Determination of homocysteine in plasma by precolumn derivatization-high performance liquid chromatography with fluorescence detection

Abstract

A precolumn derivatization-high performance liquid chromatographic method for the determination of homocysteine (Hcy) in plasma was established. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and N-(1-pyrenyl) maleimide (NPM) were used as the reduced reagent and derivatization reagent, respectively. The separation was carried out on an Agilent Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) in gradient elution mode. The mobile phase consisted of A (15 mmol/L sodium acetate solution), B (acetonitrile) and C (300 mL water containing 1 mL acetic acid and 1 mL phosphoric acid). The eluate was monitored by the fluorescence detector at an excitation wavelength of 330 nm and an emission wavelength of 380 nm. The mean recovery of Hcy was (102.08 +/- 4.94)%. The linear range was from 0.500 micromol/L to 100 micromol/L, with a detection limit of 0.016 micromol/L. The intra-day and inter-day relative standard deviations (RSDs) for Hcy were less than 5%. Seven plasma samples of patients with hypertension and seven plasma samples of healthy controls were tested, and the results demonstrated that the Hcy in the plasma from the hypertension group was significantly different from that of the control group (p < 0.05). The developed method is simple, fast, accurate, and suitable for clinical measurement.