Waters acquity uplc hss t3

The UPLC-PDA system (WATERS Acquity H-Class, Milford, MA, USA) was used to detected L-ascorbic acid [60 (link)]. 0.5 g grounded (IKA A11 basic, IKA- Labortechnik, Staufen, Germany) pepper was mixed with 35 mL extraction solution (3%meta-phosphoric acid, 8% acetic acid, 1 mM EDTA—VWR Chemicals BDH Prolabo, Leuven, Belgium), 10 min vortexed and centrifuged (5 min, 6000 rpm). Supernatant was filtered through 0.22 μm polytetrafluoroethylene (PTFE) syringe filters. 1 mL solution was added to 1 mL eluent and injected (5 µL). Separation was performed using an WATERS Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 μm; Waters, Ireland) with pre-column BEH C18 (2.1 × 5 mm, 1.7 µm; Waters, Ireland). Mobile phase (Milli-Q water with 0.1% (v/v) formic acid) flow was 0.25 mL/min. Column thermostat temperature was 25 °C and the samples were kept at 4 °C. The spectrum at wavelength 245 nm was analyzed. Vitamin C content was calculated in relation to the calibration curve prepared for the L-ascorbic acid (VWR Chemicals BDH Prolabo, Leuven, Belgium) analytical standard (0.005–0.100 mg/mL). The analysis was performed in triplicate.

Tryptophan-targeted metabolism in mouse feces was analyzed according to a previous study [36 (link)]. Mice feces were accurately weighed (50 mg), and then 500 μL of extraction solution (methanol/acetonitrile/water = 2:2:1, pre-cooled at −40 °C, with 0.1% formic acid and an internal standard of isotope labeling) was added. The mixture was vortexed for 30 s and sonicated for 5 min in an ice-water bath (SB 25-12 DTDN, Scientz, Ningbo, China). This ultrasound-assisted extraction was repeated twice. Subsequently, the samples were left to rest at −40 °C for 1 h, followed by centrifugation (13,523× g, 15 min, 4 °C). A volume of 320 μL of the supernatant was transferred, dried under nitrogen gas, redissolved in 80 μL of an aqueous solution containing 0.1% formic acid, and then centrifuged at 13,523× g at 4 °C for 15 min. The supernatant was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed on a column (Waters ACQUITY UPLC HSS T3, 100 mm × 2.10 mm, 1.8 μm, Waters, Milford, MA, USA) maintained at 40 °C. The autosampler temperature was set at 4 °C and the injection volume was 5 μL. Analytes were eluted using a gradient of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).