HPLC-MS/MS法测定伏立康唑的血药浓度及其在生物等效性研究中的应用

目的建立测定人血浆中伏立康唑的高效液相色谱-串联质谱法（HPLC-MS/MS）,研究2种伏立康唑片的相对生物利用度。方法血浆样品经酸化后用甲醇沉淀蛋白,经Waters Atlantis C18柱分离,流动相为乙腈-0.2%冰醋酸水溶液（内含20 mmol.L-1醋酸铵）（60∶40,v/v）,流速为0.2 mL.min-1;选择多反应离子检测（MRM）方式进行定量分析,用于监测的离子为质荷比m/z350.9→281.4（伏立康唑）和m/z307.9→220.3（内标氟康唑）。18名健康志愿者以随机交叉方式分别单次口服伏立康唑分散片T或伏立康唑片R 0.2 g后于不同时间点取血,样品以新建立的HPLC-MS/MS法测定,研究比较两制剂的药动学及相对生物利用度。结果伏立康唑与血浆中内源性杂质分离度好,伏立康唑浓度在27.35~3 500 ng.mL-1与峰面积比线性良好,最低定量浓度为27.35ng.mL-1。蛋白沉淀绝对回收率为86.2%~88.8%,相对回收率为97.9%~105.7%,日内精密度（RSD）〈9.8%,日间精密度（RSD）〈9.4%。单剂量口服伏立康唑分散片T和伏立康唑片R 0.2 g后2种制剂的Cmax为（717.9±325.1）、（671.8±243.3）μg.L-1;tmax为（1.1±0.5）、（1.1±0.4）h;AUC0~24为（3 729.1±1 887.7）、（3 811.2±1 836.6）μg.h.L-1;t1/2为（6.7±1.9）、（6.7±1.7）h。与R相比,T制剂相对生物利用度为（97.3±13.0）%。结论该方法简单快速,灵敏度高,可用于伏立康唑的体内过程研究。方差分析表明伏立康唑分散片T与伏立康唑片R中伏立康唑的主要药动学参数之间均无明显差异,双单侧t检验结果表明两制剂为生物等效制剂。

Determination of voriconazole and relative bioavailability in human plasma by HPLC-MS/MS

Objective To develop an HPLC-MS/MS method for the determination of voriconazole in human plasma and evaluate the bioequivalence of voriconazole tablets.Methods After acidization and protein precipitation with methanol,analysis was performed on a Waters HPLC system using a Waters Atlantis C18 column and isocratic elution with the mobile phase of acetonitrile-0.2% glacial acetic acid（contain 20 mmol·L-1 ammonium acetate）（60∶40,v/v） at 0.2 mL·min-1,and a tandem mass spectrometer Micromass,equipped with an electrospray ionization interface,operated in a positive mode.The multiple reaction monitor（MRM） was used to detect voriconazole（m/z 350.9→281.4） and fluconazol（m/z 307.9→220.3,as the internal standard）.In a randomized crossover design,18 healthy male volunteers were given 0.2 g voriconazole tablet T（test drug） or tablet R（reference drug）.The blood samples were obtained and determined by the newly-developed HPLC-MS/MS method and the pharmacokinetics and bioavailability were studied.Results The calibration curve of voriconazole was linear over 27.35~3 500 ng·mL-1 with the limit of quantity 27.35 ng·mL-1.The absolute recoveries were at 86.2%~88.8%,and the relative recoveries at 97.9%~105.7%.Inter-day and intra-day RSD was less than 9.8% and 9.4%,respectively.Pharmacokinetic parameters of voriconazole after a single dose of tablet T and R were as follows：Cmax was（717.9±325.1）,（671.8±243.3）μg·L-1,tmax was（1.1±0.5）,（1.1±0.4）h,AUC0~24 was（3 729.1±1 887.7）,（3 811.2±1 836.6）μg·h·L-1,t1/2 was（6.7±1.9）,（6.7±1.7）h,and the relative bioavailability was（97.3±13.0）%.Conclusion The method is selective,sensitive and rapid for voriconazole determination.ANOVA and two one-sided t-tests analysis show that there is no significant difference between the pharmacokinetic parameters of the two formulations and they are bioequivalent.