Matrix effects in metabolite quantification for MIST assessment: the impact of phospholipid removal and HPLC column particle size

Background: This Research article investigates the impact of phospholipid removal and high-performance liquid chromatography column particle size on the accuracy of determining the relative abundance of human metabolites using mass spectrometry peak areas in the context of assessing metabolite abundance for Metabolites in Safety Testing assessment. Results/Methodology: Plasma samples spiked with 20 compounds, representing ten pairs of drugs and metabolites, were prepared using phospholipid removal plates (Ostro (TM)) or standard protein precipitation techniques and analyzed by liquid chromatography-tandem mass spectrometry using high-performance liquid chromatography columns containing either 2.5 or 3.5 mu m particles. Removal of phospholipids significantly reduced matrix effects for samples analyzed on the larger particle size columns while preventing phospholipid build up on the analytical columns. In addition, quantitative accuracy and linearity were not affected by phospholipid removal. Conclusion: Both sample preparation strategies and column particle sizes should be considered in order to reduce the inaccuracy as a result of matrix effects in assessing metabolite abundance using mass spectrometry peak areas.