title: Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method.
abstract: Serum steroid assays are major tools in the clinical evaluation of adrenal disorders. The main adrenal steroids are routinely measured with immunoassays. However, chromatographic methods are known to offer better specificity. We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of 15 adrenal steroids targeting the mineralo- and gluco-corticosteroid pathways. Serum steroids combined with deuterated internal standards were extracted using successive protein precipitation and solid phase extraction steps. Cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, progesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone, aldosterone, dehydroepiandrosterone sulfate, testosterone and androstenedione were resolved in fourteen minutes using a BEH C18 column coupled to a methanol-ammonium formate gradient. Detection was performed using multiple reaction monitoring quantitation. Routinely determined steroid levels by immunoassays were compared to those measured by LC-MS/MS. This method was applied to assess steroid profiles in congenital adrenal hyperplasia (CAH) patients with 21-hydroxylase deficiency. Low quantification limits depending on each steroid (ranging from 0.015ng/mL for aldosterone to 20ng/mL for DHEAS) are adapted to the clinical use. Recoveries of steroids range from 64% for 21-deoxycortisol to 101% for cortisol and are fully corrected by internal standards. A good linearity with R>0.989 is obtained for each compound. The inter-day variation coefficients ranged from 4.7% for cortisol to 16.3% for 11-deoxycorticosterone. The immunoassay for cortisol (Immulite 2000, Siemens) showed acceptable agreement with LC-MS/MS (bias +7.2%). However, Bland-Altman plots revealed large negative bias for aldosterone (-33.4%, AldoCT, CisBio international), for 17-hydroxyprogesterone at concentrations below 2ng/mL (-74.1%, OHP-CT MP Biomedical), for androstenedione (-80.3%, RIA D4, Beckman Coulter) and for 11-deoxycortisol (-125.3%, Diasource Immunoassays). Finally, the analysis of samples from 21-hydroxylase defective patients demonstrated the potential usefulness of multiplexed steroid profiling for the diagnosis and/or monitoring of different forms of congenital adrenal hyperplasia. This LC-MS/MS method provides highly sensitive and specific assessments of mineralo- and glucocorticoids pathways from a small volume sample and is therefore a promising potent tool for clinical and experimental endocrine studies.
authors.affiliation: Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital de Bicêtre, Hôpitaux Universitaires Paris Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Travers
authors.affiliation: Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Service d'Endocrinologie Pédiatrique, Hôpital Robert Debré, Assistance Publique Hôpitaux de Paris, Paris, F-75019, France; PremUp Foundation, Paris, F-75005, France; Université Paris Diderot, Sorbonne Paris Cité, Paris, F-75019, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Martinerie
authors.affiliation: Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; Département d'Endocrinologie Pédiatrique, Hôpital de Bicêtre, Hôpitaux Universitaires Paris Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France; Centre de référence des maladies rares du développement sexuel, Le Kremlin Bicêtre, F-94275, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Bouvattier
authors.affiliation: PremUp Foundation, Paris, F-75005, France; Service de Réanimation Néonatale, CH Poissy St-Germain en-Laye, Poissy, F-78303, France; EA 7285, UFR des Sciences de la Santé, Simone Veil. Université Versailles St-Quentin en Yvelines, Montigny le Bretonneux F-78180, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Boileau
authors.affiliation: Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; PremUp Foundation, Paris, F-75005, France; Service d'Endocrinologie et Maladies de la Reproduction, Hôpital de Bicêtre, Hôpitaux Universitaires Paris Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France; UMS 32, Institut Biomédical de Bicêtre, Le Kremlin-Bicêtre F-94276, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Lombès
authors.affiliation: Inserm, U1185, Le Kremlin-Bicêtre, F-94276, France; Fac Med Paris-Sud, Univ. Paris-Sud, Université Paris Saclay, UMR-S 1185, Le Kremlin-Bicêtre, F-94276, France; Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital de Bicêtre, Hôpitaux Universitaires Paris Sud, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, F-94275, France; UMS 32, Institut Biomédical de Bicêtre, Le Kremlin-Bicêtre F-94276, France. Electronic address: eric.pussard@bct.aphp.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Pussard
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000097:blood / Q000378:metabolism*; D002648:Child; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D005938:Glucocorticoids / Q000097:blood*; D006801:Humans; D007118:Immunoassay; D057230:Limit of Detection; D008901:Mineralocorticoids / Q000097:blood*; D009154:Mutation; D012044:Regression Analysis; D015203:Reproducibility of Results; D013256:Steroids / Q000097:blood*; D053719:Tandem Mass Spectrometry
chemical_list: D005938:Glucocorticoids; D008901:Mineralocorticoids; D013256:Steroids
keywords: Aldosterone; Congenital adrenal hyperplasia; Glucocorticoids; LC–MS/MS; Mineralocorticoids; Steroid profiling