Uplc ms ms

Prepared samples were analyzed by an Agilent UPLC-MS/MS (Infinity ultraperformance liquid chromatograph, Agilent, Beijing, China) with an Eclipse plus C₁₈ column (50 mm × 2.1 mm, i.d. 1.8 μm particle size). The mobile phase consisted of 0.01% formic acid in water (solvent A) and acetonitrile (solvent B) applied at a flow rate of 0.2 mL/min under the following gradient conditions: (1) 0.05 min (A-B, 95:5, v/v); (2) 2 min (A-B, 5:95, v/v); (3) 5.5 min (A-B, 95:5, v/v); (4) 8 min (A-B, 95:5, v/v). Injection volume and column temperature were set at 5 μL and 30 °C. The mass spectrometer was operated with ESI source in the positive ionization mode, and sulfoxaflor was detected by multiple reaction monitoring (MRM) with 1 precursor ion and 2 product ions. Optimized MRM parameters of sulfoxaflor were as follows: Qualifying ion pairs were 278.1/174.0 m/z and 278.1/153.9 m/z, and the quantifying ion pair was 278.1/174.0 m/z; collision energy was 12 V and 20 V, declustering potential was 100 V. The LOQ (S/N = 10) and LOD (S/N = 3) were 3.87 μg·kg⁻¹, 1.16 μg·kg⁻¹ in pollen and 2.26 μg·kg⁻¹, 0.68 μg·kg⁻¹ in nectar, respectively. The mean recoveries of sulfoxaflor in pollen and nectar were within 85.67–92.33% and 83.00–96.18%, respectively. Additionally, the RSDs ranged within 2.46–3.06% and 1.17–4.04%, respectively.

The sulfation reaction system and incubation procedures were the same as those in a previous study [19 (link)]. In brief, the incubation mixture, with a total volume of 200 μL, consisted of 10 μL of enzymes (liver S9 fractions), 5 μL of MgCl₂ (1 mM), 5 μL of PAPS (0.05 mM), 178 μL of potassium phosphate buffer (KPI) (44.5 mM, pH 7.4) and 2 μL of magnolol dissolved in dimethyl sulfoxide (DMSO) (100 mM) and then diluted by 50% ice-cold acetonitrile to different concentrations. The mixture, prepared in triplicates, was incubated in a shaking water bath at 37 °C for 90 min. The reactions were stopped by the addition of an acetonitrile solution containing IS. Incubations without PAPS were used as control.
The sulfation of magnolol at different concentrations was investigated using seven recombinant human SULTs (SULT1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1E1, and 2A1). All assays were conducted at 37 °C for 90 min with a final protein concentration of 0.01 mg/mL. All samples were centrifuged at 14,000× g for 30 min at 4 °C, and the supernatant was analyzed directly by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) (Agilent technologies, Palo Alto, Santa Clara, CA, USA).

Extracts were filtered through 0.2 μm filters (Millipore, Etobicoke, Canada) before UPLC–MS/MS (Agilent, Montreal, Quebec, Canada) analysis equipped with a Hypercarb column (100×2.1 mm, 5 μm) and a Hypercarb pre-column (2.1×10 mm, 5 μm) (Thermo Fisher, Burlington, Canada), as previously described [43] , [44] . Mobile phase consisted in Buffer A: 20 mM ammonium acetate at pH 7.5, and Buffer B: 10% (v/v) methanol in water. Flow rate was set at 0.3 mL min⁻¹ using the following gradient: 0–5 min at 10% A, 5–10 min at linear gradient from 10% to 20% A, 10–20 min at linear gradient from 20% to 100% A, 20–30 min at 100% A, 30–32 min at linear gradient from 100% to 10% A and 32–40 min at 10% A. The Agilent 6460 triple quadruple mass spectrometer (Agilent technologies, Quebec, Canada), equipped with a Jet stream source (Agilent technologies, Quebec, Canada), was used for the analysis of sugar phosphates and low molecular organic acids in negative ion mode. The mass spectrometer parameter were 100 ms scan time; 300°C gas temperature; 7 L min⁻¹ gas flow rate; 35 PSI nebulizer pressure; 400°C sheath gas temperature; 12 L min⁻¹ heath gas flow rate and 3500 V capillary voltage. Data were recorded in MRM mode with the mass spectrometer conditions listed in Table S3. The external standard curve was used for quantification.