blood, and tissues for 24–48 h at room temperature with Soluene-350 (Packard
Instrument Co., Meriden, CT). The digested aliquots were then bleached with
20% benzoyl peroxide in toluene, neutralized with a mixture of saturated
sodium pyruvate in methanol/glacial acetic acid, mixed with scintillation
cocktail and counted using LSC (Krishna et al., 2002). Each sample is
homogenized before radioanalysis. In the muraglitazar study, the radioactivity
in plasma and excreta (urine, bile, and feces) was determined after combustion
of the samples (Zhang et al., 2006). HPLC effluent was collected at 0.26 min
intervals after sample injection into 96-deep-well Lumaplates^1 with a Gilson
fraction collector (Gilson Medical Electronics, Middleton, WI). The effluent in
the plates was dried with a Speed-Vac^1 (Savant, Holbrook, NY) and the plates
were counted for 10 min per well with a TopCount^1 scintillation analyzer
(Packard Instrument Company, Meriden, CT).
18.3.3 LC/MS/MS Quantification and Pharmacokinetic Analysis
The unchanged drug in plasma and urine samples from mass balance studies
can be quantified using LC/MS technique. In muraglitazar study, plasma
concentrations of the unchanged drug were determined by a validated protein
precipitation and LC/MS/MS method (Wang et al., 2006). The internal
standard was dissolved into 0.1% formic acid in acetonitrile, which also served
as a protein precipitation reagent. Human plasma samples (0.1 mL) and the
internal standard solution (0.3 mL) were added to a 96-well plate. The plate
was vortexed for 1 min and centrifuged for 5 min, then the supernatant was
directly injected into the LC/MS/MS. Chromatographic separation was
achieved isocratically on a Phenomenox Luna C18 column (2 50 mm, 5m).
The mobile phase contained 20% of 1 mM formic acid in water and 80% of
1 mM formic acid in acetonitrile. Detection was by positive ion electrospray
tandem mass spectrometry on a Sciex API 4000. The standard curve, which
ranged from 1 to 1000 ng/mL, was fitted to a 1/xweighted quadratic regression
model. All plasma samples were analyzed within a total of four analytical runs.
QC samples were analyzed along with the study samples to assess the accuracy
and precision of the assay. The acceptance criteria established for the analysis
of muraglitazar in plasma specified that the predicted concentrations of at least
three fourths of the standards and two thirds of the QC samples be within
15% of their individual nominal concentration values (20% for the lowest
concentration standard). In addition, at least one QC sample at each
concentration had to fall within15% of its individual nominal concentration
value. Values for the between-run precision and the within-run precision for
analytical quality control samples were no greater than 0% and 11.1%
coefficient of variation (CV), respectively, with deviations from the nominal
concentrations of no more than5.2%.
18.3.3.1 Pharmacokinetic Analysis The plasma concentration versus time
data for radioactivity and unchanged muraglitazar (Wang et al., 2006) were
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