九香虫HPLC特征图谱构建和8个核苷类成分含量测定

Establishment of HPLC chromatogram and content determination of 8 nucleoside components of Aspongopus

目的通过HPLC建立九香虫药材特征图谱,并测定尿嘧啶、腺嘌呤、尿苷、尿酸、次黄嘌呤、腺苷、黄嘌呤和犬尿喹啉酸8个核苷类成分含量,为其质量控制及评价提供参考。方法采用Agilent ZORBAX SB-Aq色谱柱(4.6 mm×250 mm,5µm),以甲醇(A)-0.05%磷酸溶液(B)为流动相,梯度洗脱,柱温25℃,流速0.8 ml/min,检测波长254 nm,建立九香虫药材HPLC特征图谱,并对8个指标成分含量进行测定。结果建立了15批九香虫药材特征图谱,确定10个共有特征峰,并指认其中8个,各样品特征图谱与对照图谱相似度在0.969~0.997之间。含量测定结果表明,尿嘧啶、腺嘌呤、尿苷、尿酸、次黄嘌呤、腺苷、黄嘌呤和犬尿喹啉酸线性范围分别为0.0020~0.6440µg、0.0014~0.4480µg、0.0010~1.2570µg、0.0054~6.2210µg、0.0010~0.7240µg、0.0010~0.6440µg、0.0020~1.1130µg、0.0038~2.0590µg,线性关系良好(r≥0.999);重复性和稳定性RSD均<2.0%,平均加样回收率在99.36%~103.40%之间。结论本研究建立的特征图谱及核苷类成分含量测定方法简便、可靠、重复性好、精密度高,可用于九香虫药材的定性、定量分析,可为九香虫质量评价提供参考。

Objective To establish HPLC chromatogram for Aspongopus;To determine 8 nucleoside components of uracil,adenine,uridine,uric acid,hypoxanthine,adenosine,xanthine and canine quinolinic acid;To provide reference for quality control and evaluation.Methods The Agilent ZORBAX SB-Aq chromatographic column(4.6 mm×250 mm,5μm)was used for gradient elution with mobile phases consisting of a methanol(A)and 0.05%phosphoric acid(B).The column temperature was 25℃,the flow rate was 0.8 ml/min,and the detection wavelength was 254 nm.HPLC chromatograms for Aspongopus were established and the contents of 8 components were determined.Results The characteristic chromatogram of 15 batches aspongopus herbs was established.A total of 10 common characteristic peaks were identified and 8 were identified.The similarity between the characteristic chromatogram of samples and the control chromatogram was 0.969-0.997.The content determination showed that the linear range of uracil,adenine,urin,uric acid,hypoxanthine,adenosine,xanthine and xanuric acid was among 0.0020-0.6440,0.0014-0.4480,0.0010-1.2570,0.0054-6.2210,0.0010-0.7240,0.0010-0.6440,0.0020-1.1130,0.0038-2.0590μg,respectively,with a good linear relationship(r≥0.999);the repeatability and stability of RSD were<2.0%,and the average sampling recovery rate was between 99.36%and 103.40%.Conclusion The characteristic chromatogram and content determination method established in this study are simple,reliable,reproducible and accurate,and can be used for the qualitative and quantitative analysis of Aspongopus and can provide a reference for the quality evaluation method of the Aspongopus.