Determination of michellamine B in biological fluids by high-performance liquid chromatography with fluorescence detection.

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 (alpha-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-microns Nova-Pak phenyl column (3.9 mm x 15 cm). Chromatography was performed at ambient temperature using an isocratic mobile phase composed of 10 mM octyl sodium sulfate and 15 microM 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 nm, 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 min, respectively (SD, n = 52). Employing a sample volume of 50 microliters, 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%.(ABSTRACT TRUNCATED AT 250 WORDS)