A Combination of Isocratic and Gradient Elution Modes in HPLC with the Aid of Time-Overlapping Process for Rapid Determination of Methyldopa in Human Urine

Abstract

A new rapid time-overlapping high-performance liquid chromatography method using coupled-column double-injection technique with fluorescence detection has been developed and validated to determine methyldopa (MTD) in human urine. The method was based on injecting a new sample onto the second column before finalizing the cleanup and the re-equilibration of the first column for the former sample. A combination of isocratic and gradient elution was employed according to a pre-set program. At the beginning, isocratic step of acetate buffer solution (0.1 M, pH 2.4) was set until 7 min. Subsequently, a gradient elution step using acetate buffer (0.1 M, pH 2.4) as mobile phase A and acetonitrile as mobile phase B was employed. After the end of each gradient step, the column was re-equilibrated with 4 mL of the starting isocratic elution system before the next analysis. The overall cycle time was 7 min per each sample. The calibration curve was linear over the concentration range of 0.1–40 µg/mL MTD. The overall mean recoveries were in the range of 98.29–101.39%. The applicability of the method was successfully evaluated by monitoring the incremental urinary excretion of MTD in human urine over 12 hr after a single oral administration of 250 mg.