Vitamins B2 and B6 serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large-scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B6 forms (pyridoxal 5'-phosphate, pyridoxal, 4-pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, xanthurenic acid and 3-hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope-labelled internal standards was added to 60 microL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable-bond C8 column by a gradient-type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75-123% and within-day and between-day coefficients of variance (CVs) were 2.5-9.5% and 5.4-16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B-vitamin status and inflammation, and may prove useful in large-scale epidemiological studies.
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