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

¹ Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN 55905.

^aAddress correspondence to this author at: Hilton 730, Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. Fax 507-284-9758; e-mail singh.ravinder{at}mayo.edu.

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 = 0.81x - 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 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.

The following articles in journals at HighWire Press have cited this article:

				A. Algeciras-Schimnich, C. M. Preissner, W. F. Young Jr., R. J. Singh, and S. K. G. Grebe Plasma Chromogranin A or Urine Fractionated Metanephrines Follow-Up Testing Improves the Diagnostic Accuracy of Plasma Fractionated Metanephrines for Pheochromocytoma J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 91 - 95. [Abstract] [Full Text] [PDF]

				W. H.A. de Jong, K. S. Graham, J. C. van der Molen, T. P. Links, M. R. Morris, H. A. Ross, E. G.E. de Vries, and I. P. Kema Plasma Free Metanephrine Measurement Using Automated Online Solid-Phase Extraction HPLC Tandem Mass Spectrometry Clin. Chem., September 1, 2007; 53(9): 1684 - 1693. [Abstract] [Full Text] [PDF]

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				Y. C. Kudva, A. M. Sawka, and W. F. Young Jr. The Laboratory Diagnosis of Adrenal Pheochromocytoma: The Mayo Clinic Experience J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4533 - 4539. [Full Text] [PDF]

				G. Eisenhofer Biochemical Diagnosis of Pheochromocytoma--Is it Time to Switch to Plasma-Free Metanephrines? J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 550 - 552. [Full Text] [PDF]

				R. L. Taylor, D. Machacek, and R. J. Singh Validation of a High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Method for Urinary Cortisol and Cortisone Clin. Chem., September 1, 2002; 48(9): 1511 - 1519. [Abstract] [Full Text] [PDF]