Determination of Metyrosine and Its Metabolite in Serum by Reversed Phase High Performance Liquid Chromatography Using Solid Phase Extraction and Fluorescence Detection

Abstract

A novel and rapid method for the separation and determination of metyrosine and its major metabolite alpha-methyldopa in serum by high performance liquid chromatography with fluorescence detection is reported. The methods involved a solid-phase extraction of the two analytes and the internal standard dopamine using a Bond-Elut strong cation-exchange (SCX) column. The eluate obtained from the SCX column is then chromatographed on a reversed phase octadecylsilane column (Spherisorb 0DS2, 250 × 4.6 mm I.D.) with a 92.5:5:2.5 v/v/v 0.1 M aqueous phosphate buffer pH 3-acetonitrile - methanol mobile phase containing EDTA and heptane sulfonate. The flow rate was 1.0 mL/min with excitation at 282 nm and a 370 nm emission filter. The detection and quantitation limits were 1.0 mg/mL and 0.1 mg/mL for metyrosine and alpha-methyldopa, respectively, using 1 mL of serum. Linear calibration curves of 5–35 mg/mL and 0.2–2.5 mg/mL for metyrosine and alpha-methyldopa, respectively, show coefficients of determination of more than 0.9995. Precision calculated as %RSD and accuracy calculated as % error were within 2.5 –6.5% and 2.8–4.2%, respectively, for metyrosine and 4.1–6.3% and 1.3–1.5%, respectively, for alpha-methyldopa.