Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry

Since vitamin B6 is essential for normal functioning of the central nervous system, there is growing need for sensitive analysis of B6 vitamers in cerebrospinal fluid (CSF). This manuscript describes the development and validation of a rapid, sensitive and accurate method for quantification of the vitamin B6 vitamers pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxic acid (PA), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP) in human CSF.The method is based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a simple sample preparation procedure of protein precipitation using 50gL^-1 trichloroacetic acid containing stable isotope labeled internal standards: PL-D₃ for PL and PM, PN-¹³C₄ for PN, PA-D₂ for PA and PLP-D₃ for the phosphorylated vitamers. B6 vitamers were separated (Acquity HSS-T3 UPLC column) with a buffer containing acetic acid, heptafluorobutyric acid and acetonitrile. Positive electrospray ionization was used to monitor transitions m/z 168.1→150.1 (PL), 169.1→134.1 (PM), 170.1→134.1 (PN), 184.1→148.1 (PA), 248.1→150.1 (PLP), 249.1→232.1 (PMP) and 250.1→134.1 (PNP).The method was validated at three concentration levels for each B6 vitamer in CSF. Recoveries of the internal standards were between 93% and 96%. Intra- and inter-assay variations were below 20%. Accuracy tests showed deviations from 3% (PN) to 39% (PMP). Limits of quantification were in the range of 0.03-5.37. nM. Poor results were obtained for quantification of PNP.The method was applied to CSF samples of 20 subjects and two patients on pyridoxine supplementation. Using minimal CSF volumes this method is suitable for implementation in a routine diagnostic setting.