Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection

Abstract

A simple, rapid isocratic liquid chromatographic procedure with ultraviolet and fluorimetric detection is described for the separation and quantification of L-tryptophan (Trp) and six of its kynurenine metabolites (kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic, kynurenic, xanthurenic and anthranilic acids). Using the Perkin Elmer LC 200 system, a reverse phase Synergi 4 μ fusion-RP80 A column (250 × 4.6 mm) (Phenomenex), and a mobile phase of 10 mM sodium dihydrogen phosphate: methanol (73:27, by vol) at pH 2.8 and a flow rate of 1.0–1.2 ml/min at 37 °C, a run took ∼13 min. The run took <7 min at 40 °C and a 1.4 ml/min flow rate. Limits of detection of all 7 analytes were 5–72 nM and their recoveries from human plasma and rat serum and liver varied between 62% and 111%. This simple method is suitable for high throughput work and can be further developed to include quinolinic acid and other Trp metabolites.

A liquid chromatography – tandem mass spectrometry method to measure a selected panel of uremic retention solutes derived from endogenous and colonic microbial metabolism

“…Previously reported LC-MSMS methods allow for quantification of a lesser number of analytes in one chromatographic run, and/ or have longer total run times [26,30,31,33,35,[38][39][40]. The LC-MSMS method as described by Itoh et al [29] also allows for quantification of a significant number of relevant analytes.…”

“…A C C E P T E D ACCEPTED MANUSCRIPT 5 Several of these metabolites can be measured using existent methodologies, using gas chromatographic mass spectrometry [24], high performance liquid chromatography (HPLC) coupled to fluorescence and ultraviolet detection [21,[25][26][27] or mass spectrometry [28][29][30][31][32][33][34][35].…”

Metal Ion‐Assisted Infrared Optical Sensor for Selective Determination of Tryptophan in Urine Samples

“…[4][5][6] Several methods have been developed to determine the concentration of tryptophan in a variety of matrices. Chromatographic separation, including liquid chromatography 7,8 and capillary electrophoresis chromatography, 9,10 has been investigated extensively. Mass spectrometry is also becoming increasingly more popular because of its sensitivity.…”

Novel inhibitor of brain indoleamine 2,3 dioxygenase, docking and experimental studies

“…Transfer the filtrate into an HPLC vial using a pasteure pipette. Serum TRP and KYN concentration were determined by HPLC method of Badawy and Morgan (2010).…”

“…A 0.5 ml portion of each homogenate was filtered and used or stored. IDO activity was determined by calculating KYN/TRP ratio in serum and brain as described by (Badawy and Morgan, 2010).…”