title: Free cortisol method comparison: ultrafiltation, equilibrium dialysis, tracer dilution, tandem mass spectrometry and calculated free cortisol.
abstract: BACKGROUND
  Free cortisol (FC) can be calculated from measurements of total cortisol and binding proteins or measured after mechanical separation of unbound and bound fractions by equilibrium dialysis or ultrafiltration. FC can then be measured indirectly by 3H-cortisol dilution or directly by immunologic or tandem mass spectrometry assays.
  
  
  METHODS
  We compared FC measured with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) with 3H-cortisol dilution in ultrafiltrates and dialysates and also with calculated FC (Coolens equation). An adult FC reference interval was established.
  
  
  RESULTS
  The UHPLC MS/MS and 3H-cortisol dilution methods were non-linearly related (Cusum linearity test p<0.001) but well correlated (R2=0.984). FC calculated with Coolens equation agreed with the UHPLC MS/MS method. Impurity of 3H-cortisol and non-specific adsorption were excluded as causes on non-linearity. Ultrafiltration was linearly related to equilibrium dialysis, simpler to perform and more repeatable. A gender non-specific FC reference interval of 2.1-19.1 nmol/L was established.
  
  
  CONCLUSIONS
  In view of the non-linearity between measuring techniques and the variability of reported reference ranges, care should be exercised in adopting a reference range. The ultrafiltration UHPLC MS/MS method we described is robust and suitable for use in a routine laboratory.
authors.affiliation: Department of Chemical Pathology, Pathology Queensland, Brisbane, Queensland, Australia. carel_pretorius@health.qld.gov.au
authors.forename: Carel J
authors.initials: CJ
authors.lastname: Pretorius
authors.forename: John P
authors.initials: JP
authors.lastname: Galligan
authors.forename: Brett C
authors.initials: BC
authors.lastname: McWhinney
authors.forename: Scott E
authors.initials: SE
authors.lastname: Briscoe
authors.forename: Jacobus P J
authors.initials: JP
authors.lastname: Ungerer
mesh_terms: D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods*; D055815:Young Adult
chemical_list: D006854:Hydrocortisone