The Method For The Determination Of Alprostadilin Animal Plasma And Tissues And Comparative Pharmacokinetics Of Alprostadil Micelle For Injection In Animals

AIM:To develop a rapid and sensitive method for the determination of alprostadil in Beagle dog plasma and rat tissues by liquid chromatography-mass spectrometry (LC-MS/MS). The essay has been successfully applied to a comparative pharmacokinetics study of alprostadil in Beagle dogs and a comparative tissue distribution study in rats after intravenous administration of alprostadil micelle and lipo-alprostadil.STUDY METHOD: Biological specimen of alprostadil was acidifed by acetic acid and extracted with liquid-liquid extraction, and then separated on a Zarbax XDB-C18 column employing an isocratic mobile phase consisting of acetonitrile and 0.5 % formic acid (50:50, v/v). Alprostadil was detected by an API 4000 LC-MS/MS system with multiple reaction monitoring (MRM) in the negative ion mode of an ESI interface. The mass transition ion-pairs of alprostadil and dexamethasone were performed using m/z 353.4→m/z 317.3 and m/z 391.1→m/z 361.0, respectively. The accuracy, precision, specificity, linearity, sensitivity, extraction recovery, matrix effects and stability were estimated for the validations of the essay.A comparative pharmacokinetics study was performed in Beagle dogs after intravenous administration of alprostadil micelle and lipo-alprostadil. After the plasma concentration-time profile was shown, the data obtained was used to calculate pharmacokinetic parameters, and determine statistically significant differenes between alprostadil micelle and lipo-alprostadil preparations. And a comparative tissue distribution study was performed in rats after intravenous administration of alprostadil micelle and lipo-alprostadil. The data obtained was analyzed with SPSS version 13.0 to determine statistically significant differences between the praparations, and evaluated for the accumulation and distribution of alprostadil in tissues.RESULTS: A highly sensitive, selective, rapid and reproducibility method for the determination of alprostadil in dog plasma and rat tissues by using LC-MS/MS has been developed and validated. The linearity range of alprostadil was 0.01-3 ng/mL, with the lower limit of quantitation (LLOQ) of 3 ng/mL and stable recovery for sample preparing procedure and stable matrix effects. The accuracy and presision in the intra- and inter-day were between 85-115 %. Because of unstable structure, the biological specimen of alprostadil was only stable in storage at -80 ?C for 14 days, and at room temperature for 0.5 h. And the sample after the preparing procedure was stable in autosampler ar room temperature for 1.5 h. The method was suited for the study of a comparative pharmacokinetics and tissue distribution of alprostadil, which kept the conformance to the relevant standards of Pharmacopoeia of People's Republic of China.The validated assay was applied to the comparative pharmacokinetics study of 12 healthy beagle dogs after intravenous administration of alprostadil micelle and lipo-alprostadil preparation. The pharmacokinetics parameters were as follows: Tmax: 0.0417±0.144 (mean±SD) and 0.417±0.359 min; C_max: 0.555±0.170 and 0.268±0.131 ng/mL; t1/2: 7.00±1.37 and 7.81±1.93 min; AUC_0-t: 1.27±0.487 and 0.843±0.244 ng·min/mL; AUC_0-∞: 1.41±0.482 and 1.03±0.256 ng·min/mL, respectively. The results of pharmacokinetics study showed that: there was significant difference in t1/2, and no significant difference in C_max and AUC_0-t. The validated assay was applied to the comparative tissue distribution study of 60 healthy rats after intravenous administration of alprostadil micelle and lipo-alprostadil preparation. The results showed that: alprostadil was distributed in cardiac, spleen, lung, kidney, fat, muscle and ovary tissues, mainly. And there was no alprostadil in large intestine, small intestine, stomach, brain, uterus and testicles.Conclusion: The results of comparative pharmacokinetic study of alprostadil show that: there was significant difference in C_max,T_max和AUC_0-t, and no significant difference in t1/2. And alprostadil was distributed in cardiac, spleen, lung, kidney, fat, muscle and ovary tissues, mainly. In different times, there was no significant difference in tissues between alprostadil micelle and lipo-alprostadil preparation.