Exploring the use of the desirability function to optimize the separation of oligonucleotide impurities by ion pair-RP LCMS

The use of small alkyl amines as ion pair reagents permits enhanced separation of impurities of phosphate diester oligonucleotides, which can be beneficial to quality control applications, and aid elucidation of the mechanisms of impurity formation. In general, however, separation of the individual components that comprise the majority of oligonucleotide impurities requires development of several independent chromatographic methods. Ideally, a single method capable of separating the individual components of all impurity classes would be developed. The mathematical concept of the desirability function has been explored here for its ability to determine the combination of experimental factors that result in a single, globally optimized chromatographic method. The optimized mobile phase, consisting of 1 mM propylamine (PA), 30 mM ammonium bicarbonate (ABC), and 1 mM octanoic acid (C8A), produced excellent agreement between measured and predicted peak resolution values for a set of n - 1 impurities. The relative importance of the mobile phase constituents on the mechanism of separation has been discussed. The approach holds great promise for the improved separation of components in complex chromatographic systems.