Simultaneous determination of total homocysteine and cysteine in human plasma by capillary zone electrophoresis with pH-mediated sample stacking

Abstract

An accurate, simple and sensitive method for simultaneous determination of total homocysteine (Hcy) and total cysteine (Cys) in human plasma has been developed and validated. Hcy and Cys play a major role in metabolism and cellular homeostasis. An elevated level of plasma Hcy is considered an important risk factor or marker for several diseases, in particular cardiovascular disease, while Cys is involved in a variety of important cellular functions such as protein synthesis, detoxification and metabolism. The proposed analytical procedure consists of reduction of title thiol dimers and unsymmetrical disulfides with tris(2-carboxyethyl)phosphine, and derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by pH-mediated stacking capillary electrophoresis separation and ultraviolet-absorbance detection of Hcy-CMQT and Cys-CMQT derivatives at 355 nm. Effective baseline electrophoretic separation was achieved using a standard fused-silica capillary (effective length 91.5 cm, 75 μm id) and 0.1 mol L⁻¹, lithium acetate buffer adjusted to pH 4.75. The limit of quantification (signal to noise ratio of 9) for derivatized Hcy and Cys in plasma was 2 μmol L⁻¹. The calibration curves obtained for Hcy and Cys in plasma showed linearity in the range 2–20 μmol L⁻¹, with R² = 0.9986 and 20–300 μmol L⁻¹, with R² = 0.9998, respectively. The relative standard deviation of the points of the calibration curve was lower than 7%. The method can be used for a routine monitoring of total Hcy and total Cys levels in plasma of human subjects.