麻黄-杏仁药对有效成分在大鼠体内组织分布的定量分析

目的:建立并验证同时测定大鼠组织中去甲基麻黄碱、去甲基伪麻黄碱、麻黄碱、伪麻黄碱、甲基麻黄碱、苦杏仁苷和野樱苷的UPLC-MS/MS,分析大鼠口服麻黄-杏仁药对水提物后有效成分在主要脏器组织中的分布情况。方法:参照FDA生物样本分析方法的方法学验证指南完成该测定的方法验证。采用盐酸苯海拉明和葛根素2种内标物分别对5种麻黄生物碱和苦杏仁苷、野樱苷进行定量分析。结果:该方法灵敏可靠,9种化合物(含3对差向异构体)在18 min内快速分离。麻黄生物碱在各组织中广泛分布;苦杏仁苷在体内分布较少,组织中(脑除外)可检测到其代谢产物野樱苷。与体外药物原形不同,D-野樱苷在组织中峰浓度(心、肝、脾、肺、肾中质量分数分别为170.5,112.8,98.4,152.3,381.7 ng·g~(~(-1)))低于L-野樱苷(心、肝、脾、肺、肾依次为906.4,652.3,177.4,500.9,2 060.4 ng·g~(~(-1)))。结论:建立的LC-MS/MS适用于大鼠口服麻黄-杏仁药对水提物后的组织分布研究。D-野樱苷在组织分布减少可能是麻黄-杏仁药对毒性拮抗作用的内在体现。麻黄生物碱和苦杏仁苷、野樱苷在肺组织中均有较高分布,这可能与两药协同平喘的药效学机制有关。

Quantitative Analysis of Tissue Distribution of Bioactive Compounds in Rats After Oral Administration of Ephedrae Herba-Amygdalus Communis Vas Extract

Objective: To develop a simple UPLC-MS/MS method for simultaneous determination of norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methylephedrine, amygdalin and prunasin in rat tissues and to analysis tissue distribution of these compounds in rats after oral administration of Ephedrae Herba-Amygdalus Communis Vas extract. Method: The current method was validated according to the FDA guidelines for the validation of biological sample analysis method. Diphenhydramine hydrochloride and puerarin were selected for quantitative analysis of alkaloids in Ephedrae Herba, amygdalin and prunasin, respectively. Result: Nine active compounds, including 3 pairs of epimers, were separated and quantified within 18 min by a sensitive and reliable UPLC-MS/MS method. Alkaloids in Ephedrae Herba were widely distributed in major tissues. Poor tissue distribution of amygdalin was observed, and prunasin (metabolite of amygdalin) can be detected in tissues except the brain. Contrary to D-amygdalin in vivo, D-prunasin had lower concentration (its concentrations in heart, liver, spleen, lung, kidney were 170.5, 112.8, 98.4, 152.3, 381.7 ng·g^-1) in tissues by comparing with L-prunasin (its concentrations in heart, liver, spleen, lung, kidney were 906.4, 652.3, 177.4, 500.9, 2 060.4 ng·g^-1). Conclusion: The method is successfully applied to tissue distribution study in rats after intragastric administration of Ephedrae Herba-Amygdalus Communis Vas extract. Reduction of D-prunasin in tissues may be the reason of toxicity antagonism in vivo. High concentration of alkaloids in Ephedrae Herba, amygdalin and prunasin are found in lung, which may be correlated with its synergistic anti-asthmatic effects.