By Martin Koos
Purification and structure confirmation of newly synthesized compounds before biological testing to determine activity has been the mainstay of analytical departments in the pharmaceutical industry for many years. Purification is usually achieved by high performance liquid chromatography (HPLC) which pairs well with mass spectrometry (MS) used for simple structure verification. While MS is a very sensitive method, it does not provide much structural information unless fragments are analyzed and cannot differentiate between molecules with the same mass or m/z (isobaric isomers) — a gap that could be addressed by nuclear magnetic resonance spectroscopy (NMR). NMR offers a huge amount of additional information, however, NMR has very different sample requirements and is not easily coupled to LC online.
Traditional offline NMR analysis has a reputation of having low sensitivity and requiring large sample amounts. NMR is non-destructive but recovering compounds from samples on a large scale, especially with dimethylsulfoxide (DMSO) as solvent, poses a significant effort. Chemists, consequently, are weary of using precious reaction product except in special cases where NMR analysis is requested. This prevents NMR from becoming a default method applied to all compounds that get isolated.
Appreciating the value of by-default acquisition of NMR data, we implemented a high-throughput offline NMR platform to complement HPLC-MS purification at the Pfizer research site in Groton, CT, that is presented here. After purification, roughly 1 mg of dissolved leftover material that would otherwise be discarded is combined, its solvent removed, and prepared into NMR samples in 96-tube-rack format. The sensitivity of modern cryogenically cooled NMR micro-probes allows acquisition on more than 100 such samples per day.