[HPLC determination of plasma/serum homocysteine and cysteine with UV detection and solid-phase extraction on a...

Isocratic HPLC determination of plasma/serum homocysteine and cysteine with separation on reversed-phase column and UV detection at 330 nm is proposed. The mobile phase consist of acetonitrile - 0.05 M citrate-phosphate buffer with pH 2.4 - isopropanol (15:85:1, v/v/v). Full separation of cysteine, cysteamine (IS), glutathione and homocysteine was achieved within less than 10 minutes. Reduction of thiols from disulfides was performed by 1,4-dithioerithreitol, and derivatization by with Ellman's reagent [5'5-dithiobis-(2-nitrobenzoic acid)]. After that plasma/serum, containing derivatives of thiols, is cleared and concentrated on cartridge packed with 10 mg of hypercross-linked polystyrene (Purosep-200). Elution from cartridge is made with water-organic solvent (without evaporation and concentration, but without dilution), as well as waterless solvents (with evaporation and concentration). Simplicity, reproducibility in combination with high cleanliness of extracts and sufficient sensitivity (0.4 ng for homocysteine, 2 ng for glutathione and 0.2 ng for cysteine and cysteamine at a signal/noise ratio > 3), make this method suitable for routine clinical application.