A rapid high performance liquid chromatographic determination of methyldopa in human serum with fluorescence detection and alumina extraction: Application to a bioequivalence study

Abstract

A simple and ultra rapid high performance liquid chromatographic (HPLC) method coupled with alumina extraction and fluorescence detection was described for determination of methyldopa in human serum. The drug and an internal standard were adsorbed onto alumina and eluted using acidic methanol. The eluate was directly injected onto ODS reverse phase column using a mixture of phosphate buffer (0.05 M) containing triethylamine (100 microl/l, v/v; pH 2.3) and methanol (92:8, v/v) at a flow rate of 2.1 ml/min as the mobile phase. The fluorescence detector excitation and emission wavelengths were set at 270 and 320 nm, respectively. No interference in the assay from any endogenous substances or other concurrently used drugs was observed and the retention times of I.S. and the drug were 1.7 and 2.4 min, respectively with total run time (injection to injection) of less than 3.5 min. The limit of quantification was evaluated to be 2 ng/ml. Validity of the method was studied and the method was precise and accurate with a linearity range from 20 ng/ml to 5000 ng/ml. This method has been used in a randomized crossover bioequivalence study of two different methyldopa preparations in 24 healthy volunteers.