AMS quantification is independent of the starting matrix or internal standards, allowing definitive comparisons of metabolite profiles across species and matrices. It is one method for the mitigation of "MIST" liabilities early in a development program. Metabolite characterizations (profiling) can quickly generate 1000’s of time fractions when using off-line fraction collection prior to AMS analysis. As a result, strategies for efficient metabolite profiling have been a high priority. In this talk we will discuss some of the methods used at BioCore (and Vitalea Science) for efficient metabolic profiling. The general features of our strategy have as follows: -Develop high resolution UPLC chromatography separations linked to high precision fraction collector (Waters Fraction Manager) -Perform prestudy analysis of incurred radiolabeled animal samples on well characterized clinical samples using similar UPLC systems -Optimize gradient condition to ensure separation of closely eluting components in a minimal run-time -Create representative pooling schemes of study samples by AUC and subject/time point pools -Collect multiple 96-well fraction microtiter plates in a single operator session -Scan first plates for regions of &gt;10% of injected radioactivity at low temporal resolution (e.g. 2 min fractions) -Fill-in regions of &gt;10% activity using backup wells down to minimal (dt) resolution (usually 6-sec) Off-line graphite production has the benefit of allowing independent scheduling of the AMS uncomplicated by chromatographic issues. Graphite sample definition ensures low carryover with high ion source currents. UPLC systems (Waters Acquity™) , provide excellent injection-to-injection reproducibility, high isotope transmission, and robust operation over a wide range of flowrates. Collecting multiple backup plates ensures the security of the data for reliable inter-species comparisons. We present data from several human radiolabel investigations after microtrace doses that enlist some or all of the above strategies. Variable-width fractions down to 6-sec at key regions analyzed for 14C contents by AMS with reliable limits of quantification of &lt;10 amol of 14C-drug. These same methods were applied to specimens collected from multiple safety species exposed to the radioactive drug. An overlaying of the LC plots revealed multiple metabolic differences not only between the human and the animals but also between the two animal species.

Stategies for efficient metabolite characterization by UPLC + AMS

253rd National Meeting (2017)

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