title: Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for LC-MS/MS analysis of plasma free metanephrines.
abstract: BACKGROUND
  LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for quantifying target analytes in complex matrices. However, the technique lacks selectivity when plasma free metanephrines are measured. We propose the use of multistage fragmentation (MRM(3)) to improve the analytical selectivity of plasma free metanephrine measurement.
  
  
  METHODS
  Metanephrines were extracted from plasma with weak cation exchange solid-phase extraction before separation by hydrophilic interaction liquid chromatography. We quantified normetanephrine and metanephrine by either MRM or MRM(3) transitions m/z 166→134→79 and m/z 180→149→121, respectively.
  
  
  RESULTS
  Over a 6-month period, approximately 1% (n = 21) of patient samples showed uncharacterized coeluting substances that interfered with the routine assay, resulting in an inability to report results. Quantification with MRM(3) removed these interferences and enabled measurement of the target compounds. For patient samples unaffected by interferences, Deming regression analysis demonstrated a correlation between MRM(3) and MRM methods of y = 1.00x - 0.00 nmol/L for normetanephrine and y = 0.99x + 0.03 nmol/L for metanephrine. Between the MRM(3) method and the median of all LC-MS/MS laboratories enrolled in a quality assurance program, the correlations were y = 0.97x + 0.03 nmol/L for normetanephrine and y = 1.03x - 0.04 nmol/L for metanephrine. Imprecision for the MRM(3) method was 6.2%-7.0% for normetanephrine and 6.1%-9.9% for metanephrine (n = 10). The lower limits of quantification for the MRM(3) method were 0.20 nmol/L for normetanephrine and 0.16 nmol/L for metanephrine.
  
  
  CONCLUSIONS
  The use of MRM(3) technology improves the analytical selectivity of plasma free metanephrine quantification by LC-MS/MS while demonstrating sufficient analytical sensitivity and imprecision.
authors.affiliation: Department of Clinical Chemistry and Endocrinology, South Eastern Area Laboratory Services (SEALS), Prince of Wales Hospitals, Sydney, Australia; michael.wright2@sesiahs.health.nsw.gov.au.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wright
authors.affiliation: Department of Clinical Chemistry and Endocrinology, South Eastern Area Laboratory Services (SEALS), Prince of Wales Hospitals, Sydney, Australia;
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Thomas
authors.affiliation: Department of Clinical Chemistry and Endocrinology, South Eastern Area Laboratory Services (SEALS), Prince of Wales Hospitals, Sydney, Australia;
authors.forename: Phoebe E
authors.initials: PE
authors.lastname: Stanford
authors.affiliation: Department of Clinical Chemistry and Endocrinology, South Eastern Area Laboratory Services (SEALS), Prince of Wales Hospitals, Sydney, Australia; School of Medical Sciences, University of New South Wales, Sydney, Australia.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Horvath
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016477:Artifacts; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008676:Metanephrine / Q000097:blood*; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008676:Metanephrine