HPLC法同时测定穿山甲炮制品中3个主要环二肽成分的含量

目的：建立高效液相色谱法同时测定炮山甲中3个成分L-丝-L-酪氨酸环二肽、D-丝-L-酪氨酸环二肽、L-甘-L-酪氨酸环二肽的含量。方法：采用赛分Sepax Bio-C₁₈色谱柱（4.6 mm×250 mm，5μm），以0.1%TFA乙腈-0.1%TFA水（2：98）为流动相，流速1.0 mL·min^-1，检测波长210 nm。并以3种环二肽为指标，使用SPSS 22.0软件对40批样品进行聚类分析。结果：3个环二肽分离良好，L-丝-L-酪氨酸环二肽、D-丝-L-酪氨酸环二肽、L-甘-L-酪氨酸环二肽进样量分别在0.056~2.244μg（r=0.9999）、0.061~2.452μg（r=0.9997）、0.059~2.348μg（r=0.9999）范围内呈良好的线性关系。加样回收率分别为101.8%、98.1%、95.7%，相应的RSD分别为0.76%、2.2%、2.6%；40批样品中3个成分的含量范围分别为0.0070%~0.23%，0.0050%~0.14%，0.0080%~0.18%，盐类增重对环二肽含量有显著影响，聚类分析将其分为3类。结论：本方法准确可靠，适用于炮山甲中环二肽成分的含量测定。  

Objective: To establish an HPLC method for simultaneous determination of L-serine-L-tyrosine cyclic dipeptide, D-serine-L-tyrosine cyclic dipeptide and L-glycine-L-tyrosine cyclic dipeptide in pangolin scales. Methods: Separation was performed on a Sepax Bio-C₁₈ column (4.6 mm×250 mm, 5 μm)with 0.1% TFA acetonitrile-0.1% TFA water (2:98)as the mobile phase at a flow rate of 1.0 mL·min^-1. The detection wavelength was 210 nm. Hierarchical clustering analysis was performed to evaluate and classify 40 batches of samples based on the contents of the 3 cyclic dipeptides using SPSS 22.0 software. Results: Three cyclic dipeptides were well separated. L-serine-L-tyrosine cyclic dipeptide, D-serine-L-tyrosine cyclic dipeptide and L-glycineL-tyrosine cyclic dipeptide were linear in the ranges of 0.056-2.244 μg (r=0.999 9), 0.061-2.452 μg (r=0.999 7), 0.059-2.348 ug (r=0.999 9), respectively. The recovery rates were 101.8%, 98.1%, and 95.7%, respectively, with RSDs of 0.76%, 2.2%, and 2.6%, respectively. The contents of the three components in 40 batches of samples were 0.007 0%-0.23%, 0.005 0%-0.14% and 0.008 0%-0.18%, respectively. Sulphate in illegal processed pangolin had a significant influence on cyclic dipeptides. All samples were classified into 3 types by clustering analysis. Conclusion: This method is accurate and reliable, which is suitable for determination of cyclic dipeptides in pangolin scales. 

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