五味消毒饮口服液HPLC指纹图谱的建立及6种指标性成分定量测定

Study on chemical fingerprint of Wuwei Xiaoduyin Oral Liquid and simultaneous determination of six characteristic components

目的建立五味消毒饮口服液(WXOL)的HPLC法特征指纹图谱,并同时测定绿原酸、木犀草苷、木犀草素、蒙花苷、咖啡酸、秦皮乙素6种主要指标成分的量。方法采用Agilent 1260高效液相色谱仪进行色谱分析,色谱柱为Agilent TC-C18柱(250 mm×4.6 mm,5μm);流动相为0.5%醋酸水溶液-甲醇,梯度洗脱:0~10 min,10%~32%甲醇;10~20 min,32%甲醇;20~25 min,32%~46%甲醇;25~31 min,46%~48%甲醇;31~41 min,48%~80%甲醇;体积流量为1 m L/min;检测波长为320 nm;柱温为30℃;采用国家药典委员会出版的《中药色谱指纹图谱相似度评价系统》(2012年版)对10批WXOL化学成分指纹图谱进行相似度评价,并采用多成分同时测定法对6种指标性成分进行定量测定。结果 10批WXOL HPLC特征指纹图谱相似度均大于0.98,共17个共有峰,各峰分离度较好。通过对照品比对确定了其中绿原酸(5号峰)、秦皮乙素(7号峰)、咖啡酸(8号峰)、木犀草苷(12号峰)、蒙花苷(16号峰)、木犀草素(17号峰)6个成分,10批WXOL中绿原酸量为54.038 3~105.551 1μg/m L,秦皮乙素量为4.122 1~31.359 9μg/m L,咖啡酸量为2.413 0~4.420 7μg/m L,木犀草苷量为4.042 8~11.312 8μg/m L,蒙花苷量为3.866 3~46.271 9μg/m L,木犀草素量为0.990 8~2.126 8μg/m L。不同批次间各指标性成分量变化较小,样品质量较稳定。结论所建立的HPLC特征指纹图谱结合多指标成分定量测定的方法,稳定性、重复性较好,有利于保证WXOL质量均一,对其质量控制具有参考价值。

Objective To establish the HPLC chemical fingerprints of Wuwei Xiaoduyin Oral Liquid（WXOL） and simultaneous determination method for six major characteristic components in this herbal preparation, i.e. chlorgenic acid, luteoloside, luteolin, linarin, caffeic acid, and esculetin. Methods Both chemical fingerprint analysis and quantitative determination were implemented by Agilent 1260 high performance liquid chromatography system with an Agilent 5 TC-C18 column（250 mm × 4.6 mm, 5 μm）. A gradient elution program, with the mobile phase of 0.5% glacial acetic acid aqueous solution（A） and methanol（B）, was employed as following： 0—10 min, 10%—32% B; 10—20 min, 32% B; 20—25 min, 32%—46% B; 25—31 min, 46%—48% B; 31—41 min, 48%—80% B at the flow rate of 1.0 m L/min. The detection wavelength and column temperature were set at 320 nm and 30 ℃, respectively. Additionally, fingerprint similarity of 10 batches of WXOL was evaluated by software ＂Similarity Evaluation System for Chromatographic Fingerprint of TCM＂ published by GPC（Version 2012）. The amounts of six characteristic components in WXOL were also simultaneously determined. Results The common fingerprint pattern derived from 10 batches of samples was obtained with the similarity over 0.98 and 17 common peaks defined. Meanwhile, some common peaks were identified via peak pattern match with standard substances, showing that the peak No.5, No.7, No.8, No.12, No.16, and No.17 was chlorgenic acid, esculetin, caffeic acid, luteoloside, linarin, and lutelin, respectively. Chlorgenic acid content range of 54.038 3—105.551 1 μg/m L, esculetin content range of 4.122 1—31.359 9 μg/m L, caffeic acid content range of 2.413 0—4.420 7 μg/m L, luteoloside content range of 4.042 8—11.312 8 μg/m L, linarin content range of 3.866 3—46.271 9 μg/m L, and luteolin content range of 0.990 8—2.126 8 μg/m L. In combination with the non-significant difference of multiple characteristic components content among 10 batches of samples, the quality of home-made WXOL would be stable. Conclusion A novel quality control method, which is HPLC fingerprint in combination with simultaneous quantitative analysis of multiple components, was established in this study, with high repeatability and reliability. Therefore, this method provides an applicable approach for the quality control of WXOL.