Chemicals and Reagents
Rat plasma was purchased from Bioreclamation Inc. (Hicksville,NY, USA). Optima-grade (99.9%) acetonitrile and methanol were obtained from Fisher Scientific Co. (Pittsburgh, PA,USA), ammonium acetate from Sigma Chemical Co.(St.Louis, MO,USA), and glacial acidic acid (99.99+%) from Aldrich Chemical Company (Milwaukee, WI,USA). Water was purified using a compact ultra-pure water system (EASYpure UV, Dubuque, IA,USA).
150 μL of internal standard (IS) in acetonitrile solution (conc. 10 ng/mL) was added to 50 μL of drug discovery compound A in rat plasma (concs. 250 ng/mL). After plasma precipitation, the mixture was vortexed for 30 s, and then centrifuged for 10 min. The supernatant was transferred to a clean 96-well plate for LC/MS/MS analysis.
HPLC Column: Symmetry C18, 50 4.6 mm, 5 μm (Waters Corporation, Milford, MA,USA)
Mobile Phase A: 0.01 M ammonium acetate in MeOH/H2O (20:80, v/v)
Mobile Phase B: 0.01 M ammonium acetate in MeOH + 0.6 mL/L 10% acetic acid
Flow Rate: 0.8 mL/min Gradient: 5% B for 0.5 min, 5% B to 95%B for 0.5 min, 2.0 min hold at 95% B, return to 5% B at 3.0 min, 3.0 min run time
Divert to MS at 0.8 min Divert to waste at 2.0 min Injection Vol.: 8 μL
Column Temp.: Ambient Temperature
Autosampler Temp: 5C
Retention Time: Compound A - 1.2 min
Internal Standard - 1.5 min
Mass Spectrometer: Finnigan TSQ Quantum Discovery (ThermoElectron,San Jose,CA,US