Apomorphine

Mice were placed individually in a cylinder (diameter: 23 cm; height: 30 cm) and allowed to adapt to their environment for 5 min. They were subsequently injected i.p. with apomorphine (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 0.5 mg/kg. After 10 min, rotations were recorded for 30 min. Quantitative analyses of complete (360°) left rotations were made off-line by an investigator blinded to the experimental conditions. Results were expressed as the number of rotations to the side contralateral to the lesion.

LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nuciferine and apomorphine were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. Nuciferine and apomorphine were quantified using a NEXERA UPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a mass spectrometer (Triple quadrupole, LCMS8060, Shimadzu, Kyoto, Japan) as previously described [18 (link),25 (link)]. The detection was completed in multiple reaction monitoring mode (MRM) (Table 5). Samples were run in triplicate with external standards in between and the injection volume was 1 μL. The acquired chromatographic results were processed in LabSolutions Insight software version 3.2 (Shimadzu). For each compound, calibration curves (0.005–0.1 ppm) were created by linking the peak area and the concentration.