4.3.2. Sample Preparation
SEPARATION
Samples were thawed on ice and were vortexed for 1 min at +4 °C. Then, samples were centrifuged for 5 min at 5000× The prepared samples were vortex-mixed for 5 min at +4 °C. Afterward, the samples were centrifuged for 10 min at maximum rpm (18,000× A Sciex 4500QTRAP tandem mass spectrometer (AB Sciex Pte. Ltd., Framingham, MA, USA) with a Shimadzu Nexera 30AD chromatograph (Shimadzu, Kyoto, Japan) was used for analysis. Ion source settings were as follows: temperature = 550 °C; GS1 = 55; GS2 = 55; CUR = 25; IS = 5500. Ion detection was performed in the positive ionization mode of the sample. The chromatographic separation of the components of the test sample was performed via RPLC chromatography using an Agilent ZORBAX Eclipse Plus (Aglient Technologies, Santa Clara, CA, USA) C18 2.1 × 50 mm 1.8 μm chromatographic column: phase A (water; 5 mM ammonium formate); phase B (ACN: MeOH 1:1; 5 mM ammonium formate); sample input volume 1 µL. Chromatographic gradient: 0 min 2% B; 0.25 min 2% B; 1.2 min 98% B; 2.5 min 98% B; 2.6 min 2% B; 3.5 min 2% B; flow 0.45 mL/min, thermostat temperature 45 °C. The referent values for TMAO were presumed to be 0.5–5.0 μM/L as recommended in certain studies [