The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography–mass spectrometry methods

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.

The shift of charge states of oligonucleotide negative ions formed in electrospray ionization mass spectrometry to higher mass-to-charge ratio has been accomplished by addition of organic acids and bases to the solution to be electrosprayed. The use of acetic acid or formic acid combined with piperidine and imidazole effectively reduced charge states. Signal intensity and stability were enhanced greatly when the infused solution contained a high percentage of acetonitrile. In addition, the cocktail that contained imidazole, piperidine, and acetic acid in 80% acetonitrile not only reduced charge states, but also substantially suppressed Na adduction. Several oligonucleotides that varied in base composition and length were investigated, and studies of mixtures showed a significant reduction in spectral complexity.

Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.