An isocratic UV HPLC assay for analysis of total and free melphalan concentrations in human plasma

Abstract

Summary 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 C18 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 μg 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 app...