A Simple HPLC–MS/MS Method for Determination of Tryptophan, Kynurenine and Kynurenic Acid in Human Serum and its Potential for Monitoring Antidepressant Therapy

Abstract

The kynurenine pathway, in which tryptophan is metabolized to kynurenine and kynurenic acid, has been linked to depression. A rapid and highly reproducible liquid-chromatography-tandem mass spectrometry (LC-MS/MS) method were established for determining tryptophan, kynurenine and kynurenic acid in human serum. Biological samples were precipitated with methanol before separation on an Agilent Eclipse XDB-C18. The stable-isotope-labeled internal standards (kynurenine-CN and kynurenic acid-d) were used for quantification. Detection was performed using multiple reaction monitoring in electrospray ionization mode at m/z 205.1→188.1 for tryptophan, m/z 209.1→146.1 for kynurenine, m/z 190.1→144.1 for kynurenic acid. Good linearity of analyte to internal standard peak area ratios was seen in the concentration range 1,000-50,000 ng/mL for tryptophan, 100-5,000 ng/mL for kynurenine and 1-60 ng/mL for kynurenic acid. Pooled drug-free human serum was purified using activated charcoal and the method was shown to be linear, with validation parameters within acceptable limits. The newly developed method was successfully used to determine concentrations of tryptophan, kynurenine and kynurenic acid in serum from 26 healthy volunteers and 54 patients with depression. Concentrations of tryptophan and kynurenine were lower in serum from depressed individuals than from healthy individuals.

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

“…The representative chromatogram is shown in Figure 2. Compared with the chromatogram of the standard sample in Figure 1(a), an unknown endogenous peak occurred in front of KA in the chromatogram of the plasma sample, which was consistent with the previous developed LC-MS/MS method for the determination of KA in human serum (Hu et al, 2017). The plasma concentrations of Trp, Kyn, KA, XA and 5-HT, and the concentration ratios of Kyn/Trp and Trp/5-HT in the control and AMI groups are shown in Table 3.…”

Microglial Dysregulation and Suicidality: A Stress-Diathesis Perspective

“…Twelve studies examining KYNA, another metabolite of the KYN pathway, found no significant differences in blood KYNA concentrations between patients with depression without SB and HC ( 91 , 99 , 100 – 102 , 105 , 106 , 108 – 110 , 118 , 119 ). One study, in addition, reported no differences in levels of CSF KYNA among depressed patients and HC ( 120 ).…”

“…Eleven studies of the KYN pathway metabolite, KYN, provided no convincing evidence of differences in blood KYN levels between depressed patients without SB relative to healthy controls (91,(98)(99)(100)(101)(102)(103)(104)(105)(106)(107). On the other hand, seven studies reported lower blood KYN concentrations in the depressed group (108)(109)(110)(111)(112)(113)(114). A metaanalysis, which evaluated these studies, similarly concluded that blood KYN levels were lower in depressed patients compared to HC (115).…”

Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry with Electrospray Ionization Quantification of Tryptophan Metabolites and Markers of Gut Health in Serum and Plasma—Application to Clinical and Epidemiology Cohorts

“…Tryptophan and its metabolites have previously been quantified using LC-UV 37 and LC-MS/MS. 28,38−44 However, existing methodology either has been applied to relatively small studies (<20 individuals) for biological validation 28,40,41,43 or has only focused on the major metabolites such as kynurenine and tryptophan. 37,39,42 Because of the rise in molecular phenomics in epidemiology, metabolite analysis is increasingly being applied to large population cohorts, 1,45 and therefore the methodology presented here was designed to be sufficiently stable to allow for multiday and multibatch preparations.…”