Titration-Based Assessment of Matrix Effects in Direct Infusion Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a standard and robust technique for analysis of a large variety of biomolecules. Direct infusion approaches offer rapid, high-throughput analysis of small sample volumes but are inherently susceptible to matrix effects during electrospray ionization. These matrix effects can substantially alter analyte ionization and gas-phase response and thereby complicate the interpretation of analyte abundances in complex biological samples. Herein, we developed a titration-based workflow to identify matrix effects and distinguish true changes in endogenous analyte abundance from ionization artifacts using direct infusion probe mass spectrometry (DIP-MS). Rat brain tissue extracts, rat insulinoma cell extracts, and isotopically labeled internal standards were used to model different matrix complexities to define condition-based differences in matrix effects. Thereafter, by mixing paired extracts in different ratios and fitting linear regression models, we assessed analyte-specific responses to sample-specific matrices. Moreover, we uncovered ion suppression or enhancement of hundreds of analytes by comparing regression slopes using interaction tests. We find that using endogenous analytes in our titration-based workflow gives a good indication of matrix effects for certain analyte classes. However, using a preselected panel of internal standards is strongly favorable for reliably identifying matrix effects during ionization. Altogether, combining our titration workflow, a selected panel of internal standards, and regression modeling provides a sensitive way of detecting matrix effects and for distinguishing true metabolite abundance changes from ionization artifacts.