First HPLC-UV method for the determination of homocysteine thiolactone in human urine after derivatization with 1-benzyl-2-chloropyridinium bromide.

The report presents first high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) based method for the determination of homocysteine thiolactone (HTL) in human urine. Sample preparation procedure chiefly involves chloroform liquid-liquid extraction of HTL and its re-extraction with hydrochloric acid, followed by evaporation to dryness and treatment of the residue with 1-benzyl-2-chloropyridinium bromide and perchloric acid. The chromatographic separation of obtained UV-absorbing 2-S-pyridinium derivative is achieved within 6 min at 25 °C on Zorbax SB-C18 (150 × 4.6 mm, 5 μm) column using gradient elution. The eluent consists of 0.1% acetic acid in a mixture with acetonitrile delivered at a flow rate of 1 mL/min. The analyte is quantified by monitoring at 316 nm. The assay linearity was observed within 0.1-1.0 µmol/L in urine. The limit of quantification was found to be 100 nmol/L. The accuracy ranged from 92.56 to 112.51% and 92.56-114.31%, while precision varied from 5.89 to 11.27% and 3.85-14.25% for intra- and inter-assay measurements, respectively. The method was successfully applied to urine samples delivered by apparently healthy donors. The presence of HTL was confirmed in twelve of eighteen study samples. The HTL concentration ranged from 36.67 to 693.12 nmol/L.