A Radiocalibration Method with Pseudo Internal Standard to Estimate Circulating Metabolite Concentrations

Abstract

Background: It has become important for metabolism scientists to identify and quantify prominent circulating human metabolites in order to develop a metabolite safety-qualification package that meets regulatory standards. Often these metabolites cannot be analyzed using traditional bioanalytical methods because a standard is not available. Results: A radiocalibration method is described that can estimate circulating metabolite concentrations in nonradioactive human and animal plasma. The key to this method is application of a pseudo internal standard (PIS) that is present in both radioactive reference and nonradioactive (i.e., unknown) samples. Metabolite exposure in the unknown samples is estimated from measured PIS exposure using a relative molar ratio established between the metabolite and PIS (usually parent drug). Conclusion: Two case studies demonstrate that the method can be used to establish human metabolite safety coverage in animal plasma and method validation is demonstrated by comparing estimated metabolite concentrations in human plasma with concentrations obtained directly using a metabolite calibration curve.

Ethical conduct of research

The authors state that they have adhered to the principles of Good Laboratory Practices in the conduct of the chronic toxicity animal studies described herein. For human studies, appropriate institutional review board approval was obtained and the principles outlined in the Declaration of Helsinki were followed for all human experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Acknowledgements

The authors would like to thank Dr Shelby Anderson and Dr Mary Pat Knadler at Eli Lilly and Company for helpful discussions during the conduct of this work.