title: LC-MS/MS Method for the Simultaneous Determination of Free Urinary Steroids.
abstract: Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC-MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap®4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1-120 ng/mL for cortisol and cortisone, and 1-120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85-105 %, respectively. Our LC-MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11β-HSD2 and 11β-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.
authors.affiliation: Departamento de Laboratorios Clínicos, Facultad de Medicina, Pontificia Universidad Católica de Chile, Vicuña Mackenna 4686, Piso 3, Macul, 7820436 Santiago, Chile.
authors.forename: Fidel
authors.initials: F
authors.lastname: Allende
authors.affiliation: Departamento de Laboratorios Clínicos, Facultad de Medicina, Pontificia Universidad Católica de Chile, Vicuña Mackenna 4686, Piso 3, Macul, 7820436 Santiago, Chile.
authors.forename: Sandra
authors.initials: S
authors.lastname: Solari
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile ; Instituto Milenio de Inmunología e Inmunoterapia, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: Carmen
authors.initials: C
authors.lastname: Campino
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile ; Instituto Milenio de Inmunología e Inmunoterapia, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: Cristian A
authors.initials: CA
authors.lastname: Carvajal
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Lagos
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile.
authors.forename: Andrea
authors.initials: A
authors.lastname: Vecchiola
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile.
authors.forename: Carolina
authors.initials: C
authors.lastname: Valdivia
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile ; Instituto Milenio de Inmunología e Inmunoterapia, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: René
authors.initials: R
authors.lastname: Baudrand
authors.affiliation: Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile ; Instituto Milenio de Inmunología e Inmunoterapia, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: Gareth I
authors.initials: GI
authors.lastname: Owen
authors.affiliation: Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5 Piso, Casilla 114-D, Santiago, Chile ; Instituto Milenio de Inmunología e Inmunoterapia, Avda. Libertador Bernardo O'Higgins 340, Santiago, Chile.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Fardella
keywords: 11β-Hydroxysteroid dehydrogenase; Column liquid chromatography; Cortisol; Hypertension; Mass spectrometry; Metabolic syndrome