Dionex chromeleon 6

Two different mobile phases were finally used after several trials to obtain the best mobile phase mixture for the targeted VAs detection. The first mobile phase consisted of methanol (A), acetonitrile (B), and 0.05 M oxalic acid, as recommended by Abbasi et al. [22 (link)], while the second mobile phase was acetonitrile (B), 0.01M oxalic acid (C), and methanol (A) for washing only (Table 2). The prepared mobile phase was filtered through a 0.45 µ filter and then degassed through sonication for 10 min before application. Detection was carried out at 260 nm for TC, 270 nm for Norf and Enorf, and 277 nm for SDZ and OTC. The on-line SPE system was used. A C18 reverse-phase column was used at 30 °C with 35 min as a total run time and an injection volume of 50 µL, with a flow rate of 0.8 mL min⁻¹. The software used for control and data acquisition was Dionex™ Chromeleon™ 6.8 (Thermo Fisher Scientific, Sunnyvale, CA, US). External calibration curves were constructed by preparing the standard solutions at six known concentrations (0.5–100 μg mL⁻¹ for all VAs). The analytes’ concentration in the sample matrices was determined using peak areas that corresponded to the unknown concentrations in the calibration curve.

FBEZF and its degradation products in water were separated on an UltiMate 3000 ultra-high-performance liquid chromatography (UPLC) system (Thermo Scientific Transcend, Thermo Fisher Scientific, San Jose, CA, USA) coupled with a single-stage Orbitrap high-resolution mass spectrometer (MS/MS) (Q-Exactive, Thermo Fisher Scientific, Bremen, Germany). The experiment sample was detected with a heated electrospray interface (ESI, Thermo Fisher Scientific,) in positive ion mode (ESI⁺). Xcalibur program version 3.0.63 (Thermo Fisher Scientific) with Qual and Quanbrowser was used to process the data. Thermo Scientific Dionex Chromeleon 6.8 was employed to screen the target compounds. Optimized tuning parameters were as follows: aux gas heater temperature at 300 °C; capillary temperature at 300 °C; spray voltage at 3.70 kV; and sheath, auxiliary, and sweep gas flow rates at 35, 10, and 2 a.u., respectively. UPLC separations were obtained using an ACQUITY UPLC BEH Shield RP18 column (50.0 mm × 2.1 mm i.d., 1.7 μm film thickness). The mobile phase comprised component A accounting for 70% (H₂O + 0.1% formic acid) and component B was 30% (CH₃CN). The injection volume was 5 µL and the flow rate was set at 0.2 mL min⁻¹. Data were collected in a positive mode within the range of 150 m/z to 500 m/z using full scan and t-SIM/ddMS² analysis with resolution 140,000 during the entire process.