Background: Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. Methods: We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. Results: Total run-time including sample cleanup was 8 min. Intra-and interassay analytical variation (CV)
Dietary restrictions should be considered to minimize false-positive results for urinary and plasma deconjugated MNs during diagnosis of pheochromocytoma. Similar considerations appear warranted for plasma and urinary free 3-MT, but not for free NMN or MN, indicating advantages of measurements of the free compared to deconjugated metabolites.
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