Specific determination of pentamethylmelamine and its demethylated metabolites in human plasma by high pressure liquid chromatography.

Abstract

Pentamethylmelamine (PMM) and its demethylated metabolites tetra-N2246, tri-N246 and tri-N224 have comparable relative antitumor potencies in animal systems. We have developed a sensitive and specific high pressure liquid chromatography (HPLC) assay for these compounds in the plasma of patients receiving pentamethylmelamine, a cytotoxic S-triazine derivative now undergoing clinical trials. The method involves analysis of an ethyl acetate extract of plasma by a high pressure liquid chromatograph containing a reverse-phase column and ultraviolet detection at 240 nm. The mobile phase is acetonitrile and KH2PO4 buffer (pH 5.5); gradient elution over 25 min yields relative retention times to hexamethylmelamine as internal standard of 0.36, 0.40, 0.54 and 0.70 for tri-N246, tri-N224, tetra-N2246 and PMM. The lower limit of sensitivity for each is 300 ng/ml. Pharmacokinetic studies on four patients receiving a 24-h infusion of PMM (doses 900-3000 mg/m2) showed a mean terminal half-life of PMM of 101 +/- 28 (SD) min, a mean volume of distribution of 2036 +/- 75 ml/kg, and a metabolic clearance rate of 14.2 +/- 3.7 (ml/min)/kg. Mean areas under the plasma-vs-time curves of tri-N246, tri-N224 and tetra-N2246 were 126%, 41% and 56% of PPM, demonstrating extensive formation of these metabolites in man.