Xevo tq s micro

One 3.2mm disc of DBS was incubated with 100 µL of 2.5 nmoL/L of lysoSM-d7 in a 96-well polypropylene plate at 45 °C at 500 RPM for 1 h. After the incubation, the plate was centrifuged for 5 min at 3000 G. The supernatant was transferred to a new 96-well plate and 50 µL of MilliQ H₂O was added [26 (link)]. Then, 10 µL were injected into the ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) [26 (link)]. The mass spectrometer was a Xevo TQ-S micro from Waters (Milford, CT, USA) with an XSelect® CSH™ C18 3.5 µm (2.1 × 50 mm) from Waters. The column was kept at 55 °C.
The mobile phase was a gradient elution of 70:30 H₂O with 0.1% formic acid [solution a] to 65:35 isopropanol/acetonitrile with 0.1% formic acid [solution b]. The flow rate was 0.8 mL with a gradient of: 99.5% A at 0 min, 75% A at 0.75 min, 40% solution A at 1 min, 25% solution A at 1.5 min, 100% solution B at 1.80 min, 100% solution B at 2.15 min, 99.5% solution A at 2.20 min. The mass spec was operated with electrospray on positive mode with multiple reaction monitoring (MRM). Precursor and product ions (m/z) were used to quantify lysoSM509 509,184; lysoSM-d7 472.4,184 with 30 V for cone and 22 V for collision. The injection volume was 10 µL per sample with a running time of 2.20 min. The levels of lysoSM509 were expressed as nmoL/L.

Metabolites analyses were performed liquid chromatography (ACQUITY I-Class Plus UPLC FL, Waters Corp) coupled to electrospray mass spectrometry (XEVO TQ-S Micro, Waters Corp). For each analysis, 5 μL of sample was injected on an Agilent InfinityLab Poroshell 120 HILIC-Z P 2.7 μm, 2.1×50 mm (Agilent Technologies Inc., Santa Clara, CA USA); using a flow rate of 0.5 mL/min at 25°C. Mobile phase A consisted of water with 10 mM ammonium acetate + 5 μM medronic acid (Agilent), pH 9. Mobile phase B consisted of acetonitrile:water 9:1 with 10 mM ammonium acetate + 5 μM medronic acid (Agilent), pH 9. The gradient was programmed as follows: 0.0–1.0 min at 95% B; 1.0–6.0 min to 50% B; 6.0–6.5 min to 95% B; and 6.5–10.0 min at 95% B. Electrospray ionization was performed in negative ion mode. The following source settings were applied: capillary voltage at 1.9 kV; source temperature at 150°C; desolvation temperature at 600°C; desolvation gas flow at 1000 L/hr; cone gas flow at 50 L/hr; cone voltage at 20 V; nebulizer gas at 7 bar. Data acquisition was performed in multiple reaction monitoring mode (MRM) with the collision energy (CE) values reported in Supplementary Table 6. Quantification was performed using Skyline (v19.1;University of Washington).

The rice bran-extracted samples thermally treated were analyzed for the residual GF2, GF3, and GF4 contents using an ultra-performance liquid chromatography system equipped with an ESI (Waters Corporation, Milford, MA, USA) coupled to an MS/MS system (Xevo TQS Micro, (Waters Corporation, Milford, MA, USA). The chromatographic separation was performed on a Luna amino—NH₂ column (150 mm × 2 mm × 5 µm) (Phenomenex, Torrance, Los Angeles, CA, USA) [30 (link)]. The elution was performed at a constant flow rate of 0.45 mL/min following the program presented in Table 1, with an injection volume of 10 µL and an overall run time of 5 min per sample injection. The Xevo TQS Micro MS/MS system runs in negative ionization mode. The initial optimization parameters are as follows: ionization potential (capillary) of 2.5 kV, source temperature of 150 °C, desolvation temperature of 500 °C, and nitrogen gas rate of 800 mL/h. The fragmentation conditions for measurement of GF2, GF3, and GF4 are also reported in Table 1.