Spherisorb s3 ods 2 c18

The obtained extracts were re-dissolved in 20% aqueous ethanol at 10 mg/mL, filtered through a 0.22 µm nylon syringe filter, and analyzed by HPLC-DAD-ESI/MSn in a Dionex Ultimate 3000 UPLC system (Thermo Scientific, San Jose, CA, USA). The equipment consisted of a diode array detector coupled to an electrospray ionization mass detector, a quaternary pump, an auto-sampler (kept at 5 °C), a degasser, and an automated thermostated column section (kept at 35 °C). A Waters Spherisorb S3 ODS-2 C18 (3 µm, 4.6 mm × 150 mm, Waters, Milford, MA, USA) column was used. Data were collected simultaneously with DAD and in negative mode detection on a Linear Ion Trap LTQXL mass spectrometer (Thermo Scientific, San Jose, CA, USA), following a previously described procedure [22 (link)]. An Xcalibur^® data system (Thermo Scientific, San Jose, CA, USA) was used in data acquisition. Phenolic compounds were identified by comparing their retention times and UV–Vis and mass spectra with standards, when available. Otherwise, compounds were identified tentatively by comparing data obtained from available information published in the literature. For quantitative analysis, calibration curves obtained from available standards or closely related standards were used. The results were expressed as mg/g of extract.

The phenolic profile was evaluated (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) after redissolving each lyophilized extract in ethanol/water (80:20; v/v) to a final concentration of 10 mg/mL (for decoctions and hydromethanolic extracts) or 20 mg/mL (for hydroethanolic and ethanolic extracts). A DAD detector (280 and 370 nm as the preferred wavelengths) coupled to an electrospray ionization mass detector (LC-DAD-ESI/MSn) was used. The selected column was a Waters Spherisorb S3 ODS-2 C18 (3 µm, 4.6 × 150 mm, Waters, Milford, MA, USA) functioning at 35 ℃. Compounds were eluted with a gradient mixture of 0.1% formic acid in water and acetonitrile. MS analysis was performed in negative mode (Linear Ion Trap LTQ XL mass spectrometer, ThermoFinnigan, San Jose, CA, USA) using an electrospray ionization source (ESI). Phenolic compounds were identified based on their chromatographic behavior, spectra, and UV–Vis mass, either by comparison with authentic standards or available data from similar studies using the Xcalibur^® software (ThermoFinnigan, San Jose, CA, USA). Each compound concentration (mg/g of lyophilized extract) was quantified through calibration curves drawn from the UV signal of the corresponding (or most similar) standard compound [22 (link)].

All the chromatographic information was obtained using a Dionex Ultimate 3000 UPLC (Thermo Scientific, San Jose, CA, USA), coupled to a diode array detector (280, 330, and 370 nm) and an electrospray ionization mass detector (Linear Ion Trap LTQ XL, Thermo Finnigan, San Jose, CA, USA), working in the negative mode. The chromatographic separation was performed using a Waters Spherisorb S3 ODS-2 C18 (4.6 × 150 mm, 3 μm, Waters, Milford, MA, USA) column at 35 °C. The compounds were identified considering the retention time, UV-Vis, and mass spectra in comparison with available standards. For quantitative analysis, calibration curves were obtained using injecting standard solutions with known concentrations (2.5–100 μg/mL): chlorogenic acid (y = 168823x − 161172), quercetin-3-O-rutinoside (y = 13343x + 76751) and p-coumaric (y = 301950x + 6966.7), based on UV-Vis signals and using the maximum absorption wavelength of each standard compound. In the case of unavailable commercial standards, the compounds were quantified via a calibration curve of the most similar standard available. The results were expressed as mg/g of extract [12 (link)].