Phenolics in crude extracts and each fraction of lingonberries were identified and quantified by HPLC-PDA (Waters e2695 Alliance system, Waters, Milford, MA, USA) according to Raudone et al. with some modifications [27 (link)]. Chromatographic separation was carried out on an ACE C18 reversed-phase column (250 mm × 4.6 mm, particle size 3 µm; ACT, UK) with a gradient elution consisting of 0.1% trifluoroacetic acid in water (eluent A) and acetonitrile (eluent B) at a flow rate of 0.5 mL/min, injection volume of 10 µL, and column temperature maintained at 35 °C. The gradient pattern was 0 min, 10% B; 0–40 min, 30% B; 40–60 min, 70% B; 60–64 min, 90% B; 64–70 min, 10% B. Prior to HPLC-PDA analysis, all samples were dissolved in 70% ethanol until complete dissolution, obtaining a concentration of 1 mg/mL, and filtered through a pore size 0.2 µm PVDF syringe filters (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
The described modified method was validated following international guidelines [28 ]. The selectivity of peaks was evaluated and phenolic compounds were identified by comparing the retention times, UV spectra of the analytes with those of the reference compounds, and on the basis of previous reports on lingonberry phenolics. The PDA detector was set at a wavelength of 280 nm for proanthocyanidins and catechins, 360 nm for flavonols, 520 nm for anthocyanins, 330 for hydroxycinnamic acids, and 260 nm for hydroxybenzoic acids. All phenolics were quantified according to 5–7 points linear calibration curves of external standards, except well-known predominant compounds of lingonberry leaves—quercetin-3-O-(4”-(3-hydroxy-3-methylglutaryl)-rhamnoside (quercetin-HMG-rhamnoside), and 2-O-caffeoylarbutin, because of commercially unavailable standards. They were tentatively quantified using calibration curves of standard substances with similar chemical structures. Limits of detection (LOD) and of quantification (LOQ) were determined via the signal-to-noise ratio method. The trueness of the method was expressed as percent recoveries of phenolics at low, medium, and high concentrations of range, each analyzed in triplicate. To assess the repeatability and intermediate precision of the method, relative standard deviation percentages (% RSD) of peak areas of each quantified phenolic compound were calculated within (six times per day) and between days (three consecutive days), respectively, resulting in total repeatability of 18 replicates.
The described modified method was validated following international guidelines [28 ]. The selectivity of peaks was evaluated and phenolic compounds were identified by comparing the retention times, UV spectra of the analytes with those of the reference compounds, and on the basis of previous reports on lingonberry phenolics. The PDA detector was set at a wavelength of 280 nm for proanthocyanidins and catechins, 360 nm for flavonols, 520 nm for anthocyanins, 330 for hydroxycinnamic acids, and 260 nm for hydroxybenzoic acids. All phenolics were quantified according to 5–7 points linear calibration curves of external standards, except well-known predominant compounds of lingonberry leaves—quercetin-3-O-(4”-(3-hydroxy-3-methylglutaryl)-rhamnoside (quercetin-HMG-rhamnoside), and 2-O-caffeoylarbutin, because of commercially unavailable standards. They were tentatively quantified using calibration curves of standard substances with similar chemical structures. Limits of detection (LOD) and of quantification (LOQ) were determined via the signal-to-noise ratio method. The trueness of the method was expressed as percent recoveries of phenolics at low, medium, and high concentrations of range, each analyzed in triplicate. To assess the repeatability and intermediate precision of the method, relative standard deviation percentages (% RSD) of peak areas of each quantified phenolic compound were calculated within (six times per day) and between days (three consecutive days), respectively, resulting in total repeatability of 18 replicates.
Free full text: Click here
