黄芩素中有关物质的检测及其降解机制初步研究

Detection of Related Substances and Preliminary Study on the Degradation Mechanism of 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?Fluoro Sep-RP Phenyl,流动相为0.3%甲酸-甲醇-乙腈(梯度洗脱),流速为1.0 m L/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?Fluoro Sep-RP Phenyl,流动相为0.3%甲酸-甲醇(梯度洗脱),流速为1.0 m L/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11L/min,干燥气体温度为350℃,毛细管电压为4.0 k V,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 100~1 000(一级质谱),50~500(二级质谱),电离电压为80~135 e V,碰撞能量为10~30 e V。结果:黄芩素检测质量浓度线性范围为2.4~480μg/m L(r=0.999 9);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。

OBJECTIVE：To establish a method for the separation and detection of related substances in baicalein,identify itsstructure 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？Fluoro Sep-RP Phenyl with mobile phase of 0.3% formic acid-methanol-acetonitrile（gradient elution）at a flow rate of 1.0 m L/min,the detection wavelength was 275 nm,the column temperature was 10 ℃,and the injection volume was 10 μL. LC-MS/MS was conducted to identify the related substances and conjecture degradation mechanism：the column was ES Industries？Fluoro Sep-RP Phenyl with mobile phase of 0.3%formic acid- methanol（gradient elution）at a flow rate of 1.0 m L/min,the detection wavelength was 275 nm,column temperature was 10 ℃,and the injection volume was 10 μL;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 k V,detection modes were full-scan first-order MS and selective ion full-scan second-order MS,scan ranges were m/z 100-1 000（first-order MS） and50-500（second-order MS）,ionization voltage was 80-135 e V,and the collision energy was 10-30 e V. RESULTS：The linear range of baicalein was 2.4-480 μg/m L（r=0.999 9）;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.