HPLC-QAMS法同时测定紫花烧伤软膏中10种有效成分的含量

Simultaneous determination of 10 active ingredients in Zihua Burn Ointment by HPLC-QAMS

目的建立同时测定紫花烧伤软膏中毛蕊花糖苷、焦地黄苯乙醇苷B_(1)、羟基-α-山椒素、羟基-β-山椒素、羟基-γ-山椒素、乙酰紫草素、β-乙酰氧基异戊酰阿卡宁、异丁酰紫草素、β,β′-二甲基丙烯酰阿卡宁和异戊酰紫草素含量的高效液相色谱-一测多评法(high performance liquid chromatography-quantitative analysis of multi-components by single-marker,HPLC-QAMS)。方法用Acclaim 120 C_(18)色谱柱;流动相为乙腈-1 mL·L^(-1)磷酸,梯度洗脱;检测波长分别为330 nm(检测毛蕊花糖苷和焦地黄苯乙醇苷B_(1))、275 nm(检测羟基-α-山椒素、羟基-β-山椒素、羟基-γ-山椒素、乙酰紫草素、β-乙酰氧基异戊酰阿卡宁、异丁酰紫草素、β,β′-二甲基丙烯酰阿卡宁和异戊酰紫草素);以乙酰紫草素为内参物,建立其与其他9种成分的相对校正因子(relative correction factor,RCF),计算各成分的含量,与外标法(external standard method,ESM)进行比较,验证所建立的HPLC-QAMS法的准确性和可行性。结果毛蕊花糖苷、焦地黄苯乙醇苷B_(1)、羟基-α-山椒素、羟基-β-山椒素、羟基-γ-山椒素、乙酰紫草素、β-乙酰氧基异戊酰阿卡宁、异丁酰紫草素、β,β′-二甲基丙烯酰阿卡宁和异戊酰紫草素质量浓度分别在3.57~178.50、1.49~74.50、2.88~144.00、1.26~63.00、0.68~34.00、4.91~245.50、2.55~127.50、2.39~119.50、3.17~158.50、7.65~382.50 mg·L^(-1)范围内与其峰面积呈良好的线性关系(r≥0.9991);所得相对校正因子耐用性良好,HPLC-QAMS法和外标法含量测定结果的差异无统计学意义(P>0.05)。结论所建立以乙酰紫草素为内参物的HPLC-QAMS法结果准确、方法可行,可用于紫花烧伤软膏中多指标成分的定量控制和综合质量评价。

Objective To establish a high performance liquid chromatography-quantitative analysis of multi-components by single-marker method(HPLC-QAMS)for the simultaneous determination of verbascoside,jionoside B_(1),hydroxy-α-sanshool,hydroxy-β-sanshool,hydroxy-γ-sanshool,acetylshikonin,β-acetoxyisovalerylalkannin,isobutylshikonin,β,β′-dimethylacrylalkannin and isovalerylshikonin in Zihua Burn Ointment.Methods An Acclaim 120 C_(18)was used as the chromatographic column with acetonitrile-1 mL·L^(-1)phosphoric acid as the mobile phase with gradient elution.The detection wavelengths were set at 330 nm for verbascoside and jionoside B_(1),and 275 nm for hydroxy-α-sanshool,hydroxy-β-sanshool,hydroxy-γ-sanshool,acetylshikonin,β-acetoxyisovalerylalkannin,isobutylshikonin,β,β′-dimethylacrylalkannin and isovalerylshikonin.The relative correction factors(RCFs)of other 9 components were established,and the content of each component was calculated.Compared with the external standard method(ESM),the accuracy and feasibility of the established HPLC-QAMS method were verified.Results Verbascoside,jionoside B_(1),hydroxy-α-sanshool,hydroxy-β-sanshool,hydroxy-γ-sanshool,acetylshikonin,β-acetoxyisovalerylalkannin,isobutylshikonin,β,β′-dimethylacrylalkannin and isovalerylshikonin showed good linear relationships with their peak areas at the mass concentrations of 3.57-178.50,1.49-74.50,2.88-144.00,1.26-63.00,0.68-34.00,4.91-245.50,2.55-127.50,2.39-119.50,3.17-158.50 and 7.65-382.50 mg·L^(-1)(r≥0.9991),respectively.Acetylshikonin as an internal reference,the relative correction factors of verbascoside,jionoside B_(1),hydroxy-α-sanshool,hydroxy-β-sanshool,hydroxy-γ-sanshool,β-acetoxyisovalerylalkannin,isobutylshikonin,β,β′-dimethylacrylalkannin and isovalerylshikonin showed good durability.There was no significant difference between ESM and HPLC-QAMS method(P>0.05).Conclusion The established HPLC-QAMS method with acetylshikonin as internal reference was accurate and feasible,which could be used for the quantitative control of multi-index components and comprehensive quality evaluation of Zihua Burn Ointment.