Acquity uplc beh shield rp18 column

The LCMS lipid mediators analysis was performed based on previous publications [26 (link),36 (link)]. Briefly, high-performance liquid chromatography (HPLC) was performed using an Agilent 1290 series chromatographer (Agilent, Santa Clara, CA, USA). Reversed-phase separation was achieved by Acquity UPLC BEH shield RP18 column (2.1 × 100 mm; 1.7 m; Waters) and maintained at 40 °C. The mobile phase was a gradient of solvents A (ACN/water/acetic acid (60/40/0.02, v/v)) and B (ACN/IPA (50/50, v/v) with a flow rate of 0.5 mL/min. The stepwise gradient conditions were carried out for 10 min as follows: 0–5.0 min, 1–55% of solvent B; 5.0–5.5 min, 55–99% of solvent B, and finally 5.5–6.0 min, 99% of solvent B. Injection volume was 10 μL, and all samples were kept at 4 °C throughout the analysis.
The HPLC system was coupled to Agilent 6495 triple-quad mass spectrometer (Agilent, Santa Clara, CA, USA). The electrospray ionization was conducted in negative mode, and the dynamic MRM option was used and performed for all compounds with optimized transitions and collision energies. The determination and integration of all peaks were manually performed using the Mass Hunter Workstation software (Agilent, Santa Clara, CA, USA).

Sample supernatants (5 µL) were injected using a Waters FTN Sample manager onto an ACQUITY UPLC BEH Shield RP18 column (Waters; particle size: 1.7 µm, length: 50 mm, internal diameter: 2.1 mm). The initial solvent was 81% A (solvent A: 100% water with 5 mmol/L ammonium acetate and 0.01% formic acid; solvent B: 100% methanol with 5 mmol/L ammonium acetate and 0.01% formic acid) at a flow rate of 0.4 mL/min at 45 °C. Gradient elution (Table 8) was performed using a Waters ACQUITY UPLC I-class pump. Mass spectrometric detection employed a Xevo TQ-XS (Waters) with Unispray ionization in negative ion mode. Individual bile salts were monitored in MRM windows, either as pseudo-transitions (for unconjugated species) or as glycine/taurine daughter ions.

The detection of FBEZF in water was performed on a Waters ACQUITY UPLC H-class system fitted with a sample manager, a quaternary solvent manager, a PDA detector, and an ACQUITY UPLC BEH Shield RP18 column (50.0 mm × 2.1 mm i. d., 1.7 μm film thickness) (Waters Corporation, Milford, MA, USA). The column temperature was at 40 °C. A total of 2 µL sample solution was injected, and the chromatography was run with acetonitrile/water (30/70, v/v) at a flow rate of 0.2 mL min⁻¹. The chromatographic conditions were determined from the trial experiments for optimal results in terms of peak shape, column efficiency, chromatographic analysis time, selectivity, and resolution. FBEZF was detected at 212 nm. Retention time of FBEZF was 3.9 min under the optimized chromatographic conditions.

Phenolic compounds present in the fermented and non-fermented avocado leaf extracts were analysed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) coupled to an electrospray ionization (ESI) source operating in the negative mode and a mass detector time of flight (TOF) micro mass spectrometer (Waters). The compounds of interest were separated on an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at 40 °C using the conditions and gradient previously stated [31 (link)]. H₂O acidified with 1% of acetic acid and acetonitrile were used as phase A and B, respectively. Analyses were performed in triplicate. The identification of the phenolic compounds was made according to the literature. For ensuring the mass accuracy, the tolerances chosen had a score higher than 90% and error lower than 5 ppm. To quantify the phenolic compounds identified in the avocado leaf extracts, calibration curves were used for vanillic acid (y = 8.1947x + 122.91; R² = 0.9976), chlorogenic acid (y = 85.138x + 135.16; R² = 0.9978), ferulic acid (y = 16.507x + 92.06; R² = 0.9980), quercetin (y = 112.8x + 287.12; R² = 0.9957), catechin (y = 41.108x + 335.6; R² = 0.9959) and rutin (y = 26.176x + 403.46; R² = 0.9924). The results are expressed as µg/g d.w.

The analysis of selected reaction intermediates was performed by the method of Vo Duy et al. (35 (link)). LC instrumentation included an ACQUITY^TM Ultra Performance Liquid Chromatography integrated system from Waters (Milford, MA). Mass spectrometry experiments were performed using a triple quadrupole mass spectrometer TSQ Quantum Ultra^TM (Thermo Fisher Scientific Inc., Waltham, MA). Metabolites were separated using an Acquity UPLC BEH Shield RP₁₈ column (150 mm × 2.1 mm, 1.7 µm; Waters, Saint-Quentin en Yvelines, France) in the positive ionization mode. Heptafluorobutyric acid was added to the mobile phase as an ion pair reagent. Quantitation was performed using one internal standard. Each metabolite was quantified against a calibration curve prepared by spiking RBC cell pellets (3 × 10⁶ cells) with appropriate working solutions of all metabolites and the internal standard. Using the method Vo Duy et al. (35 (link)) the quantities of P-Cho were overestimated because of a lack of specificity. The introduction of a second product ion for P-Cho allowed us to increase the specificity of the method and to obtain correct values.
For the water-soluble metabolites and the final PLs, the area of peaks were determined and reported as percent of the total contents of the corresponding metabolite or PL.