Quantitative Determination Of Hydralazine In Mouse Plasma And Brain: Application To Phrmacokinetic Study

Hydralazine(1-hydrazinophalazine, HYD),a potent, peripherally acting vasodilator with a general blood pressure-lowering effect, has been used as a typical hypotensor in the treatment of essential hypertension since the early 50s. Since its adverse effects and extreme variability in oral dose requirements have limited its usefulness, few attentions have been paid to HYD in the last few decades. Researches, focused on the quantitative determination of HYD, were almost published before 90s, and they are relatively poor in sensitivity. In this study, a simple and sensitive LC-MS-MS method for the quantitative determination of HYD in BALB/C mouse plasma and brain tissue has been developed and validated. This method was successfully applied to pharmacokinetic study of HYD in BALB/C mouse following a single intraperitoneal injection. In addition, the quantitative method for the determination of HYD in brain has been published for the first time.Procedures and methods:1. The content determination of HYD in BALB/C mouse plasma and brainIn this method, 2, 4-pentanedione was used as derivatization reagent. After derivatization at 50℃water bath for 1 h, solid phase extraction was done to purify and concentrate the samples. Chromatographic separation of analyte was performed with an Agilent ZORBAX SB-C18 column (150mm×2.1mm), with methanol–0.01 mol·L^-1 ammonium acetate (60:40, v/v) as mobile phase, and operated at a flow rate of 0.2 mL·min^-1. The mass spectrometer was operated in electrospray positive ionization mode. Sample analysis was performed in the multiple reaction monitoring mode (MRM). Quantification was performed using selected reaction monitoring of the transition of m/z 225.2→129.5.2. Pharmacokinetic study of HYD in BALB/C mouseAfter a single intraperitoneal injection of HYD at the dose of 20 mg·kg^-1 to BALB/C mouse, blood and brain samples were collected at control (0 min) and 5, 10, 15, 30, 45, 60, 90 and 120 min. After collection, whole blood was submitted to centrifugation and the resulting plasma was collected. The whole brain was homogenized, then the homogenate was centrifuged to obtain the supernatant. Then the derivatization reaction with 2, 4-pentanedione were done immediately, for both plasma and brain samples. The HYD concentration at each sample was determined by the above LC-MS-MS method. The concentration-time curves and pharmacokinetic parameters were all achieved by the use of WinNonlin 5.2.Results:1. A rapid, simple, sensitive and accurate LC-MS-MS method was developed in this paper for the determination of HYD in both mouse plasma and brain samples. The standard curves showed good linearity over the concentration range of 10-200 ng·mL^-1. The limit of detection were calculated to be 0.49 and 1.05 ng·mL^-1 for mouse plasma and mouse brain homogenate, respectively. The retention time of the HYD derivative was 4.2 min, with a total run time of about 6 min. Intra-day precision was 10.9% or less and inter-day precision was 18.9% or less at all levels. The accuracy of the method ranged from 96.2% to 113% at all levels.2. The results showed that HYD is able to pass through blood-brain barrier and it was rapidly absorbed by brain tissue, which is similar to the mouse plasma. In the use of pharmacokinetic software (WinNonlin5.2), the elimination phase of HYD was found to fit well to first order one compartment pharmacokinetic model. For plasma samples, the main pharmacokinetic parameters were as follows: AUC was 259 min·mg·mL^-1, t_1/2 was 14.4 min, CL was 0.0014 mL·min^-1. For brain samples, the main pharmacokinetic parameters were as follows: AUC was 296 min·mg·g^-1, t_1/2 was 9.3 min, CL was 0.0014 mg·min^-1.