HPLC-MS/MS based method for the determination of...

The report presents first high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine and active form of vitamin B6 pyridoxal 5'-phosphate. The assay employs 4-deoxypyridoxine (4-DPD) as an internal standard. Sample preparation procedure primarily involves acetonitrile (ACN) extraction of HPPTCA from plasma proteins, sample deproteinization by ultrafiltration, and dilution of ultrafiltrate with mobile phase prior to chromatographic analysis. The chromatographic separations of HPPTCA and 4-DPD are achieved within 6 min at 20 °C on X-Bridge Glycan BEH Amide (100 × 2.1 mm, 2.5 μm) column using gradient elution. The eluent consists of 0.1% formic acid in a mixture of solvent A (water and ACN (95:5, v: v)) and solvent B (water and ACN (5:95, v: v)) delivered at a flow rate of 0.3 mL/min. The assay linearity was observed within 0.25-10 µmol/L in plasma. The limit of quantification was found to be 0.25 µmol/L. The method was successfully applied to plasma samples delivered by apparently healthy donors showing that the HPLC-MS/MS assay is suitable for human plasma screening. The presence of HPPTCA was confirmed in eleven of fifteen study samples. The HPPTCA concentration ranged from 0.55 to 8.39 µmol/L.