For guanfacine, the LC–MS/MS Sirtuin inhibitor analysis was carried out with a Sciex 4000 mass spectrometer coupled with a Shimadzu LC pump (model LC-10AT) and Perkin-Elmer 200 series autosampler. The internal standard used was guanfacine (13C15N3). Guanfacine and its internal standard were extracted from 200 μL of human plasma by liquid–liquid extraction prior to LC–MS/MS analysis. The chromatographic separation was achieved on an XBridge phenyl, 3.5 μm, 4.60 × 50 mm LC column (Waters Corporation), with mobile

phase at a flow rate of 1 mL/min. The mass spectrometer was operated in positive electrospray ionization mode, and the resolution settings used were unit for Q1 and low QNZ manufacturer for Compound C in vitro Q3. The multiple reaction monitoring (MRM) transition was m/z 246 → 60 for guanfacine, and the MRM transition was m/z 250 → 159 for the internal standard, guanfacine (13C15N3). On the basis of a sample volume of 200 μL, the assay ranged from 0.05 to 50 ng/mL for guanfacine. Samples over the limit of quantitation were diluted into range with control plasma. For d-amphetamine

and lisdexamfetamine, the LC–MS/MS analysis was carried out with a Sciex API 3000 mass spectrometer coupled with a Shimadzu LC pump (model LC-10AT) and Perkin-Elmer 200 series autosampler. The internal standards used were amphetamine-D5 for d-amphetamine and lisdexamfetamine-D8 for lisdexamfetamine. Plasma samples containing d-amphetamine, lisdexamfetamine, and their internal PR-171 cost standards were extracted by liquid–liquid extraction prior to the LC–MS/MS analysis. The chromatographic separation was achieved on a Phenosphere NEXT CN, 5 μm, 4.6 × 50 mm column (Phenomenex), with mobile phase at a flow rate of 1 mL/min. The mass spectrometer was operated in positive mode, and the resolution setting used was unit for both Q1 and Q3. The MRM transitions were m/z 136 → 91 for d-amphetamine, m/z 141 → 96

for amphetamine-D5, m/z 264 → 84 for lisdexamfetamine, and m/z 272 → 92 for lisdexamfetamine-D8. On the basis of a plasma sample volume of 200 μL, the assay ranged from 2 to 200 ng/mL for d-amphetamine and from 1 to 100 ng/mL for lisdexamfetamine. 2.2 Safety Assessments Safety evaluations included AEs, vital signs, 12-lead ECGs, physical examination findings, and clinical laboratory parameters. Pulse and blood pressure (BP) were assessed in both supine and standing positions predose (within 30 min of administration) and at 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12, 24, 30, 48, and 72 h after treatment. ECGs were recorded 2, 8, and 72 h after treatment was administered. TEAEs were defined as AEs that occurred or worsened during the on-treatment period. TEAEs were assigned to the treatment received at the time of onset of the AE.