Simultaneous determination of tryptophan, kynurenine, kynurenic acid, xanthurenic acid and 5‐hydroxytryptamine in human plasma by LC‐MS/MS and its application to acute myocardial infarction monitoring

Abstract

Reliable methods for the determination of tryptophan and its metabolites are vital to the monitoring of biochemical states during the initiation and progression of cardiovascular disease. In the present study, a single-run liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of tryptophan (Trp) and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), xanthurenic acid (XA) and 5-hydroxytryptamine (5-HT), in human plasma. The plasma samples were prepared using a protein precipitation approach, and the chromatographic separation was performed by gradient elution on a C column within a total analysis time of 3.5 min. The calibration ranges were 40-20,000 ng/mL for Trp, 4-2000 ng/mL for Kyn, 0.2-100 ng/mL for KA, 0.4-200 ng/mL for XA and 1-500 ng/mL for 5-HT, and the precision and accuracy were acceptable. The evaluation of recovery and internal standard-normalized matrix effect proved that the sample preparation approach was effective and the matrix effect could be negligible. The newly developed method was successfully applied to the analysis of plasma samples from healthy individuals and myocardial infarction patients. The findings suggested that the plasma concentrations of Trp, Kyn, 5-HT as well as the concentration ratios of Kyn/Trp and Trp/5-HT might serve as biomarkers for the monitoring of acute myocardial infarction.

Kynurenine Pathway Metabolites as Potential Clinical Biomarkers in Coronary Artery Disease

“…Regarding KP metabolites, these values cover a relatively wide spectrum, which makes it difficult to optimize a given sample preparation for all components without any compromise. Generally, the protein precipitation of biological samples is before the analysis using organic solvents and/or acids such as acetonitrile, methanol, trichloroacetic acid and formic acid (57,(66)(67)(68)(69). The solid-phase extraction (SPE) procedures combined with extraction solvent evaporation provides an efficient, but labour-intensive and time-consuming approach for the enrichment of targeted kynurenines by removing interfering compounds (70)(71)(72)(73)(74).…”

The Effect of LPS and Ketoprofen on Cytokines, Brain Monoamines, and Social Behavior in Group-Housed Pigs

“…The brain samples (sample size: 20-500 mg) were homogenized with acetonitrile 1:5 (v/w) on ice and then centrifuged at 12,000 × g for 30 min at 4 • C. The plasma samples (sample size: 15 to 50 µl) were precipitated with acetonitrile 1:5 (v/v) after addition of the internal standard mixture. After being vortexed for 30 s and centrifuged at 12,000 × g for 15 min, 50 µl of plasma samples and, respectively, 100 µl of brain samples supernatants were subsequently transferred into a new 15-ml polypropylene centrifuge tube and evaporated to dryness at 40 • C under a nitrogen stream in a water bath (Zymark Turbo Vap LV, Oregon, USA) (24,25). The dry residue was reconstituted in 100 µl dilution solution of 10% methanol/water (v/v) with 0.1% formic acid and 0.05% ascorbic acid, filtered with Spin-X centrifuge tube filter, 0.22 µm (Costar, UT, USA), and transferred to a HPLC vial with insert (Agilent, Santa Clara, CA, USA).…”

Changes in tryptophan and kynurenine pathway metabolites in the blood of children treated with ketogenic diet for refractory epilepsy

“…Finally, we acknowledge that different analytical approaches to measure kynurenines in response to, for example, the KD [9], may result in different conclusions. It is therefore important to use methodologies, like the one used in the present study, that have been validated across many and varied experimental conditions [16,[31][32][33].…”