Determination of diclofensine, an antidepressant agent, and its major metabolites in human plasma by high-performance liquid chromatography with fluorometric detection

Abstract

A sensitive and selective high-performance liquid chromatographic assay was developed for the determination of diclofensine (I) and its key metabolites in human plasma. The assay involves deproteinization of plasma, overnight glusulase incubation to hydrolyze the major metabolite (I-B-glucuronide), extraction of the parent compound and its deconjugated metabolites (I-A, I-B and I-C) from the alkalinized aqueous phase into diethyl ether—ethanol (95:5), the residue of which (containing compounds I, I-A, I-B and I-C) is alkylated with 2-iodopropane dissolved in acetone, using solid potassium hydroxide as a catalyst. The compounds are extracted from the reaction mixture into diethyl ether, after adding ethanol—water—acetic acid (55:40:5), the residue of which is dissolved in 0.05 M sulfuric acid, and reacted with mercuric acetate at 100°C, which oxidizes tertiary tetrahydroisoquinolines to their 3,4-dihydroisoquinoline derivatives, followed by a photochemical reaction in the same solution to form intensely fluorescent isoquinolinium derivatives. An aliquot of this reaction mixture is injected onto a reversed-phase high-performance liquid chromatography column (5-μm Nova-Pac C 18 phase in a radial compression cartridge, 10 cm × 8 mm), using the mobile phase 0.25 M triethylammonium phosphate (pH 2.5)—0.25 M acetic acid—methanol—acetonitrile—tetrahydrofuran (150:350:125:375:25). The void volume ( V 0) is approximately 1.4 min and the retention times ( t R) of the respective isoquinolium derivatives of diclofensine (I) are ca. 3.5 min, internal standard (II) ca. 4.2 min, nordiclofensine (I-A) ca. 5 min, while the phenolic metabolites I-B and I-C give peaks at 6.4 min and 10.4 min, respectively. The derivatives are detected by fluorescence. The method was used to determine plasma concentrations of the parent drug (I) and its major phenolic metabolite I-B (aglycone) in plasma in two normal volunteers following a single oral 45-mg dose and following seven consecutive days of oral dosing of 45 mg three times a day as part of a multiple ascending dose tolerance study.