测定人血浆中齐多夫定的液相色谱-串联质谱法及生物等效性的应用

目的:建立快速、灵敏的液相色谱-串联质谱法(LC／MS／MS)测定人血浆中齐多夫定,并用于制剂生物等效性研究。方法:血浆样品经液-液萃取后,以甲醇-水(70:30,用1％氨水调节pH至6．0)为流动相, Zorbax Extend C18柱分离,采用电喷雾离子源四极杆串联质谱,以选择反应监测(SRM)方式进行正离子检测。用于定量分析的离子反应分别为m／z 268→m/z 127(齐多夫定)和m／z 225→m／z 127(内标,司他夫定)。结果:齐多夫定测定方法的线性范围为1．0-2 500μg·L-1;日内、日间精密度(RSD)均小于8．0％,准确度 (RE)在±2．0％以内。应用此法研究比较了18名健康受试者单剂量口服齐多夫定参比制剂和受试制剂 200 mg后的主要药动学参数。以AUC0-8计算受试制剂相对生物利用度。结论:该法选择性强、灵敏度高,适用于齐多夫定制剂的生物等效性评价及临床药动学研究。

Determination of zidovudine in human plasma with LC/MS/MS method and application to a bioequivalence study

AIM:To develope a sensitive and selective LC/MS/MS method for determination of zidovudine in human plasma and to study the bioequivalences with different formulations. METHODS:Zidovudine and internal standard stavudine were extracted from plasma using liquid-liquid extraction and then separated on a Zorbax Extend C18 column. The mobile phase consisted of methanol-water (70:30, v/v, adjusting pH to 6.0 with 1 % NH4OH), at a flow-rate of 0.5 mL·min-1. A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer e- quipped with electrospray ionization source was used as detector and operated in the positive ion mode. Selected reaction monitor using the precursor to produce ion combinations of m/z 268 to 127 and m/z 225 to 127 was taken to quantify zidovudine and the internal standard, respectively. The pharmacokinetic parameters of zidovudine in different formulations were calculated by non-compartment model statistics. RESULTS:The linear calibration curves were obtained in the concentration range of 1.0-2 500 μg·L-1. The lower limit of quantification was 1.0 μg·L-1. The intra-and inter-day relative standard deviation (RSD) over the entire concentration range was less than 8.0 %. Accuracy determined at three concentrations (2.5,50 and 2 000 μg·L-1 for zidovudine) ranged from -1.8 % to 2.8 %. Each plasma sample was chromatographed within 3.6 min. The method was successfully used in bioequivalence study of zidovudine in human plasma after oral administration of 200 mg zidovudine. Pharmacokinetic parameters of zidovudine reference formulation was obtained as follows: tmax was (0.6 ± s 0.3) h, cmax was(l 345 ± 656) μg·L-1, t1/2 was(1.70 ± 0.24) h,AUC0-8 was(l 603 ± 492) μg·h·L-1. For test formulation: tmax was (0.6 ± 0.3) h, cmax was (1 263 ± 637) μg·L-1,t1/2 was(1.60 ± 0.22) h,AUC0-8 was(l 555 ± 361) μg·h· L-1. Calculated with AUC0-8, the bioavailability of two formulations was (100 ± 17) %. The 90 % confidence interval of the individual ratios (test/reference formulation) for cmax and AUC0-8 were within the range of 77.0 % -119.2 % and 90.7 %-107.0 % respectively,supporting the bioequivalent of two formulations. CONCLUSION: LC/MS/MS method is rapid, selective, robust and proved to be suitable for bioequivalence evaluation of different formulations containing zidovudine.