Abstract
Background: Plasma metanephrines are the primary biomarkers used to aid in diagnosing pheochromocytoma. However, the low physiological levels of metanephrines, physicochemical properties, and potential interferences make it challenging to achieve high sensitivity and specificity. In this report, we developed and validated a sensitive and interference-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring plasma metanephrines with simple sample preparation. Methods: Plasma samples were extracted using weak cation exchange solid-phase extraction cartridges, and analyzed by LC-MS/MS with an analytical cycle time less than six minutes. Results: Absolute ion suppression and matrix effect were observed, however, were completely compensated for by the internal standards. Epinephrine, an isobaric interferent of normetanephrine, was chromatographically separated, and no interference was observed from other common interferents. The method was linear from 0.08 to 22.2 nmol/L for normetanephrine and 0.03 to 8.2 nmol/L for metanephrine with accuracy ranging from 81 to 107%. No carryover was observed up to 56.8 nmol/L for normetanephrine and 8.7 nmol/L for metanephrine. Intra-assay and total CVs were within 6.8% for normetanephrine and 5.2% for metanephrine for three levels tested. Based on Deming regression, comparison with a reference LC-MS/MS method using patient specimens (n=40) showed a slope of 0.973, intercept of 0.11 nmol/L and correlation coefficient of 0.9936 for normetanephrine and a slope of 1.039, intercept of -0.014 nmol/L, and correlation coefficient of 0.9914 for metanephrine. The mean difference was 3.5% and -1.6% for normetanephrine and metanephrine, respectively. Conclusion: This LC-MS/MS assay is sensitive and free of interference for quantitation of plasma metanephrines
Highlights
Pheochromocytomas are catecholamine producing tumors found mainly in the adrenal medulla
Assays that use High Performance Liquid Chromatography (HPLC) coupled with tandem mass spectrometry (HPLC-MS/MS) may yield high specificity with simple sample preparation and short chromatography time [7,8,9]
Several sample preparation procedures were tested for measuring plasma free metanephrines
Summary
Pheochromocytomas are catecholamine producing tumors found mainly in the adrenal medulla. Methods using high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) are commonly used [5]. Those methods require time-consuming sample preparation and relatively long chromatography time with occasional interference from other substances including certain drugs [6]. Assays that use HPLC coupled with tandem mass spectrometry (HPLC-MS/MS) may yield high specificity with simple sample preparation and short chromatography time [7,8,9]. The low physiological levels of metanephrines, physicochemical properties, and potential interferences make it challenging to achieve high sensitivity and specificity. We developed and validated a sensitive and interference-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring plasma metanephrines with simple sample preparation
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