黄芩素中有关物质的检测及其降解机制初步研究

摘要

OBJECTIVE:To establish a method for the separation and detection of related substances in baicalein,identify its structure and preliminarily explore the degradation mechanism. METHODS:HPLC was adopted to detect the baicalein,related impurities and forced destruction of degradation products in synthesis process:the column was ES Industries? FluoroSep-RP Phenyl with mobile phase of 0.3%formic acid-methanol-acetonitrile(gradient elution)at a flow rate of 1.0 mL/min,the detection wavelength was 275 nm,the column temperature was 10℃,and the injection volume was 10μL. LC-MS/MS was conducted to identify the related substances and conjecture degradation mechanism:the column was ES Industries? FluoroSep-RP Phenyl with mobile phase of 0.3%formic acid- methanol (gradient elution)at a flow rate of 1.0 mL/min,the detection wavelength was 275 nm,column temperature was 10℃,and the injection volume was 10μL;ion source was electrospray ion source,positive and negative ions,nebulizer pressure was 55 psi and the drying gas flow was 11 L/min,drying gas temperature was 350℃,capillary voltage was 4.0 kV,detection modes were full-scan first-order MS and selective ion full-scan second-order MS,scan ranges were m/z 100-1000 (first-order MS) and 50-500(second-order MS),ionization voltage was 80-135 eV,and the collision energy was 10-30 eV. RESULTS:The linear range of baicalein was 2.4-480μg/mL(r=0.9999);RSDs of precision,stability and reproducibility tests were lower than 2.0%;the limit of quantitation was 7.2 ng,the limit of detection was 2.4 ng. Baicalein was well separated with related substance and 3 major degradation products,the related substance was chemical synthesis precursor wood butterfly;the degradation products were 6,7-quinone derivatives and 7,8-quinone derivatives,which were isomers;oxidative degradation products were benzoic acid phenyl ester derivatives. CONCLUSIONS:The main mechanisms of alkali degradation and oxidative degradation of baicalein include pyran, reciprocal rearrangement and oxidation reaction;the established method is specific and sensitive,and can be used for the detection of related substances in baicalein.%目的:建立黄芩素中有关物质的分离检测方法,鉴定其结构并初步探讨降解机制.方法:采用高效液相色谱法(HPLC)检测黄芩素与合成过程中的有关杂质及强制破坏降解产物:色谱柱为ES Industries?FluoroSep-RP Phenyl,流动相为0.3%甲酸-甲醇-乙腈(梯度洗脱),流速为1.0 mL/min,检测波长为275 nm,柱温为10℃,进样量为10μL.采用LC-串联质谱法鉴定有关物质并推测降解机制:色谱柱为ES Industries?FluoroSep-RP Phenyl,流动相为0.3%甲酸-甲醇(梯度洗脱),流速为1.0 mL/min,检测波长为275 nm,柱温为10℃,进样量为10μL;离子源为电喷雾离子源,正负离子同时检测,雾化器压力为55 psi,干燥气体流速为11 L/min,干燥气体温度为350℃,毛细管电压为4.0 kV,检测模式为全扫描一级质谱和选择离子全扫描二级质谱,扫描范围为m/z 100~1000(一级质谱),50~500(二级质谱),电离电压为80~135 eV,碰撞能量为10~30 eV.结果:黄芩素检测质量浓度线性范围为2.4~480μg/mL(r=0.9999);精密度、稳定性、重复性试验的RSD<2.0%;定量限、检测限分别为7.2、2.4 ng.黄芩素与有关物质及3个主要降解产物分离良好,有关物质为化学合成前体木蝴蝶素;碱降解产物为6,7位邻醌衍生物和7,8位邻醌衍生物,两者互为异构体;氧化降解产物为苯甲酸苯酯类衍生物.结论:黄芩素碱降解和氧化降解的主要机制包括吡喃环开环、互变重排和氧化反应等;该研究所建方法专属性好、灵敏度高,可用于黄芩素有关物质的分离检测.