| PURPOSE:To determine the12healthy volunteers after single oral anagrelide hydrochloridecapsules and multiple oral administrations, anagrelide concentration in plasmasamples at different time. This study established a liquid chromatography-massspectrometry quantitative method suitable for high-throughput analysis of samples.This method has a high sensitivity, a short analysis time, a good reproducibility and aneasy sample handling. Through experimental data analyze the humanpharmacokinetics after oral anagrelide hydrochloride capsule. Provide a reliable basisfor anagrelide late clinical safety and use drug rational.METHOD:Use diethyl ether-dichloromethane (2:1, v/v) as extraction reagent. Anagrelideplasma samples will be extracted using liquid-liquid extraction method. The columntemperature was35C. The flow rate was1.0mL/min (shunt volume was1:1). Take30μL extracts for LC-MS/MS analysis. Methanol and0.1%formic acid in water(80:20, v/v) were the mobile phase in chromatographic system for anagrelide and theinternal standard glipizide. Separated after Ascetnis C18column (4.6×150mm ID,5m particle size) and using HPLC mass spectrometry analyze and detect. In multiplereaction monitoring (MRM) mode choose electrospray ionization (ESI) as ion source.Anagrelide and the internal standard glipizide used positive ion mode scanning. Ionreactions for the quantitative analysis were m/z256.3m/z199.0and m/z446.1m/z321.0.To ensure establish an effective and reliable anagrelide quantitative analysismethod. Test for the following indicators of a comprehensive methodology confirmed.Including specificity, linearity range, the lowest limit of quantification, accuracy,precision, extraction recovery, matrix effects and stability.For the recruitment of twelve healthy volunteers were oral and multiple oralanagrelide hydrochloride capsules for1mg dose. Using HPLC-MS/MS quantitativeanalysis methods established for determined the concentration of anagrelide in plasmasamples which were collected at different times. In order to understand the pharmacokinetic characteristics of anagrelide, we used DAS3.0and SPSS17.0software to calculate the corresponding pharmacokinetic parameters and usedstatistics analyze its characteristics.RESULTS:For the determination of anagrelide concentration in human plasma.In this test,we establish a rapid and sensitive, an accurate and reliable, a good reproducible and astable HPLC-MS/MS measurement method. The results show that no exogenous andendogenous substances interference at the analyte and the internal standard glipizidepeak position. Anagrelide quantitative linear range of plasma samples was0.1-100ng/mL and the LLOQ was0.1ng/mL. This method have a good linearity(r=0.9971) anda wide range of quantitative. Intra-and inter-day precisions were less than5.92and10.4. Accuracy was between3.11%and4.04%.These were within the range of15%.The recoveries of Anagrelide in low, medium, and high concentrations were96.1±3.6%,99.5±1.2%and104±0.91%. The recovery of internal standard glipizide isalso stable. Results have precision and reproducible. The matrix effects of anagrelidein low, medium, and high concentrations were95.4±3.9%,97.1±5.0%and91.1±5.6%. The matrix effect of glipizide was95.6±4.0%. These show that the matrixeffect can not affect the determination of anagrelide and glipizide. It can meet thebasic measurement requirements. Plasma samples were stable after repeated threetimes frozen at-20C, kept at room temperature for4h, left at room temperature for4h after the extraction process and placed80days after freezing at-20C. Reserveliquid were also stable after placed at room temperature for6hours and4C for7days. So anagrelide analysis method of plasma samples can be used to anagrelidehuman pharmacokinetic trial.Pharmacokinetic parameters after oral single dose and multiple dose are asfollows: Cmaxare8.47±2.05ng/mL and8.85±2.30ng/mL, respectively; t1/2are1.68±0.71h and1.82±0.70h, respectively; Tmaxare0.60±0.13h and0.77±0.13h,respectively; AUC0-tare16.49±4.30ng/mL*h and13.46±3.60ng/mL*h,respectively;AUC0-∞are16.49±4.30ng/mL*h and13.46±3.60ng/mL*h, respectively;MRT are2.25±0.40h and1.97±0.27h, respectively; Vz/F are151.31±62.20L and155.15±61.29L, respectively; CLz/F are64.78±17.99L/h and65.75±15.80L/h.respectively.By statistical analysis, major pharmacokinetic parameters of anagrelide have no significant differences between a single oral dose of1mg and continuousadministration of1mg. Such as t1/2, Cmax, CLz/F and Vz/F (P>0.05). That showcontinuous oral drug does not change the drug in vivo pharmacokineticscharacteristics. Anagrelide after multiple dosing, AUC is decreased and MRT issmaller. Small t1/2and large CLz/F of anagrelide are showed that it is excreted rapidlyafter multiple doses. Anagrelide little affinity with the organization will not lead todrug concentrations higher, so not prone to accumulate in the body. During the wholetest period, volunteers were not found to have any adverse reaction phenomenon, soanagrelide have a good safety. |
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