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[摘要]
目的 采用HPLC-DAD法同时测定拨云退翳丸中甘草苷、甘草酸铵、蔓荆子黄素、胡薄荷酮、阿魏酸、绿原酸、3,5-O-二咖啡酰基奎宁酸、盐酸小檗碱、山柰素、蒙花苷10种成分。方法 应用Shim-pack VP-ODS C18色谱柱(250 mm×4.6 mm,5 μm),流动相为甲醇-乙腈(50∶50,A)和0.05%磷酸水溶液(B),梯度洗脱,梯度洗脱程序为0~5 min,50% A;5~30 min,50%~80% A;30~32 min,80%~50% A;32~40 min,50% A;进样体积10 μL,体积流量1.0 mL/min,柱温40℃。结果 甘草苷、甘草酸铵、蔓荆子黄素、胡薄荷酮、阿魏酸、绿原酸、3,5-O-二咖啡酰基奎宁酸、盐酸小檗碱、山柰素、蒙花苷10种成分能够达到很好分离;其线性范围分别为2~20(r=0.999 2)、20~200(r=0.999 5)、3~30(r=0.999 4)、2~20(r=0.999 7)、1.2~12.0(r=0.999 5)、3.5~35.0(r=0.999 2)、8~80(r=0.999 3)、9~90(r=0.999 3)、2~20(r=0.999 6)、3~30 μg/mL(r=0.999 5);平均加样回收率分别为99.1%、101.1%、100.2%、99.4%、101.9%、98.5%、100.5%、101.7%、100.8%、99.7%,RSD分别为0.62%、0.79%、0.77%、0.83%、0.47%、0.38%、0.97%、1.05%、0.86%、1.11%(n=6)。6批次供试品中甘草苷、甘草酸铵、蔓荆子黄素、胡薄荷酮、阿魏酸、绿原酸、3,5-O-二咖啡酰基奎宁酸、盐酸小檗碱、山柰素、蒙花苷质量分数分别为0.505~0.685、1.793~2.012、0.227~0.268、0.183~0.206、1.258~1.324、0.348~0.381、0.648~0.720、1.544~1.722、1.543~1.627、3.434~3.883 mg/丸。结论 该方法快速、灵敏度高、准确度高、专属性好,为拨云退翳丸的质量控制提供依据。
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[Abstract]
Objective To develop an HPLC-DAD method for the simultaneous determination of liquiritin, ammonium glycyrrhizinate, vitexicarpin, pulegone, ferulic acid, chlorogenic acid, 3,5-dicaffeoyl quinic acid, berberine hydrochloride, kaempferol, and buddleoside in Boyun Tuiyi Pill (BTP). Methods Shim-pack VP-ODS C18 column (250 mm×4.6 mm, 5 μm) was adopted. The mobile phase was composed of methanol-acetonitrile (50:50, A) and 0.05% phosphoric acid (B) with gradient elution. Gradient elution:0-5.0 min, 50% A; 5.0-30.0 min, 50%-80% A; 30.0-32.0 min, 80%-50% A; and 32.0-40.0 min, 50% A; Injection volume was 10 μL. The flow rate was 1.0 mL/min and the column temperature was 40℃. Results liquiritin, ammonium glycyrrhizinate, vitexicarpin, pulegone, ferulic acid, chlorogenic acid, 3,5-dicaffeoyl quinic acid, berberine hydrochloride, kaempferol, and buddleoside were separated well. The linear calibration curves were obtained in 2-20 μg/mL for liquiritin, r=0.999 2; 20-200 μg/mL for ammonium glycyrrhizinate, r=0.999 5; 3-30 μg/mL for vitexicarpin, r=0.999 4; 2-20 μg/mL for pulegone, r=0.999 7; 1.2-12.0 μg/mL for ferulic acid, r=0.999 5; 3.5-35.0 μg/mL for chlorogenic acid, r=0.999 2; 8-80 μg/mL for 3,5-dicaffeoyl quinic acid, r=0.999 3; 9-90 μg/mL for berberine hydrochloride, r=0.999 3; 2-20 μg/mL for kaempferol, r=0.999 6; and 3-30 μg/mL for buddleoside, r=0.999 5. The average recoveries of the 10 constituents were 99.1%, 101.1%, 100.2%, 99.4%, 101.9%, 98.5%, 100.5%, 101.7%, 100.8%, and 99.7% with RSD of 0.62%, 0.79%, 0.77%, 0.83%, 0.47%, 0.38%, 0.97%, 1.05%, 0.86%, and 1.11%. The contents of six batches of the liquiritin, ammonium glycyrrhizinate, vitexicarpin, pulegone, ferulic acid, chlorogenic acid, 3,5-dicaffeoyl quinic acid, berberine hydrochloride, kaempferol, and buddleoside were 0.505-0.685, 1.793-2.012, 0.227-0.268, 0.183-0.206, 1.258-1.324, 0.348-0.381, 0.648-0.720, 1.544-1.722, 1.543-1.627, and 3.434-3.883 mg/pill, respectively. Conclusion The method is rapid and has high sensitivity, high accuracy, and good specificity. It can be applied to the quality control of BTP.
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