Quantification of Rosmarinic Acid in Plant Extracts

The rosmarinic acid content was assessed in extracts prepared from 100 mg of DW (dry weight) tissue in 2 mL of HPLC-grade methanol. Tissue was extracted by sonication (2 × 30 min at 23 ± 2 °C) (ultrasonic bath, Polsonic, Warsaw, Poland). Extracts were centrifuged (25,255×g for 8 min, 4 °C), with samples filtered through Millex^®GP 0.22-μm syringe filters (Millipore, Merck, Darmstadt, Germany) for analysis by diode array detector high-pressure liquid chromatography (DAD-HPLC). The validated method was applied for quantitative analyses of rosmarinic acid using Merck-Hitachi (LaChrom Elite, Tokyo, Japan) apparatus and on a RP-18 column (Purospher, 5 μm, 250 × 4 mm; Merck, Darmstadt, Germany) (Sułkowska-Ziaja et al. 2016 (link)). The parameters were as follows: flow rate 1 mL/min, A—methanol, 0.5% acetic acid 1:4, B—methanol (v/v), temp. 25 °C, injection 10 µL, λ = 254 nm. The gradient program was as follows: 0 to 20 min, 0% B; 20 to 35 min, 0–20% B; 35 to 45 min, 20–30% B; 45 to 55 min, 30–40% B; 55 to 60 min, 40–50% B; 60 to 65 min, 50–75% B; and 65 to 70 min, 75–100% B. Identification was done by comparison to Rt (retention time) and UV spectra of reference substance of rosmarinic acid (Sigma-Aldrich Co, Merck, Darmstadt, Germany). Assays were done in three replications, with results expressed in mg/100 g DW ± SD.