Two-dimensional liquid chromatography-mass spectrometry for the characterization of modified oligonucleotide impurities

A 2D-LC system coupled with a TOF mass spectrometer has been evaluated for its capabilities to provide enhanced characterization of oligonucleotide impurities. To address loop limitations in the total volume (40 μL) sampled across 1D peaks, a column trap was incorporated between the 1D and 2D columns. The main advantages of the column trap include reduction of the total number of sequential 2D runs required to fully sample broad 1D peaks, and most importantly, reduction of the error in quantitative determination of the components in broad 1D peaks by avoiding the numerical stitching of data from several 2D runs. Comprehensive RP x IP provided orthogonal separation despite its lower 1D resolution. In contrast, IP x IP did not provide orthogonal separation. RP x IP using the direct on-line extended heart-cutting system with the column trap showed additional benefits, in the elimination of off-line fractionation and sample handling errors and was successfully applied in a pH stability study of a crude oligonucleotide. SAX x IP successfully separated the isobaric “n+16” doublet of the “n + O” and “[n + S-O]” impurity species, a feat not currently possible by mass spectrometry alone or 1D-LC, demonstrating the importance of the added capabilities of the 2D-LC approach. [Display omitted] •2D-LC system evaluated for enhanced oligonucleotide impurity characterization.•SEC, RP, and SAX methods examined for 1D analysis and, IP-RP for 2D analysis.•Column trap incorporated between 2D columns to increase volume sampled, reduce run times and quantitative errors.•From all combinations (multiple heart-cutting, extended heart-cutting, comprehensive), extended 2D-LC was the most powerful.•Extended 2D-LC successfully applied to pH stability study and to separate isobaric “n + O” and “[n + S-O]” impurities.