Plasma-Free Metanephrine and Normetanephrine Quantification for Clinical Applications Validated by Combining Solid-Phase Extraction and HPLC-MS/MS

Plasma-free metanephrines are considered the most sensitive and specific biochemical markers for the diagnosis of catecholamine-secreting tumors, such as pheochromocytoma and paraganglioma. In this study, we developed and validated a liquid chromatography–tandem mass spectrometry method for quantifying metanephrine and normetanephrine in human plasma, using solid-phase extraction with a weak cation-exchange mechanism. The method was validated according to the FDA Bioanalytical Method Validation Guidance and CLSI guideline C50-A. The method showed excellent linearity over concentration ranges of 0.11–13.92 nmol/L for metanephrine and 0.14–26.43 nmol/L for normetanephrine, with correlation coefficients greater than 0.999. The accuracy, precision, and lower limit of quantification met the acceptance criteria. Matrix effect evaluation revealed a process efficiency of 121% for metanephrine at the lowest concentration, slightly exceeding the acceptable range of 100 ± 15%. This was likely because of matrix-induced ion enhancement or variability in extraction efficiency. However, all other concentrations were within acceptable limits. Overall, this method demonstrated high sensitivity, specificity, and reproducibility, making it suitable for routine clinical use. Minor deviations at low concentrations do not compromise the reliability of the method, however, suggest that future optimization, such as matrix-matched calibration, can further improve performance.