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

• Published in: Analytica chimica acta Vol. 712; pp. 108 - 114
• Main Authors: van der Ham, M., Albersen, M., de Koning, T.J., Visser, G., Middendorp, A., Bosma, M., Verhoeven-Duif, N.M., de Sain-van der Velden, M.G.M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 27.01.2012; Elsevier
• Subjects: acetic acid; acetonitrile; Analytical chemistry; B6 vitamers; Carbon Isotopes - chemistry; central nervous system; Cerebrospinal fluid; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid; Exact sciences and technology; Human; Humans; ionization; Isotope Labeling; Liquid chromatography; Liquids; Mass spectrometry; Other chromatographic methods; patients; pyridoxal; pyridoxamine; Pyridoxine; Spectrometric and optical methods; stable isotopes; Tandem Mass Spectrometry; trichloroacetic acid; Trichloroacetic Acid - chemistry; Vitamin B 6 - cerebrospinal fluid; Vitamins; Tandem mass spectrometry; Liquid chromatography; B6 vitamers; Cerebrospinal fluid; Performance evaluation; Phosphates; Phosphorylation; Chemical analysis; Internal standard; Central nervous system; Vitamin; Electrospray; Implementation; Precipitation; Accuracy; Sample preparation; Detection limit; Diagnosis; Quantitative analysis; Human; Sample; Use; Pyridoxine; Concentration; Chemical ionization; Protein; Pyridoxal phosphate; Mass spectrometry MS/MS; Acetonitrile; Transition; Acetic acid
• ISSN: 0003-2670; 1873-4324; 1873-4324
• DOI: 10.1016/j.aca.2011.11.018

[Display omitted] ► We present a sensitive UPLC–MS/MS method for quantification of B6 vitamers in human CSF. ► Our method is very accurate since stable isotope labeled internal standards are used. ► We present data on light sensitivity, temperature dependence and rostrocaudal gradient. ► With PN supplementation, concentrations of PL, PM, PN and PA in CSF are increased. ► Our fully validated method is suitable for implementation in a diagnostic setting. 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 50 g L −1 trichloroacetic acid containing stable isotope labeled internal standards: PL-D 3 for PL and PM, PN- 13C 4 for PN, PA-D 2 for PA and PLP-D 3 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.