Comprehensive studies on the development of HPLC-MS/MS and HPLC-FL based methods for routine determination of homocysteine thiolactone in human urine

The report presents a new, robust, and reproducible liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and HPLC-fluorescence (FL) based methods for the determination of urinary homocysteine thiolactone (HTL). In particular, a versatile sample preparation procedure was designed to purify urine samples, involving chloroform liquid-liquid extraction (LLE) of HTL and its re-extraction (re-LLE) with formic acid, prior to chromatographic analysis. In relation to HPLC-FL assay, the quantification of HTL additionally uses o-phthaldialdehyde (OPA) as the on-column derivatization agent, while HPLC-MS/MS assay employs homoserine lactone (HSL) as an internal standard (IS). The baseline separation of the analyte and IS (if applicable) is accomplished under hydrophilic interactions chromatography (HILIC) and reverse phase (RP)-HPLC conditions in the case of HPLC-MS/MS and HPLC-FL based method, respectively. The assays linearity was observed within 20-400 nmol/L for HTL in urine, covering the expected unknown analyte's concentration in study samples. The value of 20 nmol/L in urine was recognized as the limit of quantification (LOQ) for both methods. The assays were successfully applied to urine samples delivered by fifteen apparently healthy volunteers showing that they are suitable for screening of human urine.