Lichroprep si 60

We used 5 ml of EDTA blood for the assessment of plasma OT concentrations. After venous puncture, the 5 ml EDTA vacutainer tubes were immediately cooled in ice-chilled water at 4°C. The samples were then centrifuged at 4°C at 4000 rpm for 5 min, aliquoted and stored at -20°C. After completion of the study, plasma samples were sent on dry ice to Munich, Germany. After extraction using LiChroprep Si60 (Merck), the samples were analyzed using a sensitive radioimmunoassay (RIAgnosis, Munich, Germany; see (Martin et al., 2014 (link)). The assay detection limit was 0.1 pg/sample, and cross-reactivity with other related neuropeptides was <0.7%. The coefficient of variation for intra-assay precision was <8%, whereas inter-assay variation was eliminated by measuring all samples within the same assay.

All common chemicals and solvents are analytical reagent grade from worldwide recognized brands. Bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and Lobar prepacked column size A [LiChroprep Si 60 (40–63 μm)] were purchased from Merck. Polyamide 6 was purchased from Fluka. Pinocembrin was isolated from Bulgarian propolis as described in our earlier work [28 (link)]. Purified standards of N-acyl-homoserine lactones (AHLs) QS signals were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Abbreviations for N-acyl homoserine lactones (AHLs) include the following: C4-HSL, N-butanoyl-homoserine lactone and 3-oxo-C12-HSL, N-3-oxo-dodecanoyl homoserine lactone. Bacto agar was purchased from VWR International, Radnor, Pennsylvania. The antibiotic gentamycin and other chemicals were purchased from Sigma-Aldrich (St. Louis).

Optical rotations were measured with a Jasco P-2000 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCEII + 600 NMR spectrometer operating at 600 MHz (150 MHz for ¹³C). High-resolution electrospray ionization mass spectra (HRESIMS) were obtained on Thermo Scientific Q Extractive Plus Mass spectrometer with Orbitrap Analyser. Vacuum liquid chromatography (VLC) was performed on Silica gel 60H (15 μm, Merck, Darmstadt, Germany). Low-pressure liquid chromatography (LPLC) was carried out on LiChroprep Si 60 Merck column (40–63 μm). Column chromatography (CC) was performed on Silica gel 60 (63–200 μm, Merck), silver nitrate-impregnated silica gel (~10 wt.% loading, 230 mesh, Sigma-Aldrich, St. Louis, MO, USA) and Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemicals, Uppsala, Sweden). Preparative thin-layer chromatography (PTLC) was performed on Silica gel 60F₂₅₄ glass plates (20 × 20 cm, 0.25 mm, Merck). Detection of the spots was achieved under UV light at 254 and 366 nm, and subsequently spraying with vanillin in sulfuric acid and heating at 100 °C. All solvents used were of analytical grade.

NMR spectra were recorded on a Bruker AVANCE II+ 600 NMR spectrometer operating at 600 MHz (150 MHz for ¹³C). Optical rotation was measured on a Jasco P-2000 polarimeter. Vacuum liquid chromatography (VLC) was performed on Silica gel 60H (Merck, 15 μm). Column chromatography (CC) was performed on Silica gel 60 (Merck, 63–200 μm) normal phase, and Sephadex LH-20 (Pharmacia Fine Chemicals, 25–100 μm). Low pressure liquid chromatography (LPLC) was carried out with LiChroprep Si 60 Merck column (40–63 μm). Preparative thin-layer chromatography (prep. TLC) was performed on Silica gel 60 F₂₅₄ glass plates (Merck, 20 x 20 cm; 0.25 mm). Detection of the spots was achieved under UV light at 254 and 366 nm, and by spraying with vanillin in sulfuric acid, followed by heating at 100°C. All solvents used were of analytical grade.

Optical rotations were taken using a Jasco P-2000 polarimeter (Jasco, Kyoto, Japan). The IR spectra (ATR) were obtained with an FT/IR-6000 FTIR spectrometer (Jasco, Kyoto, Japan). The UV spectra were determined on a Hitachi-U5100 ratio beam spectrophotometer (Hitachi, Tokyo, Japan). The circular dichroism spectra were recorded on a JASCO J-810 CD spectrometer. High-resolution electrospray ionization-mass spectrometry (HRESI-MS) were obtained on a Bruker APEX II mass spectrometer (Bruker, Karlsruhe, Germany). The NMR spectra were run on a Varian Mercuryplus-400/VNMRS-600 spectrometer (Varian, Inc. Vacuum Technologies, Ave, Lexington, MA, USA). Column chromatography was carried out with glass columns packed with silica gel (LiChroprep^® Si 60, 25–40 mesh; Merck, Darmstadt, Germany) and RP-18 (LiChroprep^® RP-18, 15–25 mesh; Merck, Darmstadt, Germany). Medium-performance liquid chromatography (MPLC) was performed using a VSP-3050 EYELA ceramic pump (EYELA, Kyoto, Japan). Further purification steps were performed by high-performance liquid chromatography (HPLC) using a JASCO HPLC LC-4000 (JASCO Corporation, Tokyo, Japan) with a photodiode array detector.