黄春晖 胡军 廖乃英

【摘要】 目的 建立高效液相色譜法（HPLC）测定大黄碳酸钠片的组方药材大黄伪品中指标性成分土大黄苷的含量，并建立超高效液相色谱-串联质谱法（UPLC-MS/MS）确证方法。

方法 HPLC法采用色谱柱为CAPCELL PAK MGⅡ（4.6 mm×250 mm，5 μm），流动相为乙腈-水（20∶80），检测波长为325 nm，流速为1.0 mL·min-1，进样量为10 μL。UPLC-MS/MS确证方法采用色谱柱为Agilent Zorbax Eclipse Plus C18（2.1 mm×100 mm，1.8 μm），流动相为0.1%甲酸水溶液-乙腈（80∶20），流速为0.2 mL·min-1，柱温为40℃，进样量为2 μL;采用电喷雾离子源（ESI）在负离子模式下，利用多反应监测（MRM）模式对土大黄苷进行分析，定量及定性离子对分别为m/z 418.9→257.1和m/z 418.9→160.9。

结果 HPLC法方法学考察结果显示，土大黄苷在0.59～23.56 μg·mL-1质量浓度范围内与峰面积线性关系良好（r=0.9997），检出限为0.06 μg·mL-1，平均加样回收率为100.3%（n=9，RSD=1.5%）。12个批次的大黄碳酸氢片中均未检出土大黄苷成分。UPLC-MS/MS法检测结果显示，土大黄苷在0.024～1.178 μg·mL-1质量浓度范围内与峰面积线性关系良好（r=0.9996），检出限为0.7 ng·mL-1，平均加样回收率为99.4%（n=9，RSD=1.5%）。12个批次的大黄碳酸氢片中均未检出质荷比（m/z）418.9的准分子离子峰和m/z 257.1、160.9的碎片离子峰。

结论 所建立的HPLC法与UPLC-MS/MS法操作简便、快速灵敏，结果准确，可作为大黄碳酸钠片中非法成分土大黄苷的质量控制方法。

【关键词】 高效液相色谱法;超高效液相色谱-串联质谱法;大黄碳酸钠片;非法成份;土大黄苷;含量测定

中图分类号：R282.5   文献标志码：A   DOI：10.3969/j.issn.1003-1383.2021.07.002

【Abstract】 Objective To establish high performance liquid chromatography （HPLC） for the determination of rhaponiticin illegally added in rhubarb and sodium bicarbonate tablets， and to establish ultra performance liquid chromatography and tandem mass spectrometry （UPLC-MS/MS） for the identification of rhaponiticin.

Methods In HPLC method， chromatographic column was CAPCELL PAK MGⅡ（4.6 mm × 250 mm， 5 μm）， mobile phase was acetonitrile-water （20∶80）， detection wavelength was 325 nm， velocity of flow was 1.0 mL·min-1， and injection volume was 10 μL. In UPLC-MS/MS corroboration method， chromatographic column was Agilent Zorbax Eclipse Plus C18 （2.1 mm × 100 mm， 1.8 μm）， mobile phase was 0.1% methyl acid aqueous solution-acetonitrile （80∶20）， velocity of flow was 0.2 mL·min-1， column temperature was 40℃， injection volume was 2 μL. Electric spray ion source （ESI）  negative ion mode and multiple reaction monitoring （MRM） were used to  analyze rhaponiticin， and quantitative and qualitative ion pairs were m/z 418.9 → 257.1 and m/z 418.9 → 160.9， respectively.

Results Results of HPLC methodology investigation showed that within mass concentration range of 0.59-23.56 μg·mL-1， rhaponiticin had good relation with peak area （r=0.9997）， detection limit was 0.06 μg·mL-1， average recovery rate of adding sample was 100.3% （n = 9， RSD = 1.5%）. Results of UPLC-MS/MS detection showed that within mass concentration range of 0.024 -1.178 μg·mL-1， rhaponiticin had good relation with peak area （ r = 0.9996）， detection limit was 0.7 ng·mL-1， average recovery rate of adding sample was 99.4% （n = 9， RSD=1.5%）. No quasimolecular ions with m/z of 418.9 and fragment ions with m/z 257.1 and 160.9 were detected in 12 batches of rhubarb and sodium bicarbonate tablets.

Conclusion HPLC and UPLC-MS/MS methods are simple， rapid， sensitive and accurate， which can be used for the quality control of rhaponiticin illegally added in rhubarb and sodium bicarbonate tablets.

【Key words】 HPLC; UPLC-MS/MS; rhubarb and sodium bicarbonate tablet; illegal components; rhaponiticin; content determination

大黄碳酸氢钠片属于中西药复方制剂，其主要成分为大黄、碳酸氢钠和薄荷油[1]，用于治疗食欲不振、胃酸过多、消化不良和便秘等。大黄作为大黄碳酸氢钠片处方中的君药，其主要作用是引起唾液和胃液的分泌增多，使食欲增加，胃肠轻度充血，加强肠胃吸收功能[2]，临床主要用于治疗积滞便秘、血热呕吐、目赤咽肿、热毒疮疡、烧烫伤、血瘀证等[3～5]。……