Female cd1 mouse liver microsomes

Test compound
(0.5 μM) was incubated with female CD1 mouse liver microsomes
(Xenotech LLC; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4)
and the reaction started with addition of excess NADPH (8 mg/mL 50
mM potassium phosphate buffer, pH 7.4). Immediately, at time zero,
and then at 3, 6, 9, 15, and 30 min an aliquot (50 μL) of the
incubation mixture was removed and mixed with acetonitrile (100 μL)
to stop the reaction. Internal standard was added to all samples,
the samples centrifuged to sediment precipitated protein, and the
plates then sealed prior to UPLCMSMS analysis using a Quattro Premier
XE (Waters Corp., USA). XLfit (IDBS, UK) was used to calculate the
exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard
at each time point. The rate of intrinsic clearance (CLi) of each
test compound was then calculated using the following calculation where V (mL/mg protein) is
the incubation volume/mg protein added and microsomal protein yield
is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was
used as a positive control to confirm acceptable assay performance.
The human biological samples were sourced ethically, and their research
use was in accord with the terms of the informed consents.

Test
compound (0.5 μM) was incubated with female CD1 mouse liver
microsomes (Xenotech LLC ; 0.5 mg/mL 50 mM potassium phosphate buffer,
pH7.4) and the reaction started with addition of excess NADPH (8 mg/mL
50 mM potassium phosphate buffer, pH 7.4). Immediately, at time zero,
then at 3, 6, 9, 15, and 30 min, an aliquot (50 uL) of the incubation
mixture was removed and mixed with acetonitrile (100 uL) to stop the
reaction. Internal standard was added to all samples, the samples
centrifuged to sediment precipitated protein, and the plates then
sealed prior to UPLC-MS/MS analysis using a Quattro Premier XE (Waters
corporation, USA).
XLfit (IDBS, UK) was used to calculate the
exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard
at each time point. The rate of intrinsic clearance (Cl_i) of each test compound was then calculated using the equation Cli
(mL/min/g liver) = k × V ×
microsomal protein yield, where V (mL/mg protein)
is the incubation volume/mg protein added and microsomal protein yield
is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was
used as a positive control to confirm acceptable assay performance.

Test compound
(0.5 μM) was incubated with female CD1 mouse liver microsomes
(Xenotech LLC; 0.5 mg/mL, 50 mM potassium phosphate buffer, pH 7.4),
and the reaction started with addition of excess NADPH (8 mg/mL, 50
mM potassium phosphate buffer, pH 7.4). Immediately, at time zero,
and then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the
incubation mixture was removed and mixed with acetonitrile (100 μL)
to stop the reaction. Internal standard was added to all samples;
the samples were centrifuged to sediment precipitated protein, and
the plates were then sealed prior to UPLC-MS/MS analysis using a Quattro
Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to
calculate the exponential decay and consequently the rate constant
(k) from the ratio of peak area of test compound
to internal standard at each time point. The rate of intrinsic clearance
(Cli) of each test compound was then calculated using the following
calculation. where V (mL/mg protein) is
the incubation volume/mg protein added and microsomal protein yield
is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was
used as a positive control to confirm acceptable assay performance.