Case Study: Metabolite discovery

Experiment

Drug pharmacokinetics and metabolite profiling of a highly potent drug in development. Three canines were orally dosed at microdose levels (30 nCi [1.11 KBq], 10 µg of total drug per animal) and blood specimens drawn for 3 weeks postdose. Blood specimens at 0.5, 1, 2, & 4 hr were pooled from all dogs and extracted using an acetonitrile precipitation method.

The molecule is an aromatic amine with limited aqueous solubility under alkaline conditions. Solubility improves with increasing acidity.

Prior knowledge

A multiplicity of metabolites had been identified for high dose studies, ranging from removal of the nitrogenous group, hydroxylation, and glucuronidation.

Processing

Matrix: Plasma
Method: Precipitation of plasma proteins with 3X acetonitrile.

The acetonitrile extract was concentrated to dryness and reconstituted in 1:1 0.2% Trifluoroacetic acid (TFA) in Acetonitrile/Mobile Phase A.

Column: Acquity HSS T3 C18, 2.1x100mm, 1.8 µm, S/N 0104170291DD 01

Fraction collection: 6 sec fractions using a Waters Fractional collector III
Volume/6-sec fraction: 60 µL

¹⁴C analysis: The entire volume in the plate wells was removed and reduced to graphite after addition of carbon carrier and solvent removal. The ¹⁴C contents above background, the specific labeling of the molecule were used to calculated the concentration of the drug related material per well.

LOD: 11.9 fg/fraction (3 X SDbkg)
Imprecision: <2% machine. <6% method.

Results

Partial radiochromatogram of potent drug under development. The data points from the discrete LC effluent samplings (6-sec) are displayed in the bottom plot. A spline fit of those points is overlaid above this plot. The parent molecule is the dominant component in the profile, but at least 6 other metabolites are identifiable as distinct peak tops and characteristic elution times (orange numbers).