Systematic Assessment of Matrix Effect, Recovery, and Process Efficiency Using Three Complementary Approaches: Implications for LC-MS/MS Bioanalysis Applied to Glucosylceramides in Human Cerebrospinal Fluid.

The evaluation of matrix effect, recovery, and process efficiency is essential in the validation of LC-MS/MS bioanalytical methods, as they impact assay accuracy, precision, and sensitivity. However, guidelines on bioanalytical method evaluation are not harmonized and can occasionally be ambiguous. To address this need, the study presents an integration of three different approaches to assess these parameters within a single experiment. The first approach examined the variability of peak areas and standard-to-internal standard (IS) ratios between different matrix lots to assess the influence of the analytical system, relative matrix effects, and recovery on method precision. The second strategy evaluates the influence of the overall process on analyte quantification. The third approach calculates both the absolute and relative values of matrix effect, recovery, and process efficiency, as well as their respective IS-normalized factors, to determine the extent to which the IS compensates for the variability introduced by the matrix and recovery fraction. Applying these strategies to an LC-MS/MS method for quantifying glucosylceramides in cerebrospinal fluid addresses the challenges posed by limited sample volume and endogenous analytes, while providing a comprehensive understanding of the factors that influence method performance and promoting adherence to different guideline recommendations. This study supports the importance of a systematic evaluation of matrix effect, recovery, and process efficiency during method validation. Standardized evaluation methodologies would improve data interpretation, enhance method reliability, and contribute to harmonization in in-house bioanalysis.