Arbutin and hydroquinone determinations were performed based on the methodology described in the latest 7th edition of the European Pharmacopoeia [32 ]. A 0.800 g weighed amount of powdered plant material was placed in a 100 mL round-bottomed flask, 20 mL distilled water was added, and the flask was heated for 30 min in a water bath under a reflux condenser. After cooling, the extract was filtered through a wad of cotton wool and was extracted again, together with the residue in the flask, with another 20 mL of distilled water for 30 min in a water bath under a reflux condenser. After cooling, all the liquid was filtered through a paper filter and left to cool, then topped up with 50 mL of distilled water and filtered again, discarding the first 10 mL of the filtrate. The purified plant extracts were applied to conditioned Waters Sep-Pak (C18, 500 mg) syringe filter cartridges (Ireland). Phenolic compounds were leached from the columns with methanol in 10 mL round-bottomed flasks and were concentrated at 40 °C until the solvent was evaporated in a Hei-VAP Precision rotational vacuum evaporator from Heidolph (Schwabach, Germany). The leaf extracts in the round-bottomed flasks were dissolved with methanol and filtered through PTFE socket filters with a 0.45 µm pore size directly prior to chromatographic analysis. A standard solution was made by dissolving 50 mg of arbutin in 50 mL of mobile phase and 2.5 mg of hydroquinone in 10 mL of mobile phase, then mixed at the ratios specified in the methodology. The plant extracts were separated using a Thermo Scientific/Dionex UltiMate 3000 liquid chromatograph with a UV detector and a DAD-3000 (RS) diode matrix (ESA, Chelmsford, MA, USA). A HALO 90 A C18, 2.7 μm, 4.6 × 150 mm column was used for the separations at 25 °C. The mobile phase was methanol and water (10:90 v/w), the flow rate was 0.8 mL/min, and the assay time was 30 min, with detection at a wavelength of 280 nm. The injection volume was 10 µL. The average recovery for the highbush blueberry was 97%. The operation of the chromatographic set and processing of the obtained data were coordinated using the Thermo Scientific Dionex Chromeleon 7.2 Chromatography Data System.
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