Determination of Michellamine B in Biological Fluids by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

The National Cancer Institute is pursuing preclinical development of michellamine B (MB), a novel dimeric polyhydroxylated naphthalene-tetrahydroisoquinoline alkaloid isolated from Ancistrocladus abbreviatus, as an anti-human immunodeficiency virus (HIV) agent. MB protects human lymphoid cells from the cytopathic effects of both HIV-1 and HIV-2 in vitro. A specific, sensitive, and convenient method for assaying the compound in biological fluids has been developed. Samples were prepared for analysis by initial treatment with dilute trichloroacetic acid followed by thorough mixing with a solution of the internal standard (α-naphthoflavone) in acetonitrile to denature macromolecules. The supernatant afforded by centrifugation, upon dilution with the aqueous component of the liquid chromatographic eluent, was loaded onto a 4-μm Nova-Pak phenyl column (3.9mm × 15cm). Chromatography was performed at ambient temperature using an isocratic mobile phase composed of 10 mM octyl sodium sulfate and 15 μM tetrabutylammonium hydrogen sulfate in acetonitrile/0.05 M ammonium formate buffer, pH 4.0 (46/ 54, v/v), at a flow rate of 0.6 ml/min. The intense native fluorescence of MB, which exhibited excitation and emission maxima in the mobile phase at 232 and 393 am, respectively, provided a highly sensitive and selective means of detection. Mean values of the retention times for the drug and internal standard determined over 11 months were 10.71 ± 0.53 and 13.14 ± 0.52 mm, respectively (SD, n = 52). Employing a sample volume of 50 μl, the lowest concentration of MB included in the standard curves of mouse, dog, and human plasma, 10 ng/ml (11.4 nM), was quantified with coefficients of variation less than 10%. The assay has been extensively utilized to characterize drug stability in aqueous solutions and plasma pharmacokinetics in the mouse and dog. The use of widely available chromatographic instrumentation, commercially available reagents, the simplicity of sample preparation, minimal sample handling, and the ability to decontaminate specimens obtained from subjects infected with the acquired immune deficiency syndrome virus by heat treatment make the analytical method very well-suited for clinical drug level monitoring.