Case Study 95-22:   Isolation of a Drug Metabolite Key to an Analysis Required by Regulatory Authorities

A generic pharmaceutical manufacturer contacted Investigative Science Incorporated about conducting an analysis of a series of tissues forming part of a medicated feed withdrawal study. The analysis was to be conducted using a GC/MS method specified by the regulatory officials. At first this appeared to be similar to countless other tissue residue analyses that we had performed. These typically involve in-house validation of the chosen method, followed by analysis of the tissues from animals on a feeding trial.

In this case however, the method was based on a metabolite, not the drug itself. Unfortunately, the metabolite was not available from any commercial source. After a review of the available literature, we proposed to our client that the metabolite be isolated from the liver (where it was formed) of an animal fed the drug in question.

The animals were fed and the metabolite successfully isolated in milligram quantities from the liver. In order to use this material as the basis for a regulatory assay, it was necessary to purify the material, establish beyond doubt its identity, then determine its purity.

The material was purified using chromatographic techniques (primarily preparative HPLC) to greater than 95% purity. The structure of the material was confirmed by mass spectrometry, proton NMR, 13C NMR, and UV scan. The purity was determined using GCMS, Thin layer chromatography and HPLC employing the non-selective refractive index detector. 

The assay was successfully validated in our lab and the tissue analysis completed. The drug submission was approved.