Direct injection high-performance liquid chromatography of tetrabenazine and its metabolite in plasma of humans and rats

Abstract

A convenient, selective, and sensitive reversed-phase HPLC assay was developed to measure concentrations of the dopamine-depleting agent, tetrabenazine (1,3,4,6,7,11b-hexahydro-3-isobutyl-9,10-dimethoxy-2H-benzo(a)quinoli zin-2-one) and its dihydro metabolite in the plasma of patients with tardive dyskinesia receiving therapeutic doses of the drug and in the plasma of rats. The method involves plasma protein precipitation, oxidation of the compounds with mercuric acetate at 110 degrees C for 1 h, addition of internal standard, and injection into the instrument. Fluorescence detection was utilized at excitation and emission wavelengths of 265 and 418 nm, respectively. The peaks from the drug, its metabolite, and at least three other substances were best resolved at 60 degrees C using a mobile phase of water:acetonitrile:acetic acid:triethylamine (65:33:2:0.15) at a flow rate of 0.6 mL/min; the 4.6 mm X 10 cm column contained 5 micron of octadecylsilane packing. To assess the applicability of the assay, the drug was administered intravenously to rats, and plasma concentrations were determined before (by UV-HPLC) and after (by fluorescence-HPLC) the oxidative procedure. In addition, the MS spectra of tetrabenazine and the dihydro metabolite, isolated from biological samples, were identical to those of authentic samples. Excellent linearity was observed between the peak area ratios and concentrations over the ranges 0.5-200 and 2-1000 ng/mL of the drug and the metabolite, respectively. Minimum quantifiable concentrations of the drug and its metabolite were 0.5 and 2.0 ng/mL, respectively. The sensitivity was found to be adequate for pharmacokinetic studies of tetrabenazine in humans and rats.