6224 tof ms system

Analyses of previously prepared urine samples, with the use of capillary electrophoresis coupled with time-of-flight mass spectrometry, were performed with a 7100 CE coupled with 6224 TOF/MS system (Agilent Technologies, Germany) equipped with an ESI ion source with a sheath liquid delivery system. Separation of the compounds was performed with a fused silica capillary (50 μm × 100 cm; Agilent Technologies, USA). Before each analysis, the capillary was flushed with BGE (0.8 ml/L, formic acid in 10% methanol in water) for 5 min at 950 mbar. Prepared urine samples were injected into the capillary for 50 s under 50 mbar pressure. Next, BGE was injected for 10 s under 100 mbar pressure. Compound separation was performed under a pressure of 25 mbar, voltage of 30 kV and amperage of 22 μA. The sheath liquid was delivered to the ion source with a flow rate of 0.6 ml/min. The drying gas temperature and flow rate were 200 °C and 10 L/min, respectively, with the nebulizer pressure set to 10 psi. The capillary, fragmentor and skimmer voltages were 3500 V, 125 V and 65 V, respectively. The mass spectrometer was operated in scanning mode with a m/z range from 50 to 1000.

Untargeted metabolomics analysis of urine samples was performed with a 1200 HPLC coupled with a 6224 TOF/MS system (Agilent Technologies, Germany) equipped with a dual electrospray ionization source (Dual-ESI). A Zorbax Extend-C18 Rapid Resolution HT column (2.1 × 100 mm; 1.8 μm) was used. The injection volume was 2 μl, and the mobile phase flow rate was 0.35 ml/min. The time of analysis was 18 min, with 10 min column equilibration. RP-HPLC separation was performed in gradient elution mode using mobile phases: A – 0.1% formic acid in water and B – 0.1% formic acid in acetonitrile. The gradient elution program was as follows: 0–6 min from 98% to 80% A, 6–9 min from 80% to 55% A, 9–14 min from 55% to 2% A and from 14 to 18 min 2% A. The column temperature was set to 35 °C. The drying gas temperature and flow rate were 350 °C and 11 L/min, respectively, with the nebulizer pressure set to 50 psi. The capillary, fragmentor and skimmer voltages were 3250 V, 150 V and 65 V, respectively. Data were collected in scan mode with m/z ranging from 50 to 1200 in both positive and negative ionization modes. Additionally, the analyses were performed using the in-source fragmentation method with the fragmentor voltage set to 120 V, 160 V and 200 V, with other parameters unchanged.