18.3.1.1 Plasma The plasma sample is treated with an organic solvent such
as acetonitrile, methanol or methanol/acetonitrile (1 : 1) at a ratio of 3 volumes
of solvent to 1 volume of plasma to precipitate proteins. Then the plasma
sample is mixed by vortexing and/or sonicating for 5 min, and centrifuged at
approximately 3000 gfor 10 min. The supernatant is transferred into a clean
tube. The remaining pellets are extracted again with 3–5 mL of the organic
solvent. The supernatants are combined and the recovery of radioactivity is
determined. In general, the radioactivity recovery of>90% is acceptable. The
combined supernatant is dried and reconstituted in an appropriate solvent that
is determined by considering the initial HPLC solvent composition and
polarity of test compound and its metabolites. The reconstituted sample is
mixed by vortexing and centrifuged at 3000 gfor 5 min to remove any solid
particles. Aliquots of the reconstituted sample are counted by LSC to
determine the final recovery of radioactivity (>80% is desired).
18.3.1.2 Urine and Bile For direct injection to HPLC, an aliquot of the urine
sample (or a diluted sample, usually with acetonitrile to a final composition of
20–30% acetonitrile, v/v) is centrifuged at 3000 gfor 5 min to remove any
solid particles. In the case that the concentration of radioactivity in urine is low,
a subsample of urine may be concentrated either by passing through a C18
cartridge (e.g., OasisTM cartridge, Waters) or by direct evaporation under a
steam of nitrogen before HPLC and LC/MS analysis. The aliquots of the
reconstituted sample are counted by LSC to determine the final recovery of
radioactivity (>90% desired). The supernatant is subjected to HPLC analysis.
18.3.1.3 Feces The amount of fecal homogenate needed for the desired level
of radioactivity per injection is determined. The fecal homogenate is extracted
with three volumes of an appropriate solvent (e.g., acetonitrile, acetonitrile/
methanol, acetonitrile/acidic acid). The fecal homogenate/solvent mixture is
vortexed and sonicated and centrifuged. The extraction is repeated at least one
more time to recover a desirable >85% of radioactivity. The sample is
combined, dried, reconstituted in an appropriate solvent (e.g., 20–40%
acetonitrile in water), vortexed and centrifuged at 3000 gfor 5 min. The
aliquots of the reconstituted sample are counted by LSC to determine the final
recovery of radioactivity (>80% desired).
18.3.2 Radioactivity Determination
The radioactivity in urine, bile, plasma, HPLC fractions, and extracts is
determined by mixing aliquots of the samples with a scintillation cocktail (e.g.,
Ecolite^1 cocktail) (5 or 15 mL) and counting with LSC (e.g., Packard 2250CA
Tri-Carb Liquid Scintillation counter, Packard Instrument Company, Meriden,
CT) for 5–10 min. Radioactivity in feces, blood, and tissues is usually
determined by combustion of aliquots by an oxidizer followed by LSC.
Another method to determine total radioactivity is to digest the aliquots of feces,
SAMPLE ANALYSIS 581