Synthetic oligonucleotides have been used as critical research reagents in the biotechnology industry since its beginning in the 1970s. Their increasing use first led to the formation of many core technology groups and eventually to an entire industry that revolved around providing custom synthetic oligonucleotides.
Methods for performing quality control analysis of these oligonucleotides have also evolved over the years, from using gels to current methods that include capillary electrophoresis, high-resolution ion exchange chromatography, and MALDI mass spectrometry. However, with the advent of oligonucleotide therapeutics as well as their widespread usage for in vitro diagnostics, there has been a growing need for methods that better characterize and quantitate both the oligonucleotide of interest as well as any synthesis contaminants.
HPLC using mass spectrometry detection (LC/MS) has been the method of choice for the analysis of protein and peptide therapeutics for quite some time, but was not widely used for characterization of oligonucleotides due to incompatibilities between the ion pairing mobile phases used for reversed-phase (RP) separations of oligonucleotides and mass spectrometry.
Ion-pairing reagents are mobile-phase additives used to increase the retention and resolution of polar compounds being separated by reversed-phase HPLC. High molarity amounts (100 mM triethylamine acetate [TEAA]) are typically used for RP separation of oligonucleotides, however the high level of TEAA leads to ion suppression that makes MS detection of oligonucleotides difficult at therapeutic and reagent concentrations.
New LC/MS methods have been introduced in the last few years that use a combination of triethylamine (TEA) at low levels and hexafluoroisopropanol (HFIP) as a mobile-phase buffer. For RP separations this mixture has enabled reasonable sensitivity by MS.
An example of a typical oligonucleotide LC/MS run is shown in Figure 1; a 21 mer DNA oligonucleotide is run on an Agilent 1100 HPLC using a Clarity Oligo-RP HPLC column from Phenomenex. MS data is collected on an ABI 3500 triple quad MS system (Applied Biosystems). The MS total ion chromatogram (TIC) in Figure 1a shows the main peak for the 21 mer oligo; the raw MS spectra of the major peak is displayed in Figure 1b. Note that the spectra contain ions corresponding to the full-length oligonucleotide in the -4 through -9 charge states.
Key to determining any structural changes or quantitating any minor components in an oligonucleotide sample by LC/MS is having deconvolution software that can calculate the contributions of these multiple ions into a parent mass. Figure 1c shows the expected reconstructed mass for the full-length oligonucleotide generated by ABI Analyst software (Applied Biosystems); minor components can also be seen in the reconstructed mass.
From this example it is readily apparent why deconvolution software is needed to interpret oligonucleotide MS data; the utility of LC/MS analysis for characterizing oligonucleotides is easily seen. Although not shown here, additional information regarding the nucleotide sequence and location of any oligonucleotide modifications could be obtained by performing MS/MS analysis of any oligonucleotide signal and analyzing the resultant daughter ion spectra.
While computational solutions for dealing with complex MS data are important for characterizing oligonucleotides, a more critical part of performing LC/MS analysis is maximizing sensitivity and resolution of the minor components in an oligonucleotide sample. Whether it is for impurity analysis or pharmacokinetic metabolite studies, chromatographically separating minor components is a requirement because many low-level impurities are isobaric and need to be somewhat resolved to aid in characterization and quantitation. MS sensitivity is also important because many of these minor components are already at the limit of detection using current LC/MS methodologies.