Liquid chromatographic assessment of total and protein-bound homocysteine in human plasma

Homocysteine present in human blood plasma is derivatized with thiol select ive ultraviolet labelling reagent, 2-chloro-l-methylpyridinium iodide, and separated from other plasma thiol derivatives by high-performance liquid ch romatography (HPLC) with detection at 312 nm. The separation is carried out isocratically on LiChrospher RP-18 column using mobile phase consisting of pH 2.5 0.04 M trichloroacetic acid buffer and methanol in the ratio 9:1 (v /v) pumped at 0.5 mi min(-1) at 40 degrees C. The homocysteine S-pyridinium derivative elutes at 6.5 min. To determine total and protein-bound homocys teine it is necessary to cleave disulphide bounds by the use of tri-n-butyl phosphine in order to form free sulfhydryl group. The method provides quant itative information on total and protein-bound homocysteine based on assays with derivatization after reduction of whole plasma, and derivatization af ter reduction of acid precipitated proteins. The calibration graph is linea r over the concentration range covering most experimental and clinical case s. The assay has a low pmol sensitivity and is reproducible; intra- and int er-day, relative standard deviation range from 1.79 to 5.09% and from 2.80 to 5.60%, respectively. The method is applied to the determination of total and protein-bound homocysteine in the plasma of healthy individuals. (C) 2 000 Elsevier Science Ireland Ltd. All rights reserved.