蚂蟥药材炮制前后质量研究

Quality Study of Whitmania Pigra Whitman Before and After Processing

目的为蚂蟥药材及饮片(烫蚂蟥)的鉴别与质量评价提供依据。方法采用高效液相色谱(HPLC)法建立蚂蟥药材、饮片各15批的特征图谱。色谱柱为Agilent Zorbax SB-Aq柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.01%甲酸水溶液(梯度洗脱),流速为1.0 mL/min,检测波长为254 nm,柱温为30℃,进样量为5μL;质谱条件,加热电喷雾正离子模式(HESI+),鞘气流速为60 L/min,辅助气流速为18 L/min,喷雾电压为2.50 kV,毛细管温度为288℃,辅助气温度为475℃,扫描模式为全扫描二级质谱。指认共有峰,并采用中药色谱指纹图谱相似度评价系统(2012.0版)进行相似度评价。将样品的11个特征峰导入SIMCA 14.1软件进行聚类分析、主成分分析(PCA)、偏最小二乘法判别分析(PLS-DA),探讨蚂蟥药材及饮片的区分度。采用HPLC法测定指认出的5种成分的含量。结果蚂蟥药材及饮片的HPLC特征图谱分别确定8个和10个共有峰(7个相同,指认出其中5个)。药材和饮片样品的相似度均大于0.990。聚类分析、PCA、PLS-DA分析均能将药材和饮片分别聚为一类;PLS-DA分析结果显示,色谱峰4,7,9-11为区分蚂蟥药材与饮片的主要差异化合物。尿嘧啶、次黄嘌呤、黄嘌呤、水蛭胺C羧基衍生物、水蛭胺B质量浓度分别在0.12~12.41μg/mL,1.20~119.62μg/mL,0.92~92.08μg/mL,1.23~122.90μg/mL,1.27~126.83μg/mL范围内与峰面积线性关系良好(r>0.9999,n=5);精密度、稳定性、重复性试验结果的RSD均小于3.0%;平均加样回收率分别为100.03%,98.37%,92.06%,93.40%,99.93%,RSD分别为2.03%,1.94%,2.61%,1.86%,1.66%(n=6)。结论该研究中建立的方法操作简便、快捷、结果可靠,可为蚂蟥药材及其炮制品的区分及质量评价提供参考。

Objective To provide a basis for the identification and quality evaluation of Whitmania pigra Whitman and its decoction pieces(scalded Whitmania pigra Whitman).Methods High-performance liquid chromatography(HPLC)method was used to establish the characteristic chromatograms of 15 batches of Whitmania pigra Whitman and 15 batches of its decoction pieces.The chromatographic column was Agilent Zorbax SB-Aq column(250 mm×4.6 mm,5μm),the mobile phase was acetonitrile-0.01%formic acid aqueous solution(gradient elution),the flow rate was 1.0 mL/min,the detection wavelength was 254 nm,the column temperature was 30℃,and the injection volume was 5μL.In the mass spectrum,the heated electrospray positive ionization mode(HESI+)was adopted,the flow rate of sheath gas was 60 L/min,the flow rate of auxiliary gas was 18 L/min,the spray voltage was 2.50 kV,the capillary temperature was 288℃,the auxiliary gas temperature was 475℃,the scanning mode was full-scan secondary mass spectrometry.The common peaks were identified,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012.0 Version)was used for similarity evaluation.Eleven characteristic peaks were imported into the SIMCA 14.1 software to perform the cluster analysis,principal component analysis(PCA)and partial least squares-discrimination analysis(PLS-DA),on this basis,the differentiation of Whitmania pigra Whitman and its decoction pieces was explored.The content of five identified components was determined by the HPLC method.Results Eight and ten common peaks in the HPLC characteristic chromatograms of Whitmania pigra Whitman and its decoction pieces were obtained respectively,including seven identical common peaks,five of which were identified.The similarity between the medicinal herbs and decoction pieces was greater than 0.990.The medicinal herbs and decoction pieces could be clustered into one group separately by the cluster analysis,PCA and PLS-DA.The PLS-DA showed that the chromatographic peak 4,peak 7 and peaks 9-11 were the main differential compounds of Whitmania pigra Whitman and decoction pieces.The linear ranges of uracil,hypoxanthine,xanthine,carboxy derivatives of hirudonucleodisulfide C and hirudonucleodisulfide B were 0.12-12.41μg/mL,1.20-119.62μg/mL,0.92-92.08μg/mL,1.23-122.90μg/mL,1.27-126.83μg/mL(r>0.9999,n=5),respectively.The RSDs of precision,stability and repeatability tests were all lower than 3.0%.The average recovery rates of above five components were 100.03%,98.37%,92.06%,93.40%,99.93%,with the RSDs of 2.03%,1.94%,2.61%,1.86%,1.66%(n=6),respectively.Conclusion The established method is simple,fast and reliable,which can provide a reference for the differentiation and quality evaluation of Whitmania pigra Whitman and its decoction pieces.