应用液质联用法同时测定新西兰家兔血浆中左炔诺孕酮和炔雌醇浓度及其药动学

Simultaneous determination of levonorgestrel and ethinylestradiol in New Zealand rabbit plasma by liquid chromatography-tandem mass spectrometry and their pharmacokinetics

目的建立液相色谱-质谱联用法(HPLC-MS/MS)测定新西兰家兔血浆中左炔诺孕酮(LNG)和炔雌醇(EE)浓度的方法,并探讨LNG/EE复方避孕贴多次给药后药动学特征。方法 6只雌性新西兰家兔连续10次给予LNG/EE复方避孕贴(5 cm×4 cm,每贴含LNG 5.35 mg,EE 0.11 mg),每3 d1贴,连续给药30 d,分别于给药后不同时间耳缘静脉采血,血浆样品经丹酰氯衍生化后,采用HPLC-MS/MS测定血浆中LNG和EE浓度,并采用DAS3.0软件计算首末次给药的主要药动学参数。结果 LNG的线性范围为0.10~20.00μg·L^(-1),最低定量限为0.10μg·L^(-1),提取回收率均>78.30%,日内精密度<7.38%、日间精密度<12.89%。首次给药后LNG的主要药动学参数为:峰浓度C_(max)为(8.10±2.38)μg·L^(-1),达峰时间(T_(max))为(2.38±1.45)h,西药浓度-时间曲线下面积(AUC)(0-768)为(142.35±36.99)h·μg·L^(-1);末次给药后LNG的药动学参数:C_(max)为(7.05±1.07)μg·L^(-1),T_(max)为(2.71±1.83)h,AUC(0-768)为(141.95±22.31)h·μg·L^(-1)。EE的线性范围为0.02~5.00μg·L^(-1),最低定量限为0.02μg·L^(-1),提取回收率均>79.99%,日内精密度<3.28%、日间精密度<12.76%。首次给药后EE的药动学参数:C_(max)为(0.18±0.04)μg·L^(-1),T_(max)为(2.50±1.30)h,AUC(0-768)为(2.65±0.56)h·μg·L^(-1);末次给药后EE的药动学参数:C_(max)为(0.17±0.07)μg·L^(-1),T_(max)为(2.17±0.26)h,AUC(0-768)为(2.02±0.82)h·μg·L^(-1)。结论建立的HPLC-MS/MS法准确灵敏,可用于同时测定血浆中LNG和EE的浓度。新西兰家兔给予LNG/EE复方避孕贴后首末次主要药动学参数无显著性差异,且多次给予此避孕贴后体内血药浓度无蓄积。

OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry （LC-MS/ MS） method for simultaneous determinations of concentrations of levonorgestrel （LNG） and ethinylestradiol （EE） in New Zealand rabbit plasma, and to study their pharmacokinetics in New Zealand rabbits after multiple dosing. METHODS Six female New Zealand rabbits were given LNG/EE patches ten times （5 cm×4 cm, each patch contained LNG 5.35 mg and EE 0.11 mg）, 1 patch every 3 d, for 30 consecutie days. Blood samples were collected at different time points before and after drug administration. The plasma samples were derived with dansyl chloride and then analyzed by HPLC-MS/MS method. The main pharmacokinetic parameters were calculated using DAS3.0 software. RESULTS The linear concentration range of LNG was 0.10-20.00 μg· L-1. The lower limit of quantitation was 0.10 μg· L-1. The extraction recovery was more than 78.30%. The intra-day and inter-day precisions were both less than 12.89%. The first-dosing pharmacokinetic parameters for LNG were as follows： Cmax （8.10±2.38） μg· L-1, Tmax （2.38±1.45） h, and AUC（0-768） （142.35±36.99） h·μg· L-1. The last administration pharmacokinetic parameters for LNG were as follows： Cmax （7.05±1.07） μg· L-1, Tmax （2.71 ±1.83） h, and AUC（1-768） （141.95±22.31） h· μg· L-1. The linear concentration range of EE was 0.02-5.00 ug· L-1. The lower limit of quantitation was 0.02 μg· L-1. The extraction recovery was above 79.99%. The intra-day and inter-day precisions were both less than 12.76%. The first-dosing pharmacokinetic parameters for EE were as follows： Cmax （0.18.-0.04） μg· L-1, Tmax （2.50±1.30） h, and AUC（0-768） （2.65±0.56） h· μg· L-1. The last administration pharmacokinetic parameters for EE were as follows： Cmax （0.17±0.07） μg· L-1, Tmax （2.17±0.26）h, and AUC （0-768） （2.02±0.82） h· μg· L-1. CONCLUSION The HPLC-MS/MS determination method is accurate and sensitive, which can be used to simultaneously determine the concentration of LNG and EE. There are no significant differences in main pharmacokinetic parameters between the first dose and the last dose. After repeated administration of this contraceptive patch, there is no accumulation of blood concentration in the rabbit body.