Nexera x2 uplc

Endogenous urinary steroids and bile acids were quantified using isotopic enrichment and authentic calibration standards detected by LC–MS/MS with electrospray ionization and multiple reaction monitoring (MRM) on an API 6500QTRAP (Sciex, Framingham, MA, USA) as previously reported for plasma^{47 (link)}. Urine extraction was performed by solid-phase extraction (SPE) using Oasis-HLB SPE 96-wellplates 30 mg (Milford, MA, USA). Briefly, 100 µL urine aliquots were spiked with 10 µL of methanol containing a suite of deuterated bile acids and steroids at 12.5 nM, vortexed, and loaded onto SPE cartridges pre-conditioned with 1 mL MeOH, followed by 1 mL H₂O. Loaded cartridges were then washed with 1 mL H₂O and eluted with 2 mL MeOH. The eluate was evaporated under vacuum at room temperature and reconstituted in 100 µL of 50:50 MeOH:H₂O (v/v). Extracts were separated on a 2.1 × 100 mm, 1.7 μm Acquity C18 BEH column using a Shimadzu Nexera X2 UPLC (Shimadzu, Kyoto, Japan). Analytes were quantified using internal standard methods with surrogate/analyte associations for response ratio calculations. Data was processed with AB Sciex MultiQuant v 3.0.1.

The HPLC system used was a Shimadzu Nexera X2 UPLC (Kyoto, Japan) chromatograph equipped with a solvent delivery unit (LC-30AD), an autosampler (SIL-30AC), a column oven (CTO-20A), a degasser (DGU-20A5R), and a photodiode array detector (SPD-M20A). Separation was conducted on a Shim-pack XR-ODS column (2.0 × 75 mm, 1.6 μm; Shimadzu Cooperation, Japan). The column temperature was set at 30°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). The composition of the mobile phase was 5% (B) for 0–2 min, 5%–10% (B) for 2–4.5 min, 10%–40% (B) for 4.5–11 min, 40%–60% (B) for 11–13 min, 60%–70% (B) for 13-14 min, 70%–80% (B) for 14–16 min, 80%–90% (B) for 16-17 min, 90% (B) for 17–20 min, and it was held for 3 min and then reequilibrated to 5% (B) until the end of the analysis. The flow rate was 0.2 mL/min and the injection volume was 5 μL. The detection wavelengths of all standards and samples were in the UV at 210, 254, and 280 nm.

Abscisic acid was determined in Physcomitrium patens samples using the LC-MS/MS system which consisted of Nexera X2 UPLC (Shimadzu) coupled QTRAP 6500+ mass spectrometer (Sciex). Chromatographic separations were carried out using the Acclaim RSLC C18 column (150×2.1 mm, 2.2μm, Thermo Scientific) employing acetonitrile/water+0.1% acetic acid linear gradient. The mass spectrometer was operated in negative ESI mode. Data was acquired in MRM mode using following transitions: 1) ABA 263.2->153.1 (−14 eV), 263.2->219.1 (−18 eV); 2) ABA -D6 (IS) 269.2->159.1 (−14 eV), 269.2->225.1 (−18 eV); declustering potential was −45 V. Freeze-dried moss samples were ground using the metal beads in homogenizer (Bioprep-24) to a fine powder. Accurately weighted (about 20mg) samples were spiked with isotopically labeled ABA -D6 (total added amount was 2 ng) and extracted with 1.5 ml acetonitrile/water (1:1) solution acidified with 0.1% formic acid. Extraction was assisted by sonication (Elma S 40 H, 15 min, two cycles) and solution was left overnight for completion of extraction. Liquid was filtered through 0.2 μm regenerated cellulose membrane filters, evaporated to dryness upon a stream of dry nitrogen and redissolved in 100 μl extraction solution.