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Multianalyte Quantification of Vitamin B₆ and B₂ Species in the Nanomolar Range in Human Plasma by Liquid Chromatography–Tandem Mass Spectrometry

¹ LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Bergen, Norway.

^aAddress correspondence to this author at: LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway. Fax 47-55-97-46-05; e-mail nkjbm{at}uib.no.

Background: Homocysteine, a risk factor of cardiovascular disease, cognitive disorders, and pregnancy complications, exists at a point of metabolic convergence of several B vitamins, including vitamins B₆ and B₂ (riboflavin). Measurement of the various forms of these vitamins may be useful for the study of hyperhomocysteinemia as well as for the assessment of vitamin status.

Methods: Plasma (60 µL) was deproteinized by mixing with an equal volume of 50 g/L trichloroacetic acid that contained d₂-pyridoxal 5'-phosphate, d₃-pyridoxal, and d₈-riboflavin as internal standards. Pyridoxal (PL), pyridoxal 5'-phosphate (PLP), pyridoxine (PN), pyridoxine 5'-phosphate, pyridoxamine (PM), pyridoxamine 5'-phosphate, 4-pyridoxic acid (PA), riboflavin, flavin mononucleotide (FMN), and FAD were separated on a C₈ reversed-phase column, which was developed with an acetonitrile gradient in a buffer containing acetic acid and heptafluorobutyric acid. The analytes were detected by tandem mass spectrometry in the positive-ion mode.

Results: The chromatographic run lasted 8 min. Within- and between-day CVs were 3%–20% and 6%–22%, respectively, and recoveries were 78%–163%. Limits of detection (signal-to-noise ratio = 5) were in the range 0.1–4.0 nmol/L, and the response was linear over several orders of magnitude. In samples from 94 healthy persons, we obtained median concentrations (nmol/L) of 35.4 for PLP, 16.9 for PL, 22.4 for PA, 10.3 for riboflavin, 7.5 for FMN, and 63.1 for FAD. PN and PM were also detected in some cardiovascular patients taking B₆ supplements.

Conclusions: This method based on liquid chromatography–tandem mass spectrometry measures all known plasma forms of vitamins B₆ and B₂, which span a wide range of polarity. The assay is characterized by simple sample processing with no derivatization, low sample volume requirement, and a short run time.

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