Sugimura, N., Furuya, A., Yatsu, T. and Shibue, T. . (C) Spectral abundance adduct distribution profile between the mobile phase and borosilicate experiments for the 260 CV data set. A single quadrupole mass detector was configured in a serial configuration post‐optical detection to evaluate alkali metal salt adducts in the deconvoluted spectrum. The same experiment was repeated using the LC/MS‐grade solvents in consideration for LC/MS‐based oligonucleotide assays that require even a higher degree of sensitivity such as toxicological profiles and metabolite studies.14, 23 The results indicated spectral abundance was maintained at 100% for the neutral peak with an average retention time of 2.45 min (R.S.D. 1(A). Sodium adducts were observed to have similar relative amounts in both IP reagents whereas significant potassium adduct(s) were observed in the first injection using the 99% purity IP reagent. A single quadrupole mass spectrometer (ACQUITY QDa, Waters Corp.) was used for MS analysis post TUV detection to monitor adduct formation. The significant decrease in spectral abundance from 94% to 37% over the course of the 8‐h injection series (Fig. Quality of starting materials can impact the purity of products while random insertion/deletions can result in improper sequence generation.10-12 Furthermore, synthetic errors (undesired nucleotide order switches) and phosphate backbone modification producing chiral centers result in difficult to separate isomers and diastereomers, respectively.13 With over 100 therapeutic oligonucleotides currently in development or in clinical trials, factors such as safety, efficacy, and stability are leading concerns for pharmaceutical companies and regulatory agencies.1 To this end, characterization methods that are robust, quantitative, and offer adequate selectivity are highly desirable in the analysis of therapeutic oligonucleotides. 1(A) data, which equals the number of injections as the current experiment but represents a 8‐fold increase in column volumes (347 CV) of mobile phase passed over the column, spectral abundance was reduced to 43.1%, with multiple adducts of sodium and potassium observed. The baseline reference acquired prior to the salt blank injections (0 injections) indicated sodium adducts were present at a relative peak intensity of 6.3%, which was in line with previous experiments. Moini, M., Jones, B.L., Rogers, R.M. The current study demonstrates that the presence of trace alkali metal salts can significantly diminish spectral quality in IP‐RPLC/MS‐based analyses of oligonucleotides. The selectivities of these adducts were evaluated by a two-dimensional plot using topological polar surface area (tPSA) and molecular weight. The good agreement in adduct distribution and intensity in the current experiment with the borosilicate experiment indicates alkali metal impurities present in the mobile phase are the main LC contributing factor to adduct formation in oligonucleotide separations in the current study. Prior to sample evaluation, the systems fluidic path was cleaned as before to remove residual metal salts. Injection 1 as shown in Fig. and Tyler, A.N. These observations combined with the fact the LC system could routinely be brought back to baseline performance with minimal adducts using a low pH cleaning protocol indicated non‐specific adsorption sites located throughout the LC fluidic path perpetuate adduct formation in oligonucleotide analyses. Login failed. The gradual increase in adduct abundance over time as observed in the mobile phase and borosilicate experiments suggest that the concentrations of impurities are at trace levels either in the solvent used in the mobile phase preparation or in the IP reagents themselves. (B) Comparison of spectral abundance for the neutral peak [M] at equivalent CVs between the mobile phase and borosilicate experimental data set. Find out about Lean Library here, If you have access to journal via a society or associations, read the instructions below. 1(A)), which was observed to be opposite of the sample purity experiment (Fig. MS spectra were combined using an equal number of scans from 1.0 to 2.0 min and 2.4 to 3.4 min for peak 1 and peak 2, respectively. Adduct formation was reduced to a single adduct of sodium (≤5.0%) even after a lengthy exposure of the system (≥5 h) to buffers containing trace salts. (D) Deconvoluted spectrum of Injection 1 was used as a baseline reference with the neutral peak [M] being observed at 93.9% spectral abundance when compared to the +Na adduct form (6.1%) in a clean LC system. Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples Author: Erngren, Ida , Haglöf, Jakob , Engskog, Mikael K.R. Deconvolution results of the oligonucleotide peaks of the ssRNA strand 1 and strand 2 were used to confirm LC system salt contamination (data not shown). 1(C)). A 10‐min method was used to provide statistical figures of merit and evaluate adduct formation over time. Access to society journal content varies across our titles. Oligonucleotide quantification and metabolite profiling by high-resolution and accurate mass spectrometry. Due to the negatively charged phosphorodiester backbone the chromatographic methods of choice for oligonucleotide characterization are ion‐exchange chromatography (IEC) and ion‐pairing reversed‐phase chromatography (IP‐RPLC).14-19 Charge‐based separations such as anion exchangers are well suited in the characterization of N‐X deletions; however, depurination events, base inversion isomers, and other base modifications are not readily characterized using IEC.1 Furthermore, buffers and salt gradients typically used in IEC prevent coupling to mass spectrometry (MS) as a complementary orthogonal technique for the characterization of oligonucleotides when dealing with challenging base modifications.