An Isocratic UV HPLC Assay for Analysis of Total and Free Melphalan Concentrations in Human Plasma

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 x 4.6 mm C-18 column with 0.016 M mixed sodium 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 mu g mL(-1), with excellent interday precision (< 6% for 0.5-40 mu g 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 mu g mL(-1), with good precision (< 11% for 0.7-2.5 mu g mL(-1) melphalan) and recovery (89-93%). Detection limits were 0.1 mu g mL(-1) and 0.05 mu g mL(-1) for total and free melphalan assays, respectively. The assays were clinically applied in a study of myeloma patients.