Phenolic Profiling of PME Extract

The phenolic profile of PME was determined in the dried extract re-dissolved in a methanol:water (80:20, v/v) mixture by liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI/MSⁿ) (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA), as previously described by Bessada et al.^{20 (link)}. For the double online detection, 280 and 370 nm were used as preferred wavelengths for diode array detection (DAD) and in a mass spectrometer (MS) connected to HPLC system via the DAD cell outlet. The MS detection was performed in negative mode, using a Linear Ion Trap LTQ XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The identification of the phenolic compounds was performed using standard compounds, when available, by comparing their retention times, UV–Vis and mass spectra; and also, comparing the obtained information with available data reported in the literature giving a tentative identification. For quantitative analysis, a 7-level calibration curve for each available phenolic standard was constructed based on the UV signal [catechin (y = 84.950x − 23.200, R² = 0.999, LOD (Limit of detection) = 0.17 μg/mL; LOQ (Limit of quantification) = 0.68 μg/mL, peaks 1, 2, 3, 4, and 5), myricetin (y = 23287x − 581,708, R² = 0.9988, LOD = 61.21 µg/mL and LOQ = 185.49 µg/mL, peaks 6 and 7) and quercetin-3-O-glucoside (y = 34843x − 160,173, R² = 0.9998; LOD = 0.21 μg/mL; LOQ = 0.71 μg/mL, peak 8)]. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standard. The results were expressed as mg/g of extract DW.