Spectra system p4000 pump

A Thermo^® Scientific UHPLC with a Thermo^® Scientific Spectra System P4000 pump was used, with a Thermo^® Scientific Spectra AS3000 automatic injector and a Thermo^® Scientific Spectra System UV8000 model DAD. The software used to process the chromatographic data was ChromeleonTM 7.0. A Kinetex^® EVO C18 LC column (1.7 μm, 2.1 mm × 100 mm) connected to a SecurityGuard™ ULTRA pre-column (sub-2 µm, 2.1 mm × 2 mm). Chromatographic analyses were performed at room temperature at a flow rate of 0.12 mL/min using 25 mM NH₄CH₃CO₂:CH₃CN:HCOOH (75:25:0.1 v/v/v) as the mobile phase. The sample injection volume was 5 μL. Stock standards of 5 mM of MDPV and all solutions were prepared in HBSS (+/+) and filtered through a 13 mm nylon syringe filter with 0.22 µM pore size from Olimpeak™. Calibrators were prepared by the dilution of the stock standards with filtered HBSS (+/+) to final concentrations of 0.5, 1, 5, 10, 25, 50, 100, 250, and 500 μM. To obtain the optimal wavelength for the detection and quantification of MDPV, UV spectra were determined for MDPV (100 µM in HBSS (+/+)), the selected mobile phase and HBSS (+/+) using a UH5300 spectrophotometer.

The simultaneous separation of high-energy phosphates (ATP, ADP, AMP, GTP), purine nucleosides (inosine, adenosine, guanosine) and oxypurines (hypoxanthine, xanthine, uric acid) in the protein-free cell extracts (200 µL) was carried out using previously established ion pairing HPLC methods which utilize tetrabutylammonium hydroxide as the pairing reagent [63 (link)]. Separation was obtained using a Hypersil C-18, 250 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy). The HPLC apparatus consisted of a SpectraSystem P4000 pump system (ThermoFisher Scientific) and a highly-sensitive UV6000LP diode array detector (ThermoFisher Scientific), equipped with 5 cm light path flow cell and set up between 200 and 300 nm wavelength. Assignment and calculations of the compounds of interest in chromatographic runs of cell extracts were performed at 260 nm wavelength by comparing retention times, absorption spectra, and area of the peaks of chromatographic runs of mixtures containing known concentrations of true ultrapure standard mixtures.