Quantitative Endogenous Polyamine Analysis via Capillary Electrophoresis/Mass Spectrometry: Characterization and Practical Considerations

Abstract

Commonly used analytical techniques for polyamine analysis, including derivatization and mixed-mode liquid chromatography (LC), have inherent disadvantages. Capillary electrophoresis (CE) is uniquely suited to analyze small, highly charged molecules because analytes are separated on the basis of their electrophoretic mobility, not polarity or association with a stationary phase. Microfluidic CE–mass spectrometry (mCE–MS) is a relatively recent addition to commercially available CE offerings that streamlines traditional CE–MS interfacing and has the potential to improve upon classic CE challenges to robustness and reproducibility. MS instrument choice and scanning parameters are strongly influenced by a need for high acquisition rate to adequately sample CE peaks. Alternatively, isotachophoresis on loading can be intentionally avoided to produce sufficiently wide peaks. The mCE platform utilized here performed very well in many metrics; a limit of detection (LOD) as low as 0.25 ng/mL was achieved for spermidine, and endogenous spermidine was easily detected in blood with this method. Both of these are challenging tasks for any separation technique and demonstrate a strong use case for the platform. During experimentation, various idiosyncrasies in the commercial CE–MS interface resulted in extensive chip-to-chip variability in both peak shape and LOD, complicating the application to robust absolute quantitation. Practical guidance for similar analyses is provided.