Quantitative profiling of biomarkers related to B‐vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

TLDR

Vitamins B2 and B6 act as cofactors in tryptophan and homocysteine metabolism, and their plasma levels, which correlate with smoking, inflammation, and immune activation, have been difficult to study at scale due to inadequate analytical methods. The assay quantifies 16 analytes—including riboflavin, five B6 vitamers, tryptophan, six kynurenine pathway metabolites, cystathionine, neopterin, and cotinine—by adding isotope‑labelled standards to 60 µL plasma, centrifuging, and analyzing the supernatant on a C8 column with a 5‑min gradient of acetonitrile, heptafluorobutyric acid, and 650 mM acetic acid using ESI‑MS/MS. The optimized mobile phase achieved complete separation and high ionization efficiency, yielding recoveries of 75–123 % and within‑day and between‑day CVs of 2.5–9.5 % and 5.4–16.9 %, respectively, with limits of detection from 0.05 to 7 nmol/L, demonstrating the method’s suitability for large‑scale epidemiological studies of B‑vitamin status and inflammation. © 2009 John Wiley & Sons, Ltd.

Abstract

Abstract Vitamins B 2 and B 6 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 B 6 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 µL 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. Copyright © 2009 John Wiley & Sons, Ltd.