Case Study: Limited Sample for API Degradant Isolation

A client unexpectedly observed an API degradant in its formulation after 18 months on long-term stability studies. It had such a limited amount of formulated material available for isolation work, however, that it was unlikely to obtain sufficient purified isolate to directly enable full characterization of the API degradant by NMR and MS (see Stability Sample chromatogram, below).

We were able to successfully elucidate the structure of this degradant in detail using the following approach:

The client’s available material was chromatographed, and about 10 mcg of purified API degradant was isolated and characterized by MS and proton NMR only,
Forced degradation conditions were rapidly screened, and a best condition was identified to generate a substantial level of the degradant (see Forced Degradation chromatogram, below)
The best forced forced degradation condition was implemented on bulk API, allowing for >2 mg of the degradant to be generated and purified (see Forced Degradation Isolate chromatogram, below)
The degradant isolate was thoroughly characterized using 2D NMR techniques (COSY, HSQC, HMBC) and MS/MS
For cross-identification purposes, a perfect match of data was established between the trace material isolated and the material generated chemically (HPLC retention time, proton NMR, MS patterns).