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

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 mL 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.

Components of One-carbon Metabolism Other than Folate and Colorectal Cancer Risk

“…concentrations of riboflavin, pyridoxal 5ʹ-phosphate, methionine, choline, betaine, dimethylglycine, sarcosine, and creatinine were measured with liquid chromatography-mass spectrometry methods. 21,22 total plasma homocysteine concentrations were measured using an isotope dilution gas chromatographymass spectrometry method. 23 Folate and vitamin B12 concentrations were determined with a microbiological method using Lactobacillus casei and Lactobacillus leichmannii, respectively, and adapted to a microtiter plate format and carried out by a robotic workstation.…”

Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry

“…The analyte recoveries describing the matrix effects (MF) were calculated from the analyte peak areas as follows [25]:…”

“…In 2009, de Jong et al [24] developed an LC-MS/MS method that enabled the simultaneous extraction, concentration, separation, and mass selective detection of Trp, KYN, and HK in plasma. The most comprehensive method to date was introduced by Midttun et al [25], who employed LC-MS/ MS to quantify plasma levels of Trp, KYN, HK, KA, AA, XA, and HAA.…”

Indoleamine 2,3‐dioxygenase 1 in coronary atherosclerotic plaque enhances tissue factor expression in activated macrophages

“…The samples were separated by centrifugation at 5800  g for 15 minutes at 4°C. Levels of Trp and Kyn in the supernatants were measured using liquid chromatography/mass spectrometry (LC‐MS) with some modification 23. A Zorbax stable‐bond C8 reversed‐phase analytical column measuring 150 × 4.6 mm with a particle size of 3.5 μm (Agilent Technologies, Tokyo, Japan) was mounted in a compartment with a thermostat set at 40°C.…”