A REVERSED-PHASE HPLC METHOD FOR DETERMINING CAMPTOTHECIN IN PLASMA WITH SPECIFICITY FOR THE INTACT LACTONE FORM OF THE DRUG

Camptothecin is a pentacyclic indole alkaloid with a terminal alpha-hydroxy-delta-lactone ring, which in aqueous media at physiological pH, exists in equilibrium with the dissociated open-lactone carboxylate. The rate of equilibration between the two components is slow enough to permit their separation by reversed-phase HPLC. Selective determination of the intact lactone form of the drug was achieved by direct analysis of plasma samples immediately upon deproteinization with a solution of the internal standard in methanol chilled to -70-degrees-C. Acidification of the sample to pH 2 with perchloric acid prior to protein precipitation effected complete lactonization of the carboxylate and, therefore, provided a measure of total drug levels. Plasma concentrations of the carboxylate may be calculated from the difference between total drug and intact lactone determinations. Chromatography was performed on a 5-mu-m Ultrasphere ODS column (4.6 mm x 25 cm) preceded by a 1.5 cm RP-18 Brownlee Guard column with an eluent composed of acetonitrile-0.1 M ammonium acetate buffer, pH 5.5 (28:72, v/v) with 1 mM sodium dodecyl sulfate at a flow rate of 1.0 ml/min. The drug was monitored by fluorescence detection with excitation at 347 nm and a 418 nm emission cutoff filter. Approximately 3.0 hr was required to assay an 8 point standard curve and a drug-free plasma sample. Employing 50-mu-l of plasma, the lowest concentration on the camptothecin lactone and total drug standard curves, 2.82 nM (0.49 ng/ml), was quantified with 3.79 and 5.58% coefficients of variation, respectively. The method has been shown to be specific and reproducible.