RP_HPLC梯度洗脱测定叶下珠中没食子酸的含量

1、RP-HPLC梯度洗脱测定叶下珠中没食子酸的含量王伽伯1,2,陈雷3,王玉4,肖小河2(11解放军第302医院,北京100039;21军事医学科学院,北京100850;31锦州医学院,辽宁锦州121001;41长春中医学院,吉林长春130117叶下珠P hy llan thus u rina ria L1为大戟科叶下珠属植物叶下珠的干燥全草,具有平肝清热、利水解毒的功效。现代药理实验证明,叶下珠具有抗乙肝病毒、保肝等作用1。没食子酸为其有效成分之一,是可水解鞣质的单体,具有抗病毒作用,在多种中药材中存在。然而,已报道的没食子酸测定方法对杂质干扰耐受力不强,重现性较差。没食子酸相对分子质量小,结

2、构中带有羟基和羧基,极性很大,通常只适合用正相键和柱分析,但是正相柱使用成本高,不及反相柱普及率高,如何建立适合反相色谱柱分析的方法尤显重要。在反相柱上测定没食子酸含量,关键是增大流动相中水相比例,延长没食子酸出峰时间,同时又能在较短时间内将样品中的低极性杂质干扰去除,保证进样分析的连续性。梯度洗脱即可实现这一目的。为此,笔者采用梯度洗脱技术测定没食子酸的含量,方法简便、快速、准确、重现性好。1仪器与试药高效液相色谱仪:惠普H P1110四元梯度泵;H P1110DAD检测器;H P Chem stati on化学工作站;甲醇为色谱纯;磷酸等其他试剂均为分析纯;没食子酸对照品(08312950

3、1(供含量测定用,中国药品生物制品检定所;叶下珠药材由中国医学科学院药用植物研究所西双版纳分所高海泉研究员提供。2方法与结果211色谱条件:K rom asil C18分析柱(250mm×416 mm,5m;检测波长:280nm;流动相:A甲醇,B 011%磷酸水溶液;梯度洗脱条件:015m in,A2B (1585;1517m in,A2B(9010,1732 m in,A2B(9010;3235m in,A2B(1585;体积流量:015mL m in;柱温为室温。212供试品溶液的制备:取叶下珠药材粗粉,精密称取012g,置锥形瓶中,精密加入甲醇25mL,称定质量,冷浸24h2

4、,再称定质量,用甲醇补足减失的质量,摇匀,滤过,取续滤液作为供试品溶液。213对照品溶液的制备:精密称取经五氧化二磷减压干燥36h的没食子酸对照品适量,加甲醇制成010235m g mL的溶液,即得。214线性关系的考察:分别精密吸取没食子酸对照品溶液2、4、8、12、16、20L,依次注入高效液相色谱仪,按上述色谱条件测定峰面积,以峰面积积分值为纵坐标,没食子酸的量(g为横坐标,绘制标准曲线,回归方程为:Y=487914X-3416,r= 019999(n=6,结果表明没食子酸在01047 01470g呈线性关系,可用外标一点法测定含量。215稳定性试验:取供试品溶液,分别于配制后0、2、4

5、、8、12、24h,依样品测定法测定,其峰面积的R SD=0151%,供试品溶液在24h内基本稳定。216精密度试验:精密吸取没食子酸对照品溶液10L,重复进样6次,测定其峰面积的R SD=0137%。217重复性试验:对同一样品取6份,平行测定,以外标法计算质量分数,平均质量分数11153m g g, R SD=0133%。218加样回收率试验:精密称取已知含量的同一批样品012g,精确加入没食子酸对照品溶液适量,按样品溶液的制备方法操作,测定其含量,并计算平均回收率为98105%,R SD=1105%(n=6。219样品测定:按样品测定方法测定3批药材样品,测定结果见表1。色谱图见图1。表

6、1样品测定结果(n=3Table1D eter m i nation results of sam ples(n=3批号没食子酸 (m gg-11111412111593111643讨论311流动相的选择:在C18柱上用文献2报道的流动相甲醇2水2磷酸(4960105进行分离,没食子酸峰形良好,但是大量极性相对较低的杂质吸附在(下转第131页621中草药Ch inese T raditi onal and H erbal D rugs第36卷第1期2005年 1月收稿日期:2004204211作者简介:王伽伯(1981,男,现为军事医学科学院2002级硕士研究生,研究方向药剂学。E2m ail

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