L2130 pump

HPLC-DAD analysis was conducted using the chromatograph LaChrom Elite (Hitachi, Tokyo, Japan) equipped with an L2455 DAD detector adjusted in the range of 230 to 400 nm, L2200 injector, L2130 pump, L2300 column oven, and a C18 column (150 × 4.6 mm, 5 µm, Merck, Darmstadt, Germany). Data were obtained with EZChrom Elite software, version 3.3.2 SP1 (Santa Clara, CA, USA). Analysis conditions were according to [20 (link)]. The mobile phase consisted of a gradient of 1% phosphoric acid and acetonitrile (Table 1) with a flow rate of 0.5 mL per minute.
Samples were obtained by diluting the extract with methanol to the concentration of 4 mg/mL followed by filtration with a 0.45 μm filter (Millex, Merck KGaA, Darmstadt, Germany). Commercial standards were used in an attempt to identify the compounds present in HEMNL by comparison of retention times and ultraviolet spectra. The standards were caffeic acid, chlorogenic acid, ferulic acid, kaempferol, gallic acid, rosmarinic acid, ellagic acid, isoquercitrin, hesperetin, quercetin, resveratrol, vitexin, isovitexin, coumarin, and myricetin acquired from Sigma-Aldrich^®, hyperoside acquired from Hwi Analytik Gmbh^® (Rülzheim, Germany), and rutin from Chromadex^® (Los Angeles, CA, USA).

To determine the content of phenolic acids, flavonoids, and caffeine in the tested materials, 5 g of samples was extracted with methanol for 30 min in an ultrasonic bath at a frequency of 49 kHz (POLSONIC 2, Warsaw, Poland). The extraction process was repeated three times for each sample. The obtained extracts were concentrated by evaporation drying in a rotating vacuum oven at 22 ± 2 °C. The extracts were dissolved quantitatively in HPLC-grade methanol and re-filtered through membrane filters. The prepared methanol extracts were used for determining the content of phenolic acids using RP-HPLC according to an optimized procedure described elsewhere [21 (link)]. The process was performed using an HPLC (Merck-Hitachi, Merck KGaA, Darmstadt, Germany) apparatus equipped with the following: an L-2200 autosampler, an L-2130 pump, a LiChrospher RP-18e column (250 mm × 4 mm, 5 µm) thermostated at 25 °C, an L-2350 column oven, and an L-2455 diode array detector at a UV range of 200–400 nm. The mobile phase comprised methanol (solvent A) and methanol/0.5% acetic acid (solvent B) (1:4, v/v). The phenolic compounds were quantitatively analyzed using a calibration curve with the assumption of the linear size of the area under the peak and the concentration of the reference standard.