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

Abstract

A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1–12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.

Simultaneous Analysis of Tryptophan and Its Metabolites in Human Plasma Using Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

“…Concentrations of the metabolites in human plasma samples from six healthy volunteers (three male and three female) are described in Table 2. The results were consistent with those of previous reports [23,24] [(3HK was 14-54 nmol/L, KYN was 1.2-3.4 μmol/L, 5HT was <4-180 nmol/L, 5HTP was <2.3-8 nmol/L, 3HA was 5-51 nmol/L, Trp was 45-88 μmol/L, XA was 12-33 nmol/L, and KA was 21-66 nmol/L) and (5HIAA was 13.0 ng/mL, IAA was 291.5 ng/mL, KA was 7.9 ng/mL, PA was 5.4 ng/mL, QA was 69.0 ng/mL]. To the best of our knowledge, there is no reported data of ILA in human plasma.…”

“…Various methods of analyzing Trp metabolites have been reported [17][18][19][20][21][22][23][24]. In our previous studies, we have developed analytical methods to quantify amino acids and their metabolites in human plasma using pre-column derivatization with a 3-aminopyridyl-N-hydroxy succinimidyl carbamate (APDS) reagent and liquid chromatography tandem mass spectrometry (LC-MS/MS) [25].…”

“…Previous reports have used various columns for solid-phase extraction [18,24,26], and analysis were chosen for the selective determination of Trp metabolites. Using solid-phase extraction, contaminants can be removed from samples, but this process is time-consuming and results in recovery variation.…”

Metabolic Profiling of Human Plasma and Urine, Targeting Tryptophan, Tyrosine and Branched Chain Amino Acid Pathways

“…In order to increase MS settling time, we selected just one qualifier ion per molecule and tried to avoid MRM transitions common to many metabolites when possible. Recently, Whiley et al (2019) highlighted that the second transition of the TRP 13C-isotope shares the same MRM transition as XA (206.09 > 132.01), interfering with its quantification [51]. With our experimental setup, XA quantification was achieved by MRM transition (206.09 > 160.01) while the MRM (206.09 > 132.01) was used for qualitative purposes.…”

“…Again, the procedure for extraction of metabolites from plasma limited the processing of a large sample set. More recently, Whiley and colleagues (2019) published a thoroughly validated method for the quantitation of 18 TRP metabolites in serum and plasma based on Phenomenex PHREE SPE 96-well plate extraction that allows high-throughput sample preparation [51]. The study, which to our knowledge represents the state-of-the-art in the field, covers the quantitative analysis of 18 metabolites associated with KP and 5-HT degradation pathways, but did not cover BCAAs or gut-derived metabolites.…”