胆固醇基磷酰齐多夫定在大鼠体内的药代动力学研究

Pharmacokinetic study of cholesteryl-phosphoryl zidovudine in rats

目的建立测定大鼠血浆及组织中胆固醇基磷酰齐多夫定(CPZ)及其代谢物齐多夫定(AZT)含量的方法,并应用于CPZ在大鼠体内的药代动力学研究。方法 CPZ的测定采用HPLC-UV法,生物样品用乙腈沉淀蛋白后,经Diamonsil C18柱(200 mm×4.6 mm,5μm)分离,以甲醇-异丙醇(90∶10,V/V,含有2 mmol/L十六烷基二甲基溴化铵、2%乙酸、0.1%氨水)为流动相,1.0 ml/min流速等度洗脱,紫外检测波长为266 nm。AZT的测定采用LC-MS/MS法,血浆及组织样品用叔丁基甲醚提取后,经Poroshell 120 EC-C18柱(50 mm×2.1 mm,2.7μm)分离,以甲醇-0.2%乙酸水(35∶65,V/V)为流动相等度洗脱;用ESI源在多反应监测方式下进行正离子检测,用于定量分析的离子反应为m/z 268.2→m/z 127.0(AZT)和m/z 152.1→m/z 110.0(对乙酰氨基酚,内标)。结果 CPZ和AZT在大鼠生物样品中浓度测定方法的线性范围分别为5~400μg/ml和2~500 ng/ml,批内、批间精密度(RSD)均<15%,准确度在±13.8%之间。CPZ在大鼠体内消除较快,AZT消除较慢,半衰期约为8 h。CPZ和AZT在大鼠的肝、脾、肺中分布较多,在心、肾中分布较少。结论方法适用于CPZ自组装体在大鼠体内的药代动力学研究。药动学结果显示,CPZ能靶向控释AZT到大鼠的肝、脾和肺中。

Objective To develop and validate methods for determination of cholesteryl-phosphoryl zidovudine（CPZ）and its metabolite zidovudine（AZT）in rat plasma and tissues,and use the methods to investigate the pharmacokinetics of CPZ in rats. Methods An HPLC-UV method was applied for the determination of CPZ in biological samples. The biological samples were precipitated with acetonitrile at first,and then CPZ was separated on a Diamonsil C18 column（200 mm×4.6 mm I.D.,5 μm）with a gradient elution system comprising 90% methanol and 10% isopropanol（containing 2 mmol/L cetyl trimethylammonium bromide,2% acetic acid and0.1% ammonium hydroxide）. The eluent was monitored at 266 nm by UV detector. The determination of AZT in biological samples was performed by LC-MS/MS after it was extracted from the biological samples by liquid-liquid extraction with methyl tertbutyl ether. Chromatographic separation was performed on a Poroshell 120 EC-C18 column（50 mm×2.1 mm I.D.,2.7 μm）. Using the mobile phase consisted of 35% methanol and 65% water containing 0.2% acetic acid,the column was eluted by an isocratic elution with the flow rate of0.3 ml/min. Detection of AZT and the internal standard（IS）acetaminophen was achieved by ESI MS/MS in the positive ion mode using m/z 268.2→m/z 127.0 and m/z 152.1→m/z 110.0 transitions,respectively. Results The linear ranges for the quantitative determination of CPZ and AZT were 5-400 μg/ml and 2-500 ng/ml,respectively,when 50 μl plasma was analyzed. The lower limit for the quantification of CPZ and AZT was 5 μg/ml and 2 ng/ml,respectively. The inter-and intra-day precision values were below 15%,and the accuracy（relative error）was within ± 13.8% in all quality control samples. CPZ was quickly metabolized in rats,while AZT was opposite. The half-life of AZT was about 8 h. The distribution of CPZ and AZT was more in the liver,spleen and lungs of rats,and less in the heart and kidney. Conclusion The methods completely meet the requirements for pharmacokinetic study of CPZ in rats. The pharmacokinetic results show that CPZ could release AZT targeting liver,spleen,and lungs in rats.