Determination And Pharmacokinetic Study Of Fudosteine, Enalapril And Its Metabolite In Human Plasma By High-performance Liquid Chromatography-tandem Mass Spectrometry

Objective:To develop and validate specific, sensitive and rapid methods for the determination and pharmacokinetic study of fudosteine, enalapril and enalaprilat in human plasma by using high-performance liquid chromatography-tandem mass spectrometry.Method:After a one-step protein precipitation with methanol of 100μL plasma, fudosteine and paracetamol (internal standard) were separated on a kromasil 60-5CN column with isocratic elution at a flow-rate of 0.35 mL/min. The mobile phase was a mixture of methanol and water (80:20, v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). The quantification was performed using the transitions of m/z 178.0â†'m/z 90.6 for fudosteine and m/z 149.9â†'m/z 106.7 for paracetamol, respectively.Plasma samples (200μL) of enalapril and enalaprilat were prepared by protein precipitation with methanol after addition of benazepril (internal standard), separated on UltimateTM XB-C18 column with isocratic elution. The mobile phase was methanol-0.2% formic acid water solution (62:38, v/v) and the flow-rate was 0.20 mL/min. MRM using the precursorâ†'product ion transition of m/z 377.2â†'m/z 234.2, m/z 349.1â†'m/z 206.1 and m/z 425.2â†'m/z 351.2 was employed to quantify enalapril, enalaprilat and benazepril, respectively.Result:The linear calibration curves were obtained over the concentration range of 0.202～20.2μg/mL for fudosteine in human plasma. The intra-and inter-day precision values (RSD) were not above 14% and the accuracy (RE) was -0.2%-5.1% at all three QC levels. The main pharmacokinetic parameters obtained after an oral dose of 400 mg fudosteine to 20 healthy volunteers were as follows:Cmax was found to be 11.0±3.0μg/mL, Tmax observed was 0.38±0.14 h, the value of t1/2 was 3.19±0.70 h, AUC0-t and AUC0-∞were 23.2±6.6μg-h/mL and 25.0±6.9μg·h/mL, respectively. The standard calibration curves for enalapril and enalaprilat were both linear in the concentration range of 0.638-255 ng/mL in human plasma. The intra-and inter-day precision values (RSD) were not above 14% and the accuracy (RE) was-8.7%～-1.1% at all three QC levels. The main pharmacokinetic parameters of enalapril and enalaprilat obtained after an oral dose of 10 mg enalapril to 20 healthy volunteers were 90.5±28 ng/mL and 47.5±12 ng/mL for Cmax,0.86±0.31 h and 4.2±1.1 h for Tmax,1.35±0.61 h and 6.71±2.2 h for t1/2,136±36 ng-h/mL and 401±89 ng-h/mL for AUC0-t and 138±36 ng-h/mL and 420±91 ng-h/mL for AUC0-∞, respectively.Conclusion:The methods are proved to be of high specificity, sensitivity, simple and have been successfully utilized for the evaluation of the pharmacokinetics of fudosteine, enalapril and enalaprilat.