One-pot sample preparation procedure for the determination of protein N-linked homocysteine by HPLC-FLD based method

Abstract

The report presents robust and high throughput method, based on liquid chromatography coupled with fluorescence detection (HPLC-FLD), for the determination of total protein N-linked homocysteine (Hcy) in human plasma. The assay involves simultaneous proteins precipitation with perchloric acid and removal of any other form of Hcy, except protein N-linked Hcy, via disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and plasma protein pellet wash with perchloric acid followed by liberation of N-linked Hcy from proteins by hydrochloric acid hydrolysis, drying under vacuum and residue reconstitution in diluted hydrochloric acid. The chromatographic separation of resulting in this way Hcy-thiolactone (HTL) is achieved within 3 min at room temperature on PolymerX RP-1 (150 × 4.6 mm, 5.0 µm) column using isocratic elution with eluent, consisted of o-phthaldialdehyde (OPA) in sodium hydroxide and acetonitrile (ACN), delivered at a flow rate 1 mL/min. The analyte is quantified by monitoring fluorescence at 480 nm using excitation at 370 nm, in a linear range from 0.25 to 10 µmol/L in plasma, while the limit of quantification (LOQ) equals 0.25 µmol/L. The method was successfully applied to plasma samples delivered by fifteen apparently healthy donors showing that the HPLC-FLD assay is suitable for screening of human plasma.