Acquity uplc beh c18 column

The same chromatographic conditions were used for targeted, untargeted, and semi-quantitative analyses. For pure compound experiments, samples were injected (2 µL) into an Acquity UPLC^® BEH C18 column (1.7 µm, 2.1 × 50 mm; Waters, Milford MA, USA) and eluted (0.5 mL/min, 40 °C) with water (A) and acetonitrile (B) both with 0.1% formic acid (Fischer Chemical, Fischer Scientific AG, Reinach, Switzerland). A 2-min gradient of 10 to 98% of B was applied, followed by an isocratic step of 0.8 min at 98% of B and a re-equilibration step of 2 min.
For PLRE experiments, samples were injected (2 µL) into an Acquity UPLC^® BEH C18 column (1.7 µm, 2.1 × 100 mm; Waters, Milford, MA, USA) and eluted (0.5 mL/min, 40 °C) with water (A) and acetonitrile (B) both with 0.1% formic acid. The following gradient was used: from 10 to 17% of B from 0 to 5 min, 17 to 75% from 5 to 11 min, 75 to 98% from 11 to 12 min, an isocratic step at 98% for 2 min, and a re-equilibration step of 2 min.

AG and SAG and fermentation bacteria were separated on a Macro Porous Resin AB-8 column. Two dried products (purity > 99.0%) were produced from the eluted fraction following evaporation. The concentration of the compounds was determined with a Waters 2695 Alliance HPLC system (Waters, USA) equipped with an ACQUITY UPLC BEH C₁₈ column (1.7 μm 2.1 × 100 mm). Elution was carried out at a flow rate of 0.4 mL·min⁻¹ with 0.1% formic acid water (A)/acetonitrile (B) in the following gradient mode: 0–2 min, 95%–95% B; 2–12 min, 95%–5% B; 12–13 min, 5%–5% B; 13–13.5 min, 5%–95% B; 13.5–16 min, 95%–95% B. UPLC/ESI-MS was carried out on a Waters UPLC-TOF mass spectrometer with an ACQUITY UPLC BEH C₁₈ column (1.7 μm 2.1 × 100 mm). Pure apigenin conversion products dissolved in DMSO-d6 were analyzed by ¹H NMR and ¹³C NMR spectra with a Bruker AV-400 spectrometer (Bremen, Germany) at 400 MHZ. Additionally, heteronuclear multiple bond correlation (HMBC), heteronuclear multiple quantum coherence, and distortionless enhancement by polarization transfer were also performed.

Metabolites and products of all enzymatic assays were analyzed using an LC-MS system (Acquity ultra-performance liquid chromatography coupled to a Synapt G2-S HDMS quadrupole-ion mobility spectrometry–time of flight mass spectrometer; Waters). Extracts from Salvia tissues were separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 µm; Waters) at a flow rate of 0.45 ml min^–1 using a linear gradient of acetonitrile (B) and water (A) with 0.1% (v/v) formic acid: 0 min, 7% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 7% B; 13 min, 7% B. Dephosphorylated enzymatic products were separated on a CORTECS UPLC C18⁺ column (100 × 2.1 mm, 1.6 µm; Waters) using the same gradient and flow rate. Positive-mode electrospray ionization (ESI) was applied for ionization. The Q-TOF-MS instrument parameters were: source 3.2 kV at 100 °C; desolvation temperature of 250 °C; desolvation gas flow of 800 l h^–1. The transfer collision energy for MS^e and MS/MS fragmentation was 15 eV. Standard curves of serial dilutions of authentic standards were used for quantification. The direct SdKPS enzyme product and GGPP were separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 µm; Waters) with 10 mM ammonium bicarbonate buffer (A) and acetonitrile (B) as the mobile phase using the same linear gradient and flow rate as above, and were ionized under negative ESI mode.