A simple isocratic HPLC-UV assay for measurement of total and free melphalan concentrations in human plasma is described. Samples were prepared by methanol precipitation (total melphalan assay) and ultrafiltration (free melphalan assay). On a 25 cm × 4.6 mm C column with 0.016 M mixed soldium phosphate citrate buffer (pH 3.75)-acetonitrile 87:13 as mobile phase, at a flow rate of 1 mL min -1, the retention time of melphalan was 11.5 min. Detection was at 254 nm. For total melphalan assay response was a linear function of concentration up to 40 µg mL -1, with excellent interday precision (<6% for 0.5-40 µg mL -1 melphalan), accuracy (<2% deviation from the true concentration), and recovery (91-110%). For free melphalan assay response was a linear function of concentration up to 2.5 ?g mL -1, with good precision (<11% for 0.7-2.5 µg mL -1 melphalan) and recovery (89-93%). Detection limits were 0.1 µg mL -1 and 0.05 µg mL -1 for total and free melphalan assays, respectively. The assays were clinically applied in a study of myeloma patients.
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