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 B(6) and B(2) (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 microL) was deproteinized by mixing with an equal volume of 50 g/L trichloroacetic acid that contained d(2)-pyridoxal 5'-phosphate, d(3)-pyridoxal, and d(8)-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 C(8) 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 B(6) supplements.
Conclusions: This method based on liquid chromatography-tandem mass spectrometry measures all known plasma forms of vitamins B(6) and B(2), 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.