目的 建立金振口服液（Jinzhen Oral Liquid，JOL）高效液相色谱-紫外真空波-蒸发光散射检测器（HPLC-UVD-ELSD）特征指纹图谱，结合13个主要代表性成分（没食子酸、甘草苷、芦荟大黄素-8-O-β-D-葡萄糖苷、甘草素、黄芩苷、白杨素-7-O-β-D-葡萄糖醛酸、千层纸苷、汉黄芩苷、大黄酚-1-O-β-D-葡萄糖苷、大黄酚-8-O-β-D葡萄糖苷、甘草酸、猪去氧胆酸、胆酸）同时定量的方法，对市售制剂进行整体质量控制。方法 采用Cosmosil-C₁₈色谱柱（250 mm×4.6 mm，5 μm）；检测波长为254 nm；ELSD漂移管温度115℃；载气体积流量2.0 L/min；以甲醇-水（含0.1%甲酸）为流动相进行梯度洗脱，建立JOL的HPLC-UVD-ELSD特征指纹图谱，对主要特征峰进行明确化学指认并确定组方中药来源，运用相似度软件对15批市售制剂进行相似度评价，同时在市售制剂批间差异小的前提下测定主要代表性成分的含量。结果 建立了JOL的HPLC-UVD-ELSD指纹图谱结合13个代表性成分同时定量的方法；覆盖该复方4味组方中药的15个主要特征峰得到明确化学指认，分别为没食子酸（1号峰）、甘草苷（5号峰）、芦荟大黄素-8-O-β-D-葡萄糖苷（7号峰）、甘草素（11号峰）、黄芩苷（13号峰）、白杨素-7-O-β-D-葡萄糖醛酸（16号峰）、千层纸苷（17号峰）、汉黄芩苷（18号峰）、大黄酚-8-O-β-D-葡萄糖苷（19号峰）、大黄酚1-O-β-D-葡萄糖苷（20号峰）、大黄酸（24号峰）、甘草酸（26号峰）、（18β，20α）-甘草酸（27号峰）、猪去氧胆酸（28号峰）、胆酸（29号峰），并初步确定了29个特征峰的组方中药来源分别为8、12、13、15～18号峰归属于黄芩，3～5、10、11、25～27号峰归属于甘草，1、6、7、9、14、19、20、24号峰归属于大黄，2号峰归属于平贝母，28、29号峰归属于人工牛黄，21～23号峰归属于辅料；15批市售制剂的指纹图谱与对照指纹图谱的相似度值在0.968～1.000；13个定量成分线性关系良好（R²＝0.999 0～0.999 9），且平均回收率为96.90%～102.84%，15批市售制剂定量成分的质量浓度分别为没食子酸51.82～148.27 μg/mL、甘草苷75.04～130.00 μg/mL、芦荟大黄素-8-O-β-D-葡萄糖苷31.72～39.84 μg/mL、甘草素14.24～43.65 μg/mL、黄芩苷610.37～867.40 μg/mL、白杨素-7-O-β-D-葡萄糖醛酸12.87～34.09 μg/mL、千层纸苷62.45～101.48 μg/mL、汉黄芩苷155.41～205.86 μg/mL、大黄酚-1-O-β-D-葡萄糖苷11.56～23.72 μg/mL、大黄酚-8-O-β-D葡萄糖苷16.14～36.87 μg/mL、甘草酸222.97～310.32 μg/mL、猪去氧胆酸177.48～239.70 μg/mL、胆酸98.54～132.85 μg/mL。结论 所建立的金振口服液HPLC-UVD-ELSD特征图谱和定量测定分析方法为进一步提升制剂整体质量标准提供了重要证据。

Objective To establish the fingerprint of Jinzhen Oral Liquid (JOL) by HPLC-UVD-ELSD and determine the main 13 representative components (gallic acid, liquiritin, aloe-emodin-8-O-β-D-glucopyranoside, liquiritigenin, baicalin, chrysin-7-O-β-D-glucoronide, oroxyloside, wogonoside, chrysophal-1-O-β-D-glucopyranoside, chrysophal-8-O-β-D-glucopyranoside, rhein, glycyrrhizic acid, hyodeoxycholic acid and cholic acid) simultaneously, in order to provide reference for the overall quality control of JOL. Methods The separation was developed on Cosmosil-C₁₈ column (250 mm×4.6 mm, 5 μm) by gradient elution with methanol-water [containing 0.1% formic acid] at 254 nm, the temperature of drift tube was maintained at 115 ℃ and the carrier gas flow rate was 2.0 L/min. An HPLC-UVD-ELSD fingerprint of JOL was set up, and 15 batches of JOL were evaluated by similarity assay. Furthermore, the contents of the main 13 representative components were determined on the premise of small disparities among batches. Results The HPLC-UVD-ELSD fingerprint of JOL was established with good separation, and 13 chemical components were determined simultaneously. Fifteen main characteristic peaks [gallic acid (peak 1), liquiritin (peak 5), aloe-emodin-8-O-β-D-glucopyranoside (peak 7), liquiritigenin (peak 11), baicalin (peak 13), chrysin-7-O-β-D-glucoronide (peak 16), oroxyloside (peak 17), wogonoside (peak 18), chrysophal-8-O-β-D-glucopyranoside (peak 19), chrysophal-1-O-β-D-glucopyranoside (peak 20), rhein (peak 24), glycyrrhizic acid (peak 26), (18β,20α)-glycyrrhizic acid (peak 27), hyodeoxycholic acid (peak 28), cholic acid (peak 29)] from four formula of JOL were chemically identified and 29 main characteristic peaks were assigned to individual herbs (peaks 8, 12, 13, 15—18 originate from Scutellariae Radix, peaks 3—5, 10, 11, 25—27 originate from Glycyrrhizae Radix et Rhizoma, peaks 1, 6, 7, 9, 14, 19, 20, 24 originate from Rhei Radix et Rhizoma, peak 2 originates from Fritillariae Ussuriensis Bulbus, peaks 28, 29 originate from Bovis Calculus Artifactus, peaks 21—23 originate from auxiliary materials). The similarity of 15 batches of JOL was about 0.968 to 1.000. Moreover, good linear relationships were found (R²＝0.999 0—0.999 9), and the average recovery rates were 96.90%—102.84%. The content range of quantitative components in 15 batches of JOL (gallic acid 51.82—148.27 μg/mL, liquiritin 75.04—130.00 μg/mL, aloe-emodin-8-O-β-D-glucopyranoside 31.72—39.84 μg/mL, liquiritigenin 14.24—43.65 μg/mL, baicalin 610.37—867.40 μg/mL, chrysin-7-O-β-D-glucoronide 12.87—34.09 μg/mL, oroxyloside 62.45—101.48 μg/mL, wogonoside 155.41—205.86 μg/mL, chrysophal-1-O-β-D-glucopyranoside 11.56—23.72 μg/mL, chrysophal-8-O-β-D- glucopyranoside 16.14—36.87 μg/mL, glycyrrhizic acid 222.97—310.32 μg/mL, hyodeoxycholic acid 177.48—239.70 μg/mL, cholic acid 98.54—132.85 μg/mL) was determinated. Conclusion The qualitative and quantitative methods of HPLC-UVD-ELSD mentioned above provided important evidence for further improving the overall quality standard of JOL.

国家自然科学基金重点项目（81630097）；现代中药创新集群与数字制药技术平台“重大新药创制”科技重大专项（2013ZX09402203）