Zorbax sb c18 2.1

Marrow was prepared for extraction by homogenization at a 1:1 ratio with 50 mM potassium phosphate buffer pH 7.4. A volume of 20 μL of serum or marrow homogenate was combined with 100 μL of HPLC grade acetonitrile and vortex mixed for two minutes. Subsequent to centrifugation at 14000 rpm for five minutes, the supernatant was transferred to a 96-well plate for liquid chromatograph tandem mass spectrometry (LC/MS-MS) analysis. A calibration curve was formed in mouse plasma from 1.00–200 nM by serial dilution and extracted via the same methodology. An Agilent 1200 system consisting of a binary pump, column compartment and autosampler was used for solvent delivery and sample introduction. Chromatographic separation was performed on an Agilent Zorbax SB C18 2.1 × 50 mm 2.7 μm column via a gradient using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Gradient elution was 95% A ramping to 5% A from 0.0–3.0 minutes, with re-equilibration at initial conditions from 3.0 to 4.0 minutes. The flow rate was 0.75 mL/min, and column temperature was 30°C. Detection of propranolol was obtained using an Agilent 6460 triple quadrupole mass spectrometer, monitoring the transition 260.0 → 116.0 with a fragmentor of 86 V and a collision energy of 21 V. The retention time of propranolol was 1.89 minutes.