HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF A NOVEL POLYMER-BOUND PACLITAXEL DERIVATIVE AND FREE PACLITAXEL IN HUMAN PLASMA

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Abstract
A high-performance liquid chromatographic (HPLC) assay has been designed for the quantitative determination of polymer-bound paclitaxel (after hydrolytic release of paclitaxel from the polymer) and free paclitaxel in human plasma after intravenous administration of the antitumor polymer-drug conjugate PNU166945. Chemical stabilization with 0.2 M ammonium acetate and solid-phase extraction (SPE) were required as sample pretreatment prior to the HPLC analysis. Separation was performed on an APEX Octyl analytical column and a mobile phase of acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5.0 (4:1:5, v/v/v) and paclitaxel was detected at 227 nm. Total paclitaxel (polymer-bound pus free) levels were determined after chemical hydrolysis of the clinical samples with a mixture of 0.1 M KH2PO4 and methanol (1:1 v/v, pH 7.5) during 48 hours at room temperature. Concentrations of polymer-bound paclitaxel were calculated by subtraction of free from total drug levels. Plasma samples containing paclitaxel were stable for at least 10 months and hydrolyzed plasma samples were stable for at least 3 months at −30°C. Within-run and between-run precisions were less than 10.9% and the accuracy of the assay ranged from 94–102%. The limit of quantification for paclitaxel in plasma was established at 10 ng/mL using a 500 μL sample volume. The presented method was successfully applied in a clinical pharmacokinetic study in our Institute.