP-HPLC)method for the simultaneous determination of 17 amino acids in Cynomorii Herba from different producing areas and conduct a multivariate statistical analysis.Method:RP-HPLC with pre-column derivatization was employed,with phenyl isothiocyanate(PITC)as derivatization reagent.Separation was performed on a WondaSil C18-WR column(4.6 mm×150 mm,5μm),with 0.05 mol·L^-1 sodium acetate solution(pH 6.5)as mobile phase A,and acetonitrile-methanol-water(3∶1∶1)as mobile phase B for gradient elution at a flow rate of 0.8 mL·min^-1.The detective wave length was set at 254 nm,and the column temperature was maintained at 35℃.Principal component analysis(PCA)and systematic cluster analysis(HCA)models were established for multivariate statistical analysis and quality evaluation.Result:17 Kinds of amino acid were detected in Cynomorii Herba,7 of which were essential amino acids.The 17 amino acids showed good linearity in respective concentration range,r=0.9990-0.9999.The average recoveries were between 98.03%-103.89%with RSD<3.5%.The results of PCA and HCA were basically the same,and both methods can be used to clearly distinguish Cynomorii Herba from 12 municipal producing areas into 6 regions.PCA can be used to classify Cynomorii Herba according to different municipal or provincial production areas,and HCA can be used to classify it according to provincial production areas.It showed that the amino acid contents in Cynomorii Herba from different municipal and provincial producing areas had differences,and the content distribution showed obvious geographical clustering characteristics.PCA showed that Cynomorii Herba from Gansu province and Inner Mongolia had higher amino acid contents and better quality as compared with other producing areas.Conclusion:The established method can be used for content determination of 17 amino acids in Cynomorii Herba from different producing areas,and provide a reference for its comprehensive quality evaluation.