High-molecular weight impurity screening by size-exclusion chromatography on a reversed-phase column.

Monitoring polymerization events leading to the discovery of new high-molecular weight (MW) impurities is challenging during chemical syntheses of active pharmaceutical ingredients. Employing reversed-phase chromatography (RPC) stationary phases (SPs) in size-exclusion chromatography (SEC) mode could be a potential solution given their high efficiency, sensitivity, and extensive solvent compatibility. However, there is a lack of generalized means for trace polymeric impurities across a wide range of physicochemical properties. Herein, we developed a SEC-based approach with a C18 SP for screening such high-MW impurities. Seven polymer standards presenting a variety of functional groups, consisting of hydrophobic, heterocyclic, ionic, and neutral hydrophilic moieties, were utilized as model impurities to establish the screening conditions. Nine mobile phases (tetrahydrofuran-based, buffered methanol, and buffered acetonitrile) were proposed to cover all model polymers and a majority of potential high-MW impurities in small molecule chemical syntheses. The established screening system demonstrated a linearity of 0.05-1.0 % w/w (R2>0.99) for the selected model impurities with proper elution conditions. Two real high-MW impurities, BMT-041910 (polymeric degradation) and poly(phenyl thiirane) (by-product polymerization), were identified from the proposed high-MW impurity screening. The successful conditions yielded a quantitative limit better than 0.1 % w/w in both cases. We believe the developed screening platform is applicable to the analysis of a wide variety of unknown high-MW impurities of low abundance potentially generated during drug substance development.

Improving the efficiency of quantitative (1)H NMR: an innovative external standard-internal reference approach.

The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.