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During the formation of a tetrazole ring on an investigational drug, two in-process impurities were detected and analyzed by LC-MS, which suggested that both impurities were drug-related with the same mass-to-charge ratio. To understand and control their formation, both impurities were isolated from the mother liquor of the reaction using a multi-step isolation procedure to obtain a sufficient amount for high-resolution mass spectrometry (HRMS) and NMR structural analysis. HRMS suggested a protonated mass of 577.32 Da for both impurities; however, MS fragmentation patterns provided limited information on their structures. NMR analysis indicated the presence on an additional NH functional group in both isolates with similar spatial and bond correlations to one of the dimethylcarbamoyl moieties and the corresponding aromatic ring. A phenyldimethylcarbamoylamino moiety was supported by the NMR and HRMS data and could be explained based on the 'Schmidt-like' reaction mechanism, which was an unexpected reaction pathway. Because the reaction conditions were fixed because of safety concerns, the crystallization protocol was redesigned to reduce the levels of these impurities significantly. Copyright © 2016 John Wiley & Sons, Ltd.

The separation of R,R-, S,S-, and meso-Koga bases on derivatized amylose chiral stationary phases (CSP) has been studied using different alcohol and alcohol-hexane mixtures as eluant. Straight-chain and branched alcohols with carbon numbers from one to four were investigated. The carbon number and geometry of the alcohol impacts the separation of Koga bases. The optimal separations were obtained using a mixture of methanol with linear or branched alcohol. Also, the elution order of meso- and R,R-Koga base was switched as content of branched alcohol increases in cosolvent. The study of acidic and basic additive effects demonstrated that maintaining analytes in the free base state is crucial in order to achieve retention and separation. TEA alone or TEA and TFA mixture were used in the studies.