Kynurenine Pathway Metabolite Analysis

Tryptophan, kynurenine, and kynurenine metabolites were measured by liquid chromatography–tandem mass spectrometry, using a CTC PAL HTS-XT, Agilent 1260 Infinity liquid chromatograph and an AB SCIEX 6500 QTRAP mass spectrometer (AB Sciex UK, Warrington, United Kingdom). All results were calculated in Analyst 1.6.

Tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, picolinic acid, and quinolinic acid were obtained from Sigma Aldrich (Poole, United Kingdom). The stable isotope–labeled internal standards were ²H₅-tryptophan (QMx Laboratories, Thaxted, United Kingdom), ²H₆-kynurenine sulfate and ²H₅-kynurenic acid (CK isotopes, Ibstock, United Kingdom), and ²H₃-picolinic acid and ¹³C₃,¹⁵N-quinolinic acid (LGC standards, Teddington, United Kingdom). Acetonitrile and methanol were obtained from Rathburn Chemicals (Walkerburn, United Kingdom) and Fisher Scientific UK (Loughborough, United Kingdom), respectively.

Standards of each analyte were used to automatically tune in multiple reaction monitoring (MRM) mode. Transition optimizations were performed manually. Xanthurenic acid was significantly more sensitive in negative ion mode, but, with the chromatographic conditions used, there was massive matrix ion suppression. Consequently, xanthurenic acid was measured in positive ion mode.

Because analyte retention times on Chirobiotic-T columns tend to be matrix dependent, there is a critical requirement for the appropriate stable isotope internal standards. Consequently, when stable isotopes were unavailable, 2 separate transitions, when possible, were used to calculate and then confirm the result.

For measurement of all analytes, 60 µL of blank (deionized water), aqueous standards, and CSF were mixed with 10 mL of the stable isotope mix (20 µL of 10 mM ²H₅-tryptophan; 2 µL of 16 mM ²H₆-kynurenine sulfate; 2 µL of 2 mM ²H₅-kynurenic acid; 5 µL of 15 mM ¹³C₃,¹⁵N-quinolinic acid; and 5 µL of 40 mM ²H₃-picolinic acid) in 75 µL of methanol, mixed by vortexing, and centrifuged for 5 minutes at 20 817 × g. Supernatants were transferred to a 96-well deep well polypropylene plate, a sealing mat was applied, and the plate was placed in the CTC autosampler and cooled to 7.5°C, awaiting injection.

Isocratic chromatography of tryptophan and kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and xanthurenic acid (injection volume, 10 µL; data acquisition time, 7 minutes) was performed on a 5-µm Astec Chirobiotic-T column (10 cm [length] × 2.1 mm [internal diameter] guard column), using a 1:1 ratio of acetonitrile to water with 0.025% formic acid at a flow rate of 200 µL/minute. Tandem mass spectrometry parameters were as follows: curtain gas, 40; CAD gas, medium; ion source voltage, 5250 V; gas temperature, 400°C; gas 1, 25; and gas 2, 25.

Isocratic chromatography of quinolinic acid and picolinic acid (injection volume, 10 µL; data acquisition time, 4 minutes) was performed on a 5-µm Astec Chirobiotic-T (10 cm [length] × 2.1 mm [internal diameter] guard column), using 62.5% water/acetonitrile at a flow rate of 225 µL/minute. Tandem mass spectrometry parameters were as follows: curtain gas, 45; CAD gas, medium; ion source voltage, −4500 V; gas temperature, 400°C; gas 1, 30; and gas 2, 30. Full details of mass spectrometry conditions are described by Forrest et al [19].