title: Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma.
abstract: 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 degree 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 microg/L. Interassay CVs were 5.7-8.6% at mean concentrations of 90-4854 microg/L for MN and NMN. The detection limit was 10 microg/L. Recovery of MN and NMN (144-2300 microg/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.
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Taylor
authors.forename: Ravinder J
authors.initials: RJ
authors.lastname: Singh
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000175:diagnosis* / Q000652:urine; D015415:Biomarkers / Q000652:urine; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008676:Metanephrine / Q000652:urine*; D009647:Normetanephrine / Q000652:urine*; D010673:Pheochromocytoma / Q000175:diagnosis* / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D009647:Normetanephrine; D008676:Metanephrine; D003903:Deuterium