Ion-neutral Clustering of Bile Acids in Electrospray Ionization Across UPLC Flow Regimes

  • Patrick Brophy
  • Corey D. Broeckling
  • James Murphy
  • Jessica E. Prenni
Research Article


Bile acid authentic standards were used as model compounds to quantitatively evaluate complex in-source phenomenon on a UPLC-ESI-TOF-MS operated in the negative mode. Three different diameter columns and a ceramic-based microfluidic separation device were utilized, allowing for detailed descriptions of bile acid behavior across a wide range of flow regimes and instantaneous concentrations. A custom processing algorithm based on correlation analysis was developed to group together all ion signals arising from a single compound; these grouped signals produce verified compound spectra for each bile acid at each on-column mass loading. Significant adduction was observed for all bile acids investigated under all flow regimes and across a wide range of bile acid concentrations. The distribution of bile acid containing clusters was found to depend on the specific bile acid species, solvent flow rate, and bile acid concentration. Relative abundancies of each cluster changed non-linearly with concentration. It was found that summing all MS level (low collisional energy) ions and ion-neutral adducts arising from a single compound improves linearity across the concentration range (0.125–5 ng on column) and increases the sensitivity of MS level quantification. The behavior of each cluster roughly follows simple equilibrium processes consistent with our understanding of electrospray ionization mechanisms and ion transport processes occurring in atmospheric pressure interfaces.

Graphical Abstract


Electrospray Ion-neutral clustering Adduction Liquid chromatography Time-of-flight Mass spectrometry Bile acids Quantification 



The authors acknowledge Waters Corporation for postdoctoral research funding and support for this project. The authors also acknowledge Angela Doneanu of Waters Corporation for technical support.

Supplementary material

13361_2017_1878_MOESM1_ESM.docx (3 mb)
ESM 1 (DOCX 3046 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Patrick Brophy
    • 1
  • Corey D. Broeckling
    • 1
  • James Murphy
    • 2
  • Jessica E. Prenni
    • 1
  1. 1.Proteomics and Metabolomics FacilityColorado State UniversityFort CollinsUSA
  2. 2.Waters CorporationMilfordUSA

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