The Great Unknown
Identifying small molecules can be a challenging task as well. In the manufacture of pharmaceuticals, any impurities or degradants found above a certain threshold level—as low as 0.05%, depending on the daily dose and genotoxicity—must be identified.
Arindam Roy, a senior analytical scientist at Boehringer Ingelheim Ben Venue Laboratories, specializes in structural elucidation of impurities in pharmaceuticals. “The first thing we do is chromatography on the front end, to make sure they’re separated from any other components that we are not interested in,” he says. “Then we get this into MS.”
When dealing with potentially genotoxic compounds, the FDA wants manufacturers to be able to detect and identify down to 1–20 parts per million. The low detection limits of such contaminants means utilizing the power of accurate mass MS, sometimes combined with other even more specialized techniques.
“When you are trying to elucidate structure you want to go beyond one MS—fragment your molecule and get accurate mass, again, from the fragmented parts as well.” This can be done repeatedly up to LC-MS10, but after MS4 or MS5 you lose signal, he adds.
LC-MS spectral databases are of little use in identifying compounds, because different instruments yield different qualitative spectra, “so the statistical matching is a very difficult process in the LC-MS world.” Vendors may try to see what compounds have the same accurate mass and narrow down the search that way. But when you have no knowledge of what that structure would be, “organic chemistry is your best friend,” Roy says.
“You look at the process and see what structures are possible in this process, and then you get the mass specs and you see, out of all the structures, what this is matching.” The elucidation protocol may not end there. To confirm the identity of the contaminant, Roy sometimes resorts to deuterium-exchange experiments, LC-NMR, and other exotic techniques.