4.6. Serum Sample Processing for Determination of Kynurenine, KYNA, and SZR72 Levels

Arterial blood samples (1 mL) at selected time intervals (Figure 1) were collected and allowed to clot, followed by centrifugation (4 °C; 10 min; 13,000 rpm) to obtain serum samples that were kept at −80 °C until analysis. For targeted analytical measurements, LC–MS grade solvents were used in all cases. The method for the preparation of serum samples prior to the analysis of endogenous kynurenine and KYNA was the following. First, 50 µL serum was spiked with 7 µL internal standard (IS) solution containing SZR73 (0.5 µM) in water/methanol (50/50 v/v) [52], and 5 µL water/methanol (50/50 v/v) solution, then 600 µL ice-cold acetone was added in a 1.7 mL microcentrifuge tube (Corning-Costar 3620, Corning Inc., Corning, NY, USA). The sample was vortex mixed for 15 s, after shaking for 10 min at room temperature the sample was centrifuged at 15,000 rpm for 15 min at 4 °C degrees (Hettich 320R, Hettich Gmbh, Tuttlingen, Germany). The 600 µL of the upper layer was transferred to a microcentrifuge tube and evaporated to dryness under nitrogen at ambient temperature (MD 200, Allsheng Instruments Ltd., Hangzhou, China). For analysis, the dried extracts were dissolved in 50 µL of H₂O/MeOH/NH3 (90/10/0.1 v/v/v%), vortex mixed for 15 s, centrifuged at 15,000 rpm for 15 min at 22 °C degrees, and the upper layer transferred to a 250 µL conical insert. For calibration samples, 50 µL pulled control serum sample was spiked with 7 µL IS, then 5 µL given calibration mix containing kynurenine and KYNA in water/methanol (50/50 v/v) solution, then 5 µL IS, and then 600 µL ice-cold acetone was added in a microcentrifuge tube. Then, the above-described sample preparation protocol was applied. The calibration points were the followings: 0, 2.79, 2.89, 2.99, 3.29, 4.79, and 6.79 µM for kynurenine, and 0, 23.98, 24.98, 25.98, 28.98, 33.98, 43.98, 63.98, 123.98, and 523.98 nM for KYNA.

For the analysis of SZR72, the applied sample preparation procedure was slightly modified. In brief, 10 µL serum was spiked with 7 µL IS solution containing SZR73 (10 µM) in water/methanol (50/50 v/v) and 10 µL water/methanol (50/50 v/v) solution, then 600 µL ice-cold acetone was added in a 1.7 mL microcentrifuge tube (Corning-Costar 3620, USA). The parameters of vortex mixing, shaking, centrifugation, collection of upper phase, and evaporation were the same as used for kynurenine/KYNA analysis. The dried extracts were dissolved in 500 µL of H₂O/MeOH/NH3 (90/10/0.1 v/v/v%), vortex mixed for 15 s, centrifuged at 15,000 rpm for 15 min at 22 °C degrees, and the upper layer transferred to a 250 µL conical insert. In the case of calibration samples, calibration points were set to 0, 5, 10, 50, 100, and 200 µM for SZR72.