Agilent 1100 series apparatus

Analytical HPLC was carried out using an Agilent 1100 series apparatus equipped with a diode array detector (DAD) (Agilent Technologies), using a XBD-C18 analytical column 4.6 × 150 mm, 3.5 μm particle size (Agilent Technologies), and a column temperature of 60°C. Elution was performed at a flow rate of 1 mL/min with 0.2% formic acid (solvent A) and acetonitrile (solvent B) for the mobile phase. The samples were eluted by applying the following gradient: 100% A as the initial condition for 3 min, 90% A and 10% B for 3 min, 85% A and 15% B for 3 min, 80% A and 20% B for 3 min, 70% A and 30% B for 3 min, 60% A and 40% B for 3 min, and finally 50% A and 50% B for 15 min. At 253, 290, and 349 nm, compounds were detected and identified by comparing them to known standards (caffeic acid, chlorogenic acid, trans-cinnamic acid, trans-ferulic acid, syringic acid, caffeine, (±)-catechin, (-)-epicatechin, kaempferol, naringenin, quercetin, and rutin), based on retention times and UV spectra.

Standard chemicals were obtained from commercial sources. Purification of the compounds was performed by flash chromatography over silica gel; eluent: cyclohexane/EtOAc 85:15. The purity of the final products was analyzed by reverse-phase (RP) HPLC, performed on Agilent 1100 series apparatus (Agilent, Technologies, Waldbronn, Germany), equipped with a RP column Phenomenex (Torrance, CA, USA) No 00D-4439-Y0 Gemini 3 μm C18 110 Å, LC column 100 mm × 3.0 mm; diode-array detector (DAD) λ = 210 nm and 254 nm; mobile phase: from 9:1 H₂O/acetonitrile (ACN) containing 0.1% formic acid to 2:8 H₂O/ACN containing 0.08% formic acid in 20 min, flow rate 1.0 mL min⁻¹. ESI-MS was done on a MS single quadrupole HP 1100 MSD detector (Agilent). ¹H NMR analysis was performed on a Varian Gemini 400 MHz apparatus (Agilent Technologies); peptide samples were dissolved in CDCl₃ or in DMSO-d6 to the final concentration of 0.01 M and analyzed in 5 mm tubes at rt. Water suppression required the PRESAT presaturation procedure. Chemical shifts (δ) are expressed as p.p.m., using the residual peak of the deuterated solvent as an internal standard (δH DMSO-d6 = 2.50 p.p.m., δH CDCl₃ = 7.27 p.p.m.).
Opioid radioligands, [³H]DAMGO, [³H]deltorphin-2 and [³H]U-69593, and human recombinant opioid receptors came from PerkinElmer (Krakow, Poland). GF/B glass fiber strips were purchased from Whatman (Brentford, UK).