1100 lc ms system

On day 49 animals were injected IP with test compounds. Blood was taken
via the retro-orbital route, brain tissue was flash frozen using acetone:
CO2(s). Tissues were stored at -80 °C until analysis. For serum samples,
20 μL were added to 80 μL of methanol + 200 ng IS. This
was centrifuged for 10 min at 10,000 rpm, and 60 μL were transferred to
a LC/MS vial. Fenethylline brain samples were diluted into four volumes of
H₂O, homogenized, and 1 mL was placed into 1 mL dichloromethane
(DCM) + 200 ng IS. This was stirred for 2 hours, the DCM was collected,
centrifuged as above, dried under vacuum, and then resuspended in 60 μL
methanol. Theophylline brain tissue was homogenized in four volumes of
H₂O, and 1 mL + 200 ng IS was stirred for 2 hours,
centrifuged, dried, and resuspended as above. Amphetamine brain tissue was
homogenized in four volumes of 10 M NaOH, 1 mL was then placed into 1 mL hexane
+ 200 ng IS, stirred for 2 hours, and the hexane fraction was
counter-extracted into 200 μL of 0.1 M HCl, dried, and resuspended as
above. Analysis occurred on an Agilent 1100 LC/MS system with a Poroshell 120
SB-C8 column using H₂O/ACN (with 0.1% Formic Acid) as the
mobile phase (5–95% ACN, 10 min gradient). Using the ratio of
drug to IS integration values, the unknown tissue concentrations were determined
using a standard curve for the drug in question.