Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis‐electrospray ionization‐quadrupole time of flight‐mass spectrometry

Abstract

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.

Recent advances in the application of capillary electrophoresis with mass spectrometric detection

“…They were able to detect a cross-contaminant in the low ng/mL range. Another CE-MS method for quality control purposes was presented by Willems et al [182]. In this case, the characterization of synthetic oligonucleotides produced for the use in microbiology, medicine, and forensics was the main aim of this work.…”

CE with MS detection: A rapidly developing hyphenated technique

“…In particular the introduction of reasonably priced bench top TOF and QTOF instruments led to an enormous boost in CE‐(Q) TOF‐MS applications. A number of analytical problems previously solved and published employing CE with low‐resolution MS instruments were now revisited and new analytical data based on CE‐TOF‐MS or CE‐QTOF‐MS taking advantage of the improved capabilities of these instruments were presented 63, 65, 68–86. Major benefits of such a setup are the clearly enhanced options accurate mass determination provides when it comes to the identification of unknowns as well as the improved certainty in peak assignment.…”

A sheathless CE/ESI‐MS interface with an ionophore membrane‐packed electro‐conduction channel

“…The analytical utility of the CE-MS technique has been demonstrated in the various fields of genomics [1,2], proteomics [3][4][5], drug analysis [6,7], metabolite screening/identification [8,9], biomarker discovery [10] and food sciences [11]. A comprehensive list of applications of CE/MS techniques can be found elsewhere [12].…”