New Sensitive HPLC Method for Evaluation of the Pharmacokinetics of New Amantadine Prodrugs as Hepatic Delivery Systems to Enhance its Activity against HCV *

Abstract

To improve the efficacy of amantadine (AMD) in chronic hepatitis C therapy, various prodrugs were designed and synthesized to enhance its hepatic delivery based on the incorporation of AMD into modified bile acid or cysteine derivatives. A new sensitive and selective HPLC method with fluorescence detection has been developed and validated for determination of AMD in human plasma for evaluation of the pharmacokinetics of these prodrugs. Betaxolol hydrochloride (BTX) was used as internal standard. AMD and BTX were isolated from plasma by protein precipitation with acetonitrile and derivatized by heating with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline medium (0.01 M NaOH) at 90±5^oC for 45 min. Separations were performed in isocratic mode on Nucleosil CN column (250 mm length × 3.9 mm i.d., 5 μm particle diameter) using a mobile phase consisting of acetonitrile:10 mM sodium acetate buffer (pH 3.5):methanol (20:70:10, v/v) at a flow rate of 1.5 mL min^-1. The derivatized samples were extracted with chloroform and reduced with 0.03% potassium borohydride. The reduced fluorescent AMD-NQS derivative was monitored at emission wavelength of 382 nm after excitation at 293 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9989, n = 5) was found between the peak area ratio of AMD to BTX and AMD concentration in the range of 30–3200 ng mL^-1. The limit of detection and limit of quantification were 6.7 and 21 ng mL^-1, respectively. The intra and interassay precisions were satisfactory; the relative standard deviations did not exceed 1.57%. The accuracy of the method was proved; the recovery of AMD from spiked human plasma were 97.51100.95 ± 0.261.57%. The method had higher throughput as it involved simple sample preparation procedure and short runtime (<15 min). The results demonstrated that the proposed method would have a great value in the pharmacokinetic studies for AMD released from the synthesized produgs.