High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions

A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 mu g ml(-1)) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma and melphalan in tissue. The mean recoveries of melphalan, MOH and DOH from perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8-56.8 mu g ml(-1), for DOH in the concentration range 0.5-30.0 mu g ml(-1) and for MOH in the range 1.4-25.1 mu g ml(-1), in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C,V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (f(u)) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb