Reduction of metal adducts in oligonucleotide mass spectra in ion‐pair reversed‐phase chromatography/mass spectrometry analysis

Abstract

RationaleElectrospray ionization mass spectrometry (ESI‐MS)‐based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS‐based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI‐MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation.MethodsA UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion‐pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro‐2‐propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm × 50 mm) for all separations. A 10‐min method was used to provide statistical figures of merit and evaluate adduct formation over time.ResultsTrace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI‐MS‐based configurations. Non‐specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion‐pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study.ConclusionsImplementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non‐specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

“…Another explanation is that due to its weaker proton affinity compared with formate or acetate anions, chloride anions did not extract protons from the acidic groups, making them more likely to stay in their neutral forms, which would also minimize sodium adduct ion formation. The enhanced detection sensitivity in the presence of HCl is consistent with a report that low pH reconditioning reduced sodium adduct formation in oligonucleotides, which also contain multiple phosphate groups . Using optimized conditions to promote protonation reduced the heterogeneity in the detection of ions of intact β‐casein (Figure ).…”

“…The enhanced detection sensitivity in the presence of HCl is consistent with a report that low pH reconditioning reduced sodium adduct formation in oligonucleotides, which also contain multiple phosphate groups. 56 Using optimized conditions to promote protonation reduced the heterogeneity in the detection of ions of intact β-casein (Figure 1). Removing sodium adducts was also recently reported to decrease in the heterogeneity of the ions and aid ECD-based top-down analysis.…”

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

“…In order to increase the sensitivity of the detection of miRNA in ESI-and to decrease cation adduct formation, several BGE and sheath liquid compositions were tested. In general, an effective method to minimize alkali adduct formation is the addition of organic bases or ammonium salts 43 . In our case, appropriate results were obtained with BGEs of ammonium acetate.…”

The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

“…HPLC is an effective technique to remove adduct metal ions from oligonucleotide molecules . Coupling HPLC to ESI‐MS (LC/MS) has gained a huge popularity in the analysis of oligonucleotides, as it simultaneously provides desalting, separation and characterization of oligonucleotides .…”

“…HPLC is an effective technique to remove adduct metal ions from oligonucleotide molecules. 28,30 Coupling HPLC to ESI-MS (LC/MS) has gained a huge popularity in the analysis of oligonucleotides, as it simultaneously provides desalting, separation and characterization of oligonucleotides. [31][32][33][34] Unlike ion-exchange chromatography, sizeexclusion chromatography, and affinity chromatography, both IP-RP-HPLC and HILIC have been regularly coupled to ESI-MS to characterize oligonucleotides.…”