Lc solution 1.21 sp1 chromatography software

The obtained propolis extracts were filtered with Iso-Disc™ Filters PTFE-25-2, diameter 25 mm, pore size 0.20 μm (Supelco Analytical™, Bellefonte, PA, USA) and subjected to HPLC. A Shimadzu Prominence chromatograph equipped with an auto sampler SIL-20AC HT, photodiode array detector SPD-M20A, and LC solution 1.21 SP1 chromatography software (Shimadzu, Kyoto, Japan) were used. Separations were achieved using a 100 × 4.60 mm, C18 reversed-phase column, 2.6-μm particles with solid core and porous outer layer (Kinetex™, Phenomenex^®, Torrance, CA, USA). Binary gradient of deionized water (WCA R03 DP ECO, COBRABiD Aqua, Warsaw, Poland) adjusted to pH 2 with phosphoric acid (Merck) and filtered with 47-mm nylon membrane filter 0.20 μm (Phenex™, Phenomenex^®, Torrance, CA, USA) as mobile phase A and MeCN (acetonitrile for HPLC ≥ 99.9%, Merck) as phase B was used as follows: 0 min–12.5% B; 25.0 min–40% B; 34.0 min–60% B; 37.0 min–95% B; 37.1 min–12.5% B; 40 min–stop. The flow rate was set to 2.0 mL/min, oven temperature 45 °C, injection volume 1 μL. Peak identification was carried out by comparison of retention time as well UV spectra with standards. The content of the determined compounds was calculated in mg per 100 mL of propolis extract.

The analyses (essential oil and sesquiterpenes acid content) were carried out, according to the European Pharmacopoeia, Valerianae radix monograph 07/2015:0453 [6 ]. The analyses were performed in triplicate. In order to obtain the essential oils, 40 g of air-dried powdered raw material was used for hydrodistillation, for 4 h, using a Clevenger-type apparatus. Due to the determination of the sesquiterpenic acid content, the methanolic extract was prepared (1.5 g of air-dried powdered raw material per 50 mL of methanol). The obtained extract was separated by HPLC Shimadzu Prominence chromatograph, equipped with autosampler SIL–20AC HT, photodiode array detector SPD–M20A and LCsolution 1.21 SP1 chromatography software (Shimadzu, Kyoto, Japan).

The work was performed using a Shimadzu Prominence chromatograph equipped with a SIL-20AC HT auto sampler, SPD-M20A photodiode array detector and LC solution 1.21 SP1 chromatography software (Shimadzu, Kyoto, Japan). Separations were achieved using a 100 mm × 4.60 mm, C18 reversed-phase column, 2.6 μm particles with solid core and porous outer layer (Kinetex™, Phenomenex, Torrance, CA, USA). Binary gradient of mobile phase A (deionised water adjusted to pH 2 with phosphoric acid) and B (ACN) was used as follows: 0 min—12.5% B; 4.0 min—23% B; 6.0 min—50% B; 6.01 min—12.5% B; 8 min—stop. The HPLC conditions were as follows: flow rate 1.5 ml×min⁻¹, oven temperature 40 °C, injection volume 1 μL.