Development of an Untargeted LC-MS Metabolomics Method with Postcolumn Infusion for Matrix Effect Monitoring in Plasma and Feces

Untargeted metabolomics based on reverse phase LC-MS (RPLC-MS) plays a crucial role in biomarker discovery across physiological and disease states. Standardizing the development process of untargeted methods requires paying attention to critical factors that are under discussed or easily overlooked, such as injection parameters, performance assessment, and matrix effect evaluation. In this study, we developed an untargeted metabolomics method for plasma and fecal samples with the optimization and evaluation of these factors. Our results showed that optimizing the reconstitution solvent and sample injection amount was critical for achieving the balance between metabolites coverage and signal linearity. Method validation with representative stable isotopically labeled standards (SILs) provided insights into the analytical performance evaluation of our method. To tackle the issue of the matrix effect, we implemented a postcolumn infusion (PCI) approach to monitor the overall absolute matrix effect (AME) and relative matrix effect (RME). The monitoring revealed distinct AME and RME profiles in plasma and feces. Comparing RME data obtained for SILs through postextraction spiking with those monitored using PCI compounds demonstrated the comparability of these two methods for RME assessment. Therefore, we applied the PCI approach to predict the RME of 305 target compounds covered in our in-house library and found that targets detected in the negative polarity were more vulnerable to the RME, regardless of the sample matrix. Given the value of this PCI approach in identifying the strengths and weaknesses of our method in terms of the matrix effect, we recommend implementing a PCI approach during method development and applying it routinely in untargeted metabolomics.