title: Cortisol and cortisone ratio in urine: LC-MS/MS method validation and preliminary clinical application.
abstract: BACKGROUND
  The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing's syndrome, apparent mineralocorticoid excess, and also provides information on 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges.
  
  
  METHODS
  After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation.
  
  
  RESULTS
  The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were <5% and 6% for cortisol and 6% and 8% for cortisone, respectively. No ion suppression was observed. The non-parametric reference range for the cortisol/cortisone ratio was 0.14-1.09.
  
  
  CONCLUSIONS
  A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Antonelli
authors.forename: Carlo
authors.initials: C
authors.lastname: Artusi
authors.forename: Mariela
authors.initials: M
authors.lastname: Marinova
authors.forename: Laura
authors.initials: L
authors.lastname: Brugnolo
authors.forename: Martina
authors.initials: M
authors.lastname: Zaninotto
authors.forename: Carla
authors.initials: C
authors.lastname: Scaroni
authors.forename: Rosalba
authors.initials: R
authors.lastname: Gatti
authors.forename: Franco
authors.initials: F
authors.lastname: Mantero
authors.forename: Mario
authors.initials: M
authors.lastname: Plebani
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D003348:Cortisone / Q000302:isolation & purification / Q000592:standards / Q000652:urine*; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000302:isolation & purification / Q000592:standards / Q000652:urine*; D008297:Male; D008875:Middle Aged; D043204:Mineralocorticoid Excess Syndrome, Apparent / Q000378:metabolism / Q000473:pathology; D012016:Reference Values; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000592:standards; D016482:Urinalysis / Q000379:methods* / Q000592:standards; D055815:Young Adult
chemical_list: D003348:Cortisone; D006854:Hydrocortisone