目的: 建立2种半夏炮制品-厚朴药对的HPLC特征图谱分析方法及鸟苷、尿苷、肌苷、紫丁香苷、绿原酸、金丝桃苷、和厚朴酚、厚朴酚的含量测定的方法。方法: 采用Agilent Zorbax SB-C18色谱柱(4.6 mm × 250 nm,5 μm),以0.5%磷酸水溶液(A)-乙腈(B)为流动相,梯度洗脱,流速为1.0 mL·min-1,检测波长为210 nm,柱温为30 ℃。采用“中药色谱指纹图谱相似度评价系统”(2012版)软件建立20批样品的HPLC特征图谱并进行相似度评价,使用SPSS 24.0、SIMCA 14.1软件进行主成分分析(PCA)和偏最小二乘聚类分析(PLS-DA)。结果: 从供试品溶液中未检出肌苷,鸟苷、尿苷、紫丁香苷、绿原酸、金丝桃苷、和厚朴酚、厚朴酚在一定浓度范围内呈良好的线性关系(r=0.999 4~0.999 8),平均回收率(n=6)分别为97.1%、96.2%、97.1%、98.2%、96.1%、98.3%及96.9%(RSD<2.0%)。方法的精密度RSD均小于3.0%,重复性RSD小于3.0%,供试品溶液放置48 h内稳定。20批样品的相似度均大于0.9,表明样品相似度差异不大。主成分得分结果与聚类分析结果一致,都将样品分为4类。本研究共标定了34个共有峰,经主成分分析,主成分1～8是影响药材样品质量评价的主要因子;其中成分15、16、21、17、11、9、6、8、30、22、1、14、26、23、5、31和3(尿苷)对样品分组起关键作用,鸟苷、尿苷、紫丁香苷、绿原酸、金丝桃苷、和厚朴酚、厚朴酚的平均含量分别为0.047、0.075、0.425、0.623、0.065、4.994、8.724 mg·g-1。结论: 所建立的HPLC方法可用于同时测定半夏炮制品-厚朴药对中8个化学成分的含量,该方法高效、准确、重复性好,可用于半夏-厚朴药对的质量控制、评价。
Objective: To establish an HPLC characteristic chromatogram analysis method of herb pair of two kinds of processed Pinelliae Rhizoma-Magnoliae Officinalis Cortex, the content determination method of guanosine, uridine, inosine, syringin, chlorogenic acid, hypericin, honokiol and magnolol. Methods: Agilent Zorbax SB-C18 column (4.6 mm×250 nm, 5 μm) was used, with gradient elution of 0.5% phosphoric acid aqueous solution (A) - acetonitrile (B) at a flow rate of 1.0 mL·min-1. The detection wavelength was 210 nm and the column temperature was 30 ℃. The HPLC characteristic chromatograms of 20 batches of samples were established and evaluated by using the software of “TCM chromatographic fingerprint similarity evaluation system” (2012 Edition). Principal component analysis (PCA) and partial least squares-cluster analysis (PLS-DA) were performed by SPSS 24.0 and SIMCA 14.1 software. Results: Inosine was not detected in the test solution. Guanosine, uridine, inosine, syringin, chlorogenic acid, hypericin, honokiol and magnolol showed good linear relationship in certain concentration ranges (r=0.999 4-0.999 8). The average recoveries (n=6) were 97.1%, 96.2%, 97.1%, 98.2%, 96.1%, 98.3% and 96.9% respectively (RSD<2.0%). The RSDs for precision were below 3.0%, and the RSDs for repeatability were below 3.0%. The test solution was stable within 48 hours. The similarities of 20 batches of samples were above 0.9, which indicated little difference between the similarities of samples. The results of principal component analysis and cluster analysis were consistent, and the samples were divided into four categories. In this study, 34 common peaks were identified. Principal component analysis showed that principal components 1-8 were the main factors affecting the quality evaluation of medicinal materials. Among them, 15, 16, 21, 17, 11, 9, 6, 8, 30, 22, 1, 14, 26, 23, 5, 31 and 3 (uridine) played a key role in the sample grouping. The average contents of guanosine, uridine, syringin, chlorogenic acid, hypericin, honoliol and magnolol were 0.047, 0.075, 0.425, 0.623, 0.065, 4.994 and 8.724 mg·g-1, respectively. Conclusion: The established HPLC method can be used for the simultaneous determination of eight chemical components in Pinelliae Rhizoma-Magnoliae Officinalis Cortex pair. The method is efficient, accurate and reproducible, and can be used for the quality control and evaluation of Pinelliae Rhizoma-Magnoliae Officinalis Cortex pair.
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