不同产地蚕蛹的HPLC指纹图谱研究及多组分含量测定

Study on HPLC Fingerprint of Bombyx mori from Different Producing Areas and Content Determination of Multi-component

目的:建立蚕蛹指纹图谱和多组分含量测定方法,为全面评价蚕蛹的质量提供参考。方法:以不同产地的18批蚕蛹为样品,采用高效液相色谱法(HPLC)进行测定。色谱柱为资生堂CAPCELL PAKC18 AQ S5,流动相为甲醇-0.05 mol/L磷酸二氢钾溶液(梯度洗脱),检测波长为260 nm,柱温为30℃,流速为1.0 mL/min,进样量为10μL。利用《中药色谱指纹图谱相似度评价系统(2012年版)》进行HPLC指纹图谱分析及相似度评价,并通过与对照品色谱图比对进行色谱峰的指认;测定蚕蛹样品中尿嘧啶、鸟嘌呤、黄嘌呤、尿苷等4个核苷类成分的含量。结果:在18批蚕蛹样品的HPLC指纹图谱中确定了16个共有峰,指认了3号峰为尿嘧啶、6号峰为鸟嘌呤、7号峰为黄嘌呤、8号峰为尿苷;各样品图谱与对照指纹图谱的相似度为0.912~1.000。尿嘧啶、鸟嘌呤、黄嘌呤、尿苷分别在5.34~534、5.28~528、5.06~506、5.195~519.5μg/mL质量浓度范围内与其峰面积线性关系良好(r均不低于0.9998);检测限分别为0.0324、0.0320、0.0307、0.0312μg/mL,定量限分别为0.1068、0.1056、0.1012、0.1030μg/mL;精密度、重复性、稳定性(24 h)试验的RSD均小于1.00%(n=6);平均加样回收率为100.15%~102.95%,RSD均小于2.00%(n=9)。含量测定结果显示,不同产地蚕蛹中尿嘧啶、鸟嘌呤、黄嘌呤、尿苷的含量分别为0.41%~2.46%、0.37%~1.98%、0.72%~2.63%、0.94%~3.67%。结论:所建蚕蛹HPLC指纹图谱及其4个核苷类成分含量测定方法的专属性较强,且准确、可靠,可用于蚕蛹的质量评价及质量控制。

OBJECTIVE:To establish the fingerprint of Bombyx mori and the method for the content determination of multi components,and to provide reference for comprehensive quality evaluation of B.mori.METHODS:Using 18 batches of B.mori from different producing areas as samples,HPLC method was used.The column was Shiseido CAPCELL PAK C18 AQ S5 with mobile phase consisted of methanol-0.05 mol/L potassium dihydrogen phosphate solution(gradient elution)at the flow rate of 1.0 mL/min.The detection wavelength was 260 nm,and column temperature was set at 30℃.The sample size was 10μL.HPLC fingerprint analysis and similarity evaluation were performed by using TCM Chromatogram Fingerprint Similarity Evaluation System(2012 edition),and the chromatographic peak was identified by comparing with the chromatogram of reference substance.The contents of 4 nucleosides as uracil,guanine,xanthine,uridine were determined.RESULTS:A total of 16 common peaks were identified in HPLC fingerprint for 18 batches of B.mori,and peaks 3,6,7 and 8 were identified as uracil,guanine,xanthine and uridine.The similarity of sample chromatogram with control fingerprint were 0.912-1.000.The linear range of uracil,guanine,xanthine and uridine were 5.34-534,5.28-528,5.06-506,5.195-519.5μg/mL(r≥0.9998).The limits of detection were 0.0324,0.0320,0.0307,0.0312μg/mL,and the limits of quantitation were 0.1068,0.1056,0.1012,0.1030μg/mL.RSDs of precision,reproducibility and stability tests(24 h)were all lower than 1.00%(n=6).Average recoveries were 100.15%-102.95%,and RSDs were all lower than 2.00%(n=9).The content determination results showed that the content of uracil,guanine,xanthine and uridine of B.mori from different producing areas were 0.41%-2.46%,0.37%-1.98%,0.72%-2.63%,0.94%-3.67%,respectively.CONCLUSIONS:Established HPLC fingerprint and content determination method of 4 nucleosides were specific,accurate and reliable,which can be used for the quality evaluation and control of B.mori.