Impurity Isolation and Structure Elucidation

Isolation Strategies

In terms of isolation strategies, we force degrade the main compound to determine if the impurity is increased. This degraded material with increased amount of impurity will make the isolation by LC-UV-MS preparatory chromatography much easier. We can also analyze any existing mother liquor’s that may be enriched in desired impurities and use the mother liquors for impurity isolation if they contain larger amounts of desired impurities. These techniques have been employed in recent projects for isolating impurities that are at the 0.1 % level in the main compound.

LC-MS, NMR and LC-HR/MS Data Acquisition & Interpretation

Once a suitable amount of a relatively pure isolate (greater than roughly 85%) is obtained, 1D/2D NMR data acquired using Bruker DRX-500 500 MHz NMR spectrometer. The data sets not only encompass conventional proton-proton and proton-carbon correlation, but also proton-nitrogen correlation experiments to allow for unambiguous structural assignments.  The instrument is equipped with a 3 mm inverse probe that can be used for samples that are on the sub-milligram level.

Derivation of the Structural Formula

Once a complete NMR data set is obtained, a structural formula can be determined that fits the NMR data, as well as the mass spectrometry parent and fragment structures. Also, high resolution MS and fragmentation MS data can be used, if needed. Ultra-High Resolution Bruker Impact II QTOF LC/MS provided through our partners is the latest innovation in Bruker’s unique UHR-QqTOF mass spectrometry product with industry-leading > 50,000 Full-Sensitivity Resolution (FSR). It opens up enhanced analytical performance levels for all applications where trace analysis from complex, high-background matrices is a challenge.  A good structural formula would be consistent with all the NMR and mass spectrometry data. As an add-on bonus, we can recover the impurity from solution and provide it as an HPLC marker for analytical development.