Two dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye conjugated oligonucleotides.
Two dimensional liquid chromatography coupled to mass spectrometry (2D LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one dimension, selecting an ion pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion pairing agent for analyses requiring mass spectrometry, but the elution order of dye conjugated failed sequences relative to the main peak is not length based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion pairing for a length based separation, but MS response is compromised due to ion suppression. In this study, 2D LC/MS is used to show that dye conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion pairing agent in the first dimension and further identified using a weaker but MS compatible ion pairing agent in the second dimension, results that are not achievable in a one dimensional analysis. More specifically, a heart cut configuration using ion pair reversed phase chromatography in both the first and second dimension (IP RP IP RP) is used to transfer the n 1 impurity from a length based separation in the first dimension to a second dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n 1 impurity from the two dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high resolution instrumentation, where a more extensive set of impurities could not be evaluated using one dimensional techniques.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences