Sqd mass spectrometer

All samples were centrifuged and the supernatants were stored at 4 °C prior to injection. UPLC chromatography system consisted in an ACQUITY UPLC (Waters, Milford, MA, USA). Separation was performed using a Waters Acquity HSS T3 C18 column (150 mm × 2.1 mm, 1.8 μm) with a flow rate of 0.4 mL/min at 55 °C. The injection volume was 5 μL. The mobile phase consisted of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile). Chromatographic separation was achieved using an 8-min linear gradient from 10 to 24 % solvent B. MS detection was performed by using a SQD mass spectrometer equipped with an electrospray ionization (ESI) source controlled by Masslynx 4.1 software (Waters, Milford, MA). The capillary and sample cone voltages were 3,000 V and 30 V, respectively. The cone and desolvation gas flow rates were 60 and 800 Lh⁻¹. Data collection was carried out in negative mode for secoxyloganin ([M-H]⁻ = 403, RT = 6.42 min) and secologanin ([M + HCOOH-H]⁻ = 433, RT = 7.12 min) and in positive mode for loganin ([M + Na]⁺ = 413, RT = 5.61 min). Standard calibration curves for secoxyloganin, secologanin and loganin were prepared (1–25 μM) with known pure standards from Chemtek (Worcester, MA, USA), Phytoconsult (Leiden, The Netherlands) and Extrasynthese (Genay, France), respectively.

Heparin was delivered by Polfa Warszawa S.A. (Warsaw, Poland), while thiopental sodium was from Sandoz International (Stryków, Poland) and 5 % HP-beta-cyclodextrin from Sigma-Aldrich, USA.
Lu AE58054 was synthesized in the Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland according to a procedure described previously (Pasternak and Szymonifka 2009 ). The structure was confirmed by HNMR obtained in a Varian BB 200 spectrometer using TMS (0.00 ppm) in chloroform-d1. The purity was confirmed by UPLC/MS analysis to be > 98 %. The UPLC/MS system consisted of a Waters AQUITY UPLC® coupled to a Waters SQD mass spectrometer. Chromatographic separations were carried out using an Acquity UPLC® BEH C18 column, 2.1 × 100 mm and 1.7 μm particle size. The column was maintained at 60 °C, and eluted under gradient conditions: 4 min, a linear gradient from 80 to 0.1 % of eluent A at a flow rate of 0.5 ml/min. Eluent A: water/formic acid (0.02 %, v/v) and eluent B: methanol – acidic gradient. Eluent A: water/formic acid/ammonia solution (0.01 %/0.1 %, v/v/v) and Eluent B: methanol – alkaline gradient.