Waters 1525 binary pump

All chemicals were purchased from commercial sources and were used without purification. Water was purified (18 MΩ cm) using a standard Milli-Q system (Millipore, Bedford, MA, USA). “PE” (petroleum ether) refers to petroleum ether with a boiling point in the range of 40–60 °C. NMR spectra (including ¹H-decoupled ¹³C NMR) were recorded on a Bruker Avance III spectrometer (Bruker, Milano, Italy) operating at 11.74 T and 298 K, corresponding to a protonic resonance frequency of 499.8 MHz. ¹H and ¹³C NMR chemical shifts are reported relative to TMS and are referenced using the residual proton solvent resonances. Samples were prepared in 5 mm NMR tubes by dissolving the compounds in appropriate deuterated solvents. Splitting patterns are described as singlet (s), broad singlet (bs), doublet (d), double doublet (dd), triplet (t) or multiplet (m). ESI mass spectra were recorded on a Waters SQD 3100 (Waters Corporation, Milford, MA, USA). Analytical HPLC-MS was carried out on a Waters modular system equipped with Waters 1525 binary pump, Waters 2487 UV/Vis and Waters SQD 3100 (ESCI ionization mode) detectors using an XBridge^TM Phenyl 3.5 μm 4.6 mm × 150 mm column (Waters). Semi-preparative HPLC purifications were performed with a XBridge^TM Prep Phenyl 5 μm OBD^TM 19 mm × 100 mm column (Waters). The HPLC methods are indicated for each procedure (Table 4).

5 μL of supernatant was sampled from sunitinib DEB treated HCT116 cells at 0.25, 0.5, 1, 2, and 24 hours of exposure, and it was then diluted 1:10 in phosphate buffered saline prior to high performance liquid chromatography (HPLC) analysis. HPLC analysis was performed using a c18 Luna (150 * 4.6 mm) column (Phenomenex, Torrance, CA, USA) attached to a Waters 1525 binary pump (Waters Corporation, Milford, MA, USA) with a mixed solvent of water (20%) and acetonitrile (80%) containing 0.1% TFA, and UV detector set to 425nm. Sunitinib concentration was calculated based on a standard curve of 20 μM sunitinib in cell culture medium. To evaluate the maximal release fraction of sunitinib from DEB after 24h, loaded DEB (5 mg/ml) were incubated in a surplus of supplemented McCoy’s 5a cell culture medium so that the maximal achievable concentration could be 20 μM in case of complete eluting from DEB.

Mice were sacrificed, their brains removed and placed on ice. Dorsal striatum and nucleus accumbens were dissected and weighed, then flash-frozen and stored at −80 °C. Tissue samples were ultrasonicated in 0.1 M perchloric acid and stored at −80 °C until extraction. Upon thawing, the samples were homogenized in 0.1 M perchloric acid and centrifuged at 25,000 g for 12 min. Dopamine levels were measured by HPLC with electrochemical detection. The analytical column was a SunFire C18 5 lm (4.6–150.0 mm) from Waters (Milford, MA, USA). The mobile phase was 0.01 M sodium dihydrogen phosphate, 0.01 M citric acid, 1.2 mM sodium EDTA, 1.2 mM sodium 1- heptane sulfonic acid, 10% methanol, pH 3.5; the flow rate was 1.0 mL/min and the column temperature was 34 °C. The installation consisted of a Waters 717 Plus automated injection system, a Waters 1525 Binary pump, and an ESA Coulochem III detector (Dionex, Sunnyvale, CA, USA). Waters Breeze system was used for analysis.