目的:建立元胡止痛片的HPLC指纹图谱方法及延胡索乙素对映异构体拆分方法,评价元胡止痛片的质量现状,以提高元胡止痛系列制剂的质量可控性。方法:HPLC指纹图谱方法采用资生堂MG C18色谱柱(4.6 mm×250 mm,5 μm),以乙腈-四氢呋喃(5:3)为流动相A,0.6%冰乙酸溶液(用三乙胺调节pH至6.0)为流动相B进行梯度洗脱,检测波长为220 nm,流速为1 mL·min-1,柱温为30 ℃;基于中心切割-二维液相色谱法,建立元胡止痛片中延胡索乙素对映异构体的手性拆分方法,以左旋延胡索乙素(L-tetrahydropalmatine,L-THP)和右旋延胡索乙素(D-tetrahydropalmatine,D-THP)峰面积的比值为指标,比较不同样品之间的差异。结果:指纹图谱结果表明,可将39个生产企业的156批样品分为A类和B类,其中7家生产企业的25批样品对照指纹图谱相似度低于0.8,被归为B类样品,其指纹图谱表现为,除延胡索乙素消旋体(DL-tetrahydropalmatine,DL-THP)对应的指纹峰外,其他来自延胡索的特征峰均较小甚至缺失;中心切割-二维液相色谱的结果表明,DL-THP经中心切割阀转切到二维色谱中,被拆分成D-THP与L-THP,B类样品中D-THP与L-THP的峰面积比值小于1.0,A类样品则相反。结论:部分生产企业延胡索药材投料存在一定问题,可能通过违规添加化学药品(罗通定)的方式,以减少成本并符合《中华人民共和国药典》含量测定的规定,这可能是导致指纹图谱存在差异的主要原因。本研究建立的指纹图谱方法简便,准确,重复性好,能有效地反映元胡止痛片的质量;以延胡索乙素对映异构体的比值作为评价指标,分析和阐明了元胡止痛片的质量问题,可作为其质量控制的重要手段,同时为其他含有延胡索的处方制剂、或其他含有对映异构体化合物的中药材和中成药的质量控制提供新思路。
Objective: To establish the HPLC fingerprint method and the separation method of tetrahydropalmatine enantiomers of Yuanhu Zhitong tablets, so as to evaluate the overall quality condition and improve the quality controllability of Yuanhu Zhitong tablets. Methods: HPLC fingerprint was performed on a Shiseido MG C18 chromatographic column (4.6 mm×250 mm, 5 μm).The mobile phase consisted of acetonitrile-tetrahydrofuran(A) and 0.6% glacial acetic acid solution (using triethylamine to adjust the pH value to 6.0, B) with gradient elution. The flow rate was 1 mL·min-1, the detection wavelength was 220 nm and the column temperature was 30 ℃. A chiral separation method of tetrahydropalmatine enantiomers in Yuanhu Zhitong tablets was established based on the heart-cutting two-dimensional liquid chromatography technology.The peak area ratio of L-tetrahydropalmatine(L-THP) to D-tetrahydropalmatine(D-THP) was used as an indicator to compare the difference between samples. Results: The fingerprint results showed that 156 batches of samples from 39 manufacturing enterprises could be divided into classes of A and B. Among them, 25 batches of samples from 7 manufacturing enterprises were classified as class B samples with similarities to the reference fingerprint below 0.8. And their fingerprint differences were as follows:except the fingerprint peak corresponding to DL-THP(DL-tetrahydropalmatine), other characteristic peaks from Corydalis yanhusuo W.T.Wang were small or even missing. The results of heart-cutting two-dimensional liquid chromatography showed that DL-THP was transferred to the second-dimensional chromatography through the heart-cutting valve and was separated as D-THP and L-THP. The ratio of the peak area of D-THP to L-THP were less than 1.0 in class B samples, while the opposite was found in class A samples, which was the most important difference between samples. Conclusion: There are some problems in the raw materials from some enterprises, and they may add chemicals(rotundine) illegally to reduce costs and comply with pharmacopoeia.The HPLC fingerprint method is simple, accurate, highly reproducible and sensitive, providing scientific basis for the overall quality control of Yuanhu Zhitong tablets.Taking the ratio of tetrahydropalmatine enantiomers as the evaluation index, the quality problems of Yuanhu Zhitong tablets are analyzed and clarified, which can be used as an important mean for its quality control.At the same time, it providesa new idea for quality control of other prescription preparations containing tetrahydropalmatine, or other Chinese medicinal materials and Chinese patent medicines containing enantiomer compounds.
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