LC-MS/MS法测定家兔血浆中奥昔布宁浓度及其在生物等效性评价中的应用

Determination of oxybutynin concentration in rabbit plasma by LC-MS/MS and its application to bioequivalence evaluation

目的建立家兔血浆中奥昔布宁的液相色谱-串联质谱联用(HPLC-MS/MS)定量方法,用于评价自制盐酸奥昔布宁凝胶和国外上市品Gelnique的生物等效性。方法以盐酸苯海拉明为内标,血浆样品经0.5 mol/L NaOH碱化后采用叔丁基甲醚液-液萃取。用Kinetex C_(18)色谱柱分离,流动相采用水相(1‰甲酸-10 mmol/L乙酸铵缓冲液)-乙腈(50∶50,v/v)进行洗脱;电喷雾离子源正离子检测,离子对为m/z 358→142(奥昔布宁),m/z 256→167(苯海拉明)。用以上方法测定家兔单次经皮给予自制盐酸奥昔布宁凝胶和Gelnique后血浆中奥昔布宁的浓度,评价两者的生物等效性。结果奥昔布宁在1~200μg/L范围内线性关系良好,低中高质控浓度的批间和批内精密度(RSD)介于1.67%~9.79%,准确度在92.9%~103%。自制盐酸奥昔布宁凝胶和Gelnique经评价生物等效。结论本方法简便,专属性强,灵敏度好且准确性高,适用于透皮制剂在家兔体内的药动学研究和生物等效性评价。

Objective To develop and validate a LC-MS/MS method to quantify oxybutynin in rabbit plasma and evaluate the bioequivalence of self-prepared oxybutynin chloride gel and Gelnique. Methods The plasma sample was submitted to liquid-liquid extraction using methyl t-butyl ether after alkalified by 0. 5 mol/L NaOH,with diphenhydramine as the internal standard. Chromatographic separation was performed on a Kinetex C18 column with the mobile phase consisting of 10 mmol/L ammonium acetate（ 1‰ formic acid）-acetonitrile（ 50 ∶ 50,v/v）. Oxybutynin and diphenhydramine were ionized with an ESI source operated in positive ion mode,and the detected ions were m/z 358→142（ oxybutynin）,m/z 256→167（ diphenhydramine）. The validated method was then applied to the drug determination in rabbit plasma following single dermal topical administration of oxybutynin gel. Results Calibration curve was liner over the concentration range of 1 ~ 200 μg/L in rabbit plasma. For quality control samples,the intra-and inter-day precision was in the range of 1. 67% ~ 9. 79%,and accuracy was within 92. 9% to 103%.Self-prepared oxybutynin chloride gel and Gelnique were proved to be bioequivalent. Conclusions It was validatedthat the LC-MS/MS method is simple,strong specificity and high sensitivity,which could be successfully applied to pharmacokinetic study and bioequivalence evaluation of transdermal oxybutynin formulations in rabbit.