Validation of Liquid Chromatography–Tandem Mass Spectrometry Method for Analysis of Urinary Conjugated Metanephrine and Normetanephrine for Screening of Pheochromocytoma

摘要

Background: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines.Methods: We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 °C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column.Results: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10–5000 μg/L. Interassay CVs were 5.7–8.6% at mean concentrations of 90–4854 μg/L for MN and NMN. The detection limit was 10 μg/L. Recovery of MN and NMN (144–2300 μg/L) added to urine was 91–114%. The regression equation for the LC-MS/MS ( x ) and colorimetric ( y ) methods was: y = 0.81 x ? 0.006 ( r = 0.822; n = 110). The equation for the HPLC ( x ) and LC-MS/MS ( y ) methods was: y = 1.09 x + 0.05 ( r = 0.998; n = 40).Conclusions: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography–mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.