HPLC法测定僵蚕5个核苷类成分的含量及聚类分析

Content Determination of 5 Nucleosides in Jiangcan(Bombyx Batryticatus)by HPLC and Cluster Analysis

目的建立同时测定僵蚕药材中5种核苷类成分含量的方法,并比较不同来源僵蚕药材中上述成分的差异性,为僵蚕药材的质量控制提供参考。方法采用月旭AQ-C_(18)(250 mm×4.6 mm,5μm)色谱柱;以纯水-甲醇为流动相,梯度洗脱,流速为0.8 mL/min;检测波长为260 nm,柱温为26℃;进样量为20μL。以21批不同来源的僵蚕药材为样品,测定上述5种成分含量,并采用SPSS 25.0统计学软件对21批不同来源的僵蚕药进行聚类分析。结果尿嘧啶、次黄嘌呤、尿苷、鸟苷及腺嘌呤分别在1~32μg/mL(r=1.0000)、0.3~9.6μg/mL(r=0.9999)、2~64μg/mL(r=0.9999)、5~160μg/mL(r=0.9998)、5~160μg/mL(r=0.9999)的浓度范围内与峰面积的线性关系良好,精密度、重复性、稳定性试验结果的RSD<3.00%,平均加样回收率为98.86%~101.73%,RSD<3.00%。不同来源僵蚕之间5个成分含量差异有统计学意义。经聚类分析,21批样品共聚为3类,产地样品即S1、S4、S5、S7、S8、S9聚类第Ⅰ类,市场样品聚为第Ⅱ类,产地样品S2、S3、S6聚为第Ⅲ类。结论该方法简便准确、重复性好,可用于同时测定僵蚕药材中上述5种核苷类成分的含量。同一产区僵蚕质量较为稳定,含量较高,市场僵蚕来源复杂,含量较低。

Objective To establish a method for simultaneously determining the content of 5 nucleoside components in Jiangcan(Bombyx Batryticatus)and compare the differences of the above components in different sources of Jiangcan(Bombyx Batryticatus),providing reference for the quality control of Jiangcan(Bombyx Batryticatus).Methods Yuexu AQ-C_(18)(250 mm×4.6 mm,5μm)chromatographic column was used,taking pure water-methanol as the mobile phase.The gradient elution was performed at a flow rate of 0.8 mL/min.The detection wavelength was 260 nm and the column temperature was 26℃.The injection amount was 20μL.SPSS 25.0 statistical software was used for cluster analysis of 21 batches of Jiangcan(Bombyx Batryticatus)from different sources.Results The linear relationship between the concentration range of 1-32μg/mL(r=1.0000)for uracil,0.3-9.6μg/mL(r=0.9999)for hypoxanthine,2-64μg/mL(r=0.9999)for uridine,5-160μg/mL(r=0.9998)for guanosine and 5-160μg/mL(r=0.9999)for adenine and the peak area were good.The RSD values of the precision,repeatability and stability test results were less than 3.00%,the average sample recovery was 98.86%-101.73%,and the RSD value was less than 3.00%.There was a significant difference in the contents of 5 components among different sources of Jiangcan(Bombyx Batryticatus).After cluster analysis,21 batches of samples were clustered into 3 categories.The origin samples,namely S1,S4,S5,S7,S8 and S9,were clustered into Class I,the market samples were clustered into Class II,and the origin samples,S2,S3 and S6,were clustered into Class III.Conclusion This method is simple,accurate and has good repeatability,and it can be used to simultaneously determine the contents of the 5 nucleoside components mentioned above in the Jiangcan(Bombyx Batryticatus).The quality of Jiangcan(Bombyx Batryticatus)in the same production area is relatively stable,with a higher content while in market has a complex source with a lower content.