A sensitive high-performance liquid chromatographic assay for melphalan and its hydrolysis products in blood and plasma.

A sensitive high-performance liquid chromatographic assay has been developed for the measurement of the alkylating cytostatic drug melphalan (4-[bis(2-chloroethyl)amino]-L-phenyl-alanine, or L-phenylalanine-mustard, L-PAM) and its two hydrolysis products, monohydroxy melphalan (MOH) and dihydroxy melphalan (DOH). A reversed-phase phenyl column and a mobile phase consisting of acetonitrile/citrate buffer made possible an isocratic separation and quantification. N,N-[bis(2-hydroxy-ethyl)]toluidine has been synthesized as an internal standard structurally related to DOH. A new, accurate "kinetic" calibration procedure enabled us to determine even the concentration of the unstable MOH. The lower limit of quantification was 30 ng/ml for L-PAM and 20 ng/ml for both DOH and MOH with fluorescence detection. The use of this method is illustrated by some pharmacokinetic data in systemic and locoregional melphalan therapy.