title: Preservation of urine free catecholamines and their free O-methylated metabolites with citric acid as an alternative to hydrochloric acid for LC-MS/MS-based analyses.
abstract: BACKGROUND
  Measurements of urinary fractionated metadrenalines provide a useful screening test to diagnose phaeochromocytoma. Stability of these compounds and their parent catecholamines during and after urine collection is crucial to ensure accuracy of the measurements. Stabilisation with hydrochloric acid (HCl) can promote deconjugation of sulphate-conjugated metadrenalines, indicating a need for alternative preservatives.
  
  
  METHODS
  Urine samples with an intrinsically acidic or alkaline pH (5.5-6.9 or 7.1-8.7, respectively) were used to assess stability of free catecholamines and their free O-methylated metabolites over 7 days of room temperature storage. Stabilisation with HCl was compared with ethylenediaminetetraacetic acid/metabisulphite and monobasic citric acid. Catecholamines and metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  
  
  RESULTS
  Free catecholamines and their O-methylated metabolites were stable in acidic urine samples over 7 days of room temperature storage, independent of the presence or absence of any stabilisation method. In contrast, free catecholamines, but not the free O-methylated metabolites, showed rapid degradation within 24 h and continuing degradation over 7 days in urine samples with an alkaline pH. Adjustment of alkaline urine samples to a pH of 3-5 with HCl or 4.8-5.4 with citric acid completely blocked degradation of catecholamines. Ethylenediaminetetraacetic acid/metabisulphite, although reducing the extent of degradation of catecholamines in alkaline urine, was largely ineffectual as a stabiliser.
  
  
  CONCLUSIONS
  Citric acid is equally effective as HCl for stabilisation of urinary free catecholamines and minimises hazards associated with use of strong inorganic acids while avoiding deconjugation of sulphate-conjugated metabolites during simultaneous LC-MS/MS measurements of free catecholamines and their free O-methylated metabolites.
authors.forename: Mirko
authors.initials: M
authors.lastname: Peitzsch
authors.forename: Daniela
authors.initials: D
authors.lastname: Pelzel
authors.forename: Peter
authors.initials: P
authors.lastname: Lattke
authors.forename: Gabriele
authors.initials: G
authors.lastname: Siegert
authors.forename: Graeme
authors.initials: G
authors.lastname: Eisenhofer
mesh_terms: D002395:Catecholamines / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D002853:Chromatography, Liquid; D019343:Citric Acid / Q000737:chemistry* / Q000652:urine; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry* / Q000652:urine; D006863:Hydrogen-Ion Concentration; D008745:Methylation; D053719:Tandem Mass Spectrometry
chemical_list: D002395:Catecholamines; D019343:Citric Acid; D006851:Hydrochloric Acid