Determination Of Amlodipine, Benazepril And Benazeprilat In Human Plasma And Its Application In A Pharmacokinetic Study

Background Amlodipine is a dihydropyridine calcium channel blocker useful in the management of angina pectoris and hypertension. Benazepril is a prodrug hydrolyzed in vivo by esterase to the active metabolite benazeprilat. The latter has a long duration of action and decreases blood pressure by inhibiting angiotension II production. Combination of amlodipine and benazepril is associated with a greater decrease in blood pressure and a lower risk for edema, compared with amlodipine or benazepril monotherapy at the same doses. To date, the combination has not been available in Chinese market, and no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma has been reported.Objective To develop a sensitive and specific liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma. The method should be applied to the pharmacokinetic study of fixed-dose combination of amlodipine and benazepril.Method Amlodipine-d4(for amlodipine) and ubenimex (for benazepril and benazeprilat) were used as internal standards (ISs), respectively. Analytes and ISs were extracted from plasma by simple protein precipitation. The reconstituted samples were chromatographed on a C18(100mm×4.6mm,5μm) column with mixture of methanol-acetonitrile-5mmol·L-1ammonium acetate-formic acid (30:30:40:0.1, v/v/v/v) as mobile phase at a flow rate of0.6mL·min-1. Detection was performed by tandem mass spectrometry using an heated electrospray ionization (HESI) source in the positive ion mode, operating in the selected reaction monitoring (SRM) of the transitions of m/z409â†'m/z238+294for amlodipine, m/z425â†'m/z351for benazepril, m/z397â†'m/z351for benazeprilat, m/z413â†'m/z238+298for amlodipine-d4and m/z309â†'m/z120for ubenimex.Results The standard curves were demonstrated to be linear in the range of0.02to6.00ng·mL-1for amlodipine,0.2to1500ng·mL-1for benazepril and benazeprilat with r2>0.99for each analyte. The lower limit of quantitation was achieved at0.02,0.2and0.2ng·mL-1for amlodipine, benazepril and benazeprilat, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limit across all concentrations. The method was applied in a pharmacokinetic study of a fixed-dose combination of amlodipine besylate and benazepril hydrochloride in Chinese healthy volunteers. After an oral administration of single-dose of amlodipine benazepril capsules, Cmax and AUC of amlodipine, benazepril and benazeprilat increased proportionally with increased doses. After an oral administration of multiple-dose of amlodipine benazepril capsules, the accumulation coefficient R=3for amlodipine, while R=1for benazepril and benazeprilat.Conclusion This method has been used in the simultaneous determination of amlodipine, benazepril and benazeprilat in human plasma. It was suitable for the pharmacokinetic study of a fixed-dose combination of amlodipine and benazepril. Three analytes showed good linear pharmacokinetic characteristics in human in a single-dose study. In a multiple-dose study, amlodipine had higher Cmax and AUC, while benazepril and benazeprilat had similar pharmacokinetic characteristics, compared with single middle-dose group.