Spherisorb s3 ods 2 c18 column

Phenolic compounds were analyzed in the hydroethanolic extracts and aqueous preparations, which were re-dissolved in ethanol/water (80:20, v/v) and water, respectively, to a final concentration of 10 mg/mL and filtered thought 0.22-μm disposable filter disks. The analysis was performed in an HPLC system (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) coupled with a diode-array detector (DAD, using 280 and 370 nm as preferred wavelengths) and a Linear Ion Trap (LTQ XL) mass spectrometer (MS, Thermo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. Separation was made in a Waters Spherisorb S3 ODS-2 C18 column (3 µm, 4.6 × 150 mm; Waters, Milford, MA, USA). The equipment and operating conditions were previously described by the authors [29 (link)] as well as the identification and quantification procedures. The phenolic standards (caffeic acid, apigenin-6-C-glucoside, and quercetin-3-O-rutinoside) were acquired from Extrasynthèse, Genay, France. The results were expressed as mg per g of extract.

The abovementioned hydroethanolic extracts were redissolved in ethanol/water (80:20, v/v) to achieve the final concentration of 10 mg/mL. Extracts were analyzed in a HPLC system coupled with a diode-array detector (DAD) and a linear ion trap (LTQ XL) mass spectrometer (MS) equipped with an electrospray ionization (ESI) source. Phenolic compounds were separated in a Waters Spherisorb S3 ODS-2 C18 column (Waters Corporation, Milford, MA, USA). The operating conditions and the identification and quantification of the detected compounds was performed according to the protocol of Bessada et al. [27 (link)].

The extract solutions were five-fold diluted with water, filtered through 0.2 μm disposable filter disks, and analysed in a Dionex Ultimate 3000 HPLC system (Thermo Scientific, San Jose, CA, USA) as previously described [4 (link),29 (link)]. Chromatographic separation was made in a Waters Spherisorb S3 ODS-2 C₁₈ column (3 µm, 4.6 mm × 150 mm; Waters, Milford, MA, USA). Double online detection was carried out with a diode array detector (DAD) operating at 280 nm and a Linear Ion Trap (LTQ XL) mass spectrometer (MS, Thermo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. Aloesin was identified based on chromatographic data previously described [4 (link)] and quantified using a seven-level calibration curve (y = 3859.4x + 21770; r² = 0.9996) constructed based on the UV-Vis signal of aloin (Alfa Aesar, Ward Hill, MA, USA) at concentrations ranging from 15.62 to 500 µg/mL. Data were processed using Xcalibur Software and the results were expressed as mg of aloesin per L of extract.

Dried extracts were re-dissolved in a water/ethanol mixture (80:20, v/v) at a concentration of 20 mg/mL and filtered through a 0.22 μm nylon disposable filter. The Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Corporation, Kyoto, Japan), operating at 0.5 mL/min and equipped with a Waters Spherisorb S3 ODS2 C18 column (3 μm, 150 × 4.6 mm) thermostatted at 35 °C, was used for phenolic acids identification and quantification. A binary solvent mixture consisting of 0.1% formic acid in water (A) and acetonitrile (B) was used. The detection was performed using a photodiode array detector (PDA) at 280 nm as the preferred wavelength [27 (link)]. The phenolic acids were quantified using calibration curves obtained from commercial standards, namely: protocatechuic acid (y = 164741x; R² = 0.999), p-hydroxybenzoic acid (y = 113523x; R² = 0.999), p-coumaric acid (y = 433521x; R² = 0.998), and cinnamic acid (y = 583527x; R² = 0.998). The results were expressed as μg per g of extract.

Phenolic compounds were determined in lyophilized decoction extracts, which were re-dissolved in distillated water to a final concentration of 10 mg/mL, filtered through 0.22 μm disposable filter disks. Chromatographic separation of the compounds was achieved with a Waters Spherisorb S3 ODS-2 C18 column (3 µm, 4.6 mm × 150 mm, Waters, Milford, MA, USA), operating at 35 °C. The elution solvents, working in the gradient, were 0.1% formic acid in water and acetonitrile. Finally, to detect MS in negative mode, a Linear Ion Trap LTQ XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an electrospray ionization source (ESI) was used. The identification of phenolic compounds was based on chromatographic performance, spectra, and UV-Vis masses by comparison with standard compounds or the data previously described in the literature, using the Xcalibur^® software (ThermoFinnigan, San Jose, CA, USA). Quantitative analysis of the recognized compounds was completed using calibration curves based on the UV signal of the standard compounds. When commercial standards were not accessible, the calibration curves of the most similar standards were used. The operating conditions were previously described in detail by Bessada et al. [29 (link)] as well as the identification and quantification procedures. The results are expressed in mg per g of fw.