Simultaneous determination of levonorgestrel and ethinylestradiol in New Zealand rabbit plasma by liquid chromatography-tandem mass spectrometry and their pharmacokinetics / 中国药理学与毒理学杂志

ABSTRACT

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 concentra-tion range of LNG was 0.10-20.00μg · L-1. The lower limit of quantitation was 0.10μg · L-1. The extrac-tion 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 followsCmax (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 followsCmax (7.05±1.07)μg·L-1, Tmax (2.71±1.83) h, and AUC(0-768) (141.95±22.31) h·μg·L-1. The linear concentration range of EE was 0.02-5.00μg·L-1. The lower limit of quantitation was 0.02μg·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 parame-ters for EE were as followsCmax (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.