Quantification of cortisol and its metabolites in human urine by LC-MSn: applications in clinical diagnosis and anti-doping control

The objective of the current research was to develop a liquid chromatography-MS n (LC-MS n ) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone,...

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Published in	Analytical and bioanalytical chemistry Vol. 414; no. 23; pp. 6841 - 6853
Main Authors	Arioli, Francesco, Gamberini, Maria Cristina, Pavlovic, Radmila, Di Cesare, Federica, Draghi, Susanna, Bussei, Giulia, Mungiguerra, Francesca, Casati, Alessio, Fidani, Marco
Format	Journal Article
Language	English
Published	Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2022 Springer Nature B.V
Subjects	Analytical Chemistry analytical methods Biochemistry Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Corticoids Corticosteroids Cortisol Cortisone Cushing syndrome detection limit Drug abuse Food Science Hormones Human wastes humans Hypothalamus Laboratory Medicine Linearity Liquid chromatography liquids Metabolism Metabolites Monitoring/Environmental Analysis Negative feedback Negative ions Pituitary Prednisolone quantitative analysis Research Paper Spectrometry Urine Addison syndrome Doping control Linear ion trap mass spectrometry Cortisol metabolites Human urine Cushing syndrome
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Abstract	The objective of the current research was to develop a liquid chromatography-MS n (LC-MS n ) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS 3 and MS 4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL −1 and 0.05 ng mL −1 , for all compounds, respectively), intra- and inter-day precision (CV = 1.4–9.2% and CV = 3.6–10.4%, respectively), intra- and inter-day accuracy (95–110%), extraction recovery (65–95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison’s disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism. Graphical abstract
AbstractList	The objective of the current research was to develop a liquid chromatography-MS n (LC-MS n ) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS 3 and MS 4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL −1 and 0.05 ng mL −1 , for all compounds, respectively), intra- and inter-day precision (CV = 1.4–9.2% and CV = 3.6–10.4%, respectively), intra- and inter-day accuracy (95–110%), extraction recovery (65–95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison’s disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism. Graphical abstract The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS3 and MS4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL-1 and 0.05 ng mL-1, for all compounds, respectively), intra- and inter-day precision (CV = 1.4-9.2% and CV = 3.6-10.4%, respectively), intra- and inter-day accuracy (95-110%), extraction recovery (65-95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison's disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism.The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS3 and MS4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL-1 and 0.05 ng mL-1, for all compounds, respectively), intra- and inter-day precision (CV = 1.4-9.2% and CV = 3.6-10.4%, respectively), intra- and inter-day accuracy (95-110%), extraction recovery (65-95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison's disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism. The objective of the current research was to develop a liquid chromatography-MS n (LC-MS n ) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS 3 and MS 4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL −1 and 0.05 ng mL −1 , for all compounds, respectively), intra- and inter-day precision (CV = 1.4–9.2% and CV = 3.6–10.4%, respectively), intra- and inter-day accuracy (95–110%), extraction recovery (65–95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison’s disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism. The objective of the current research was to develop a liquid chromatography-MSⁿ (LC-MSⁿ) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS³ and MS⁴ experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL⁻¹ and 0.05 ng mL⁻¹, for all compounds, respectively), intra- and inter-day precision (CV = 1.4–9.2% and CV = 3.6–10.4%, respectively), intra- and inter-day accuracy (95–110%), extraction recovery (65–95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison’s disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism. Abstract The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15 endogenous metabolites (6β-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-β-dihydrocortisol, 20β-dihydrocortisone, prednisolone, cortisone, α-cortolone, β-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5β-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS3 and MS4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL−1 and 0.05 ng mL−1, for all compounds, respectively), intra- and inter-day precision (CV = 1.4–9.2% and CV = 3.6–10.4%, respectively), intra- and inter-day accuracy (95–110%), extraction recovery (65–95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison’s disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism.
Author	Gamberini, Maria Cristina Draghi, Susanna Bussei, Giulia Di Cesare, Federica Mungiguerra, Francesca Casati, Alessio Arioli, Francesco Pavlovic, Radmila Fidani, Marco
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Issue	23
Keywords	Addison syndrome Doping control Linear ion trap mass spectrometry Cortisol metabolites Human urine Cushing syndrome
Language	English
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PublicationDate	2022-09-01
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PublicationTitle	Analytical and bioanalytical chemistry
PublicationTitleAbbrev	Anal Bioanal Chem
PublicationYear	2022
Publisher	Springer Berlin Heidelberg Springer Nature B.V
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KamrathCHartmannMFWudySAQuantitative targeted GC-MS-based urinary steroid metabolome analysis for treatment monitoring of adolescents and young adults with autoimmune primary adrenal insufficiencySteroids20191501:CAS:528:DC%2BC1MXht1OjsbrK10.1016/j.steroids.2019.10842631228484 ProttiMMandrioliRMercoliniLMicrosampling and LC–MS/MS for antidoping testing of glucocorticoids in urineBioanalysis2020127697821:CAS:528:DC%2BB3cXhtlCmtrjP10.4155/bio-2020-004432530296 ShackletonCClinical steroid mass spectrometry: a 45-year history culminating in HPLC–MS/MS becoming an essential tool for patient diagnosisJ Steroid Biochem Mol Biol20101214814901:CAS:528:DC%2BC3cXpvFKqsLc%3D10.1016/j.jsbmb.2010.02.01720188832 de VriesLvde JongWHATouwDJBergerSPNavisGKemaIPBakkerSJLTwenty-four hour urinary cortisol excretion and the metabolic syndrome in prednisolone-treated renal transplant recipientsSteroids201712731391:CAS:528:DC%2BC2sXhsV2qsrvO10.1016/j.steroids.2017.09.00128893559 PussardETraversSBouvattierCXueQ-YCossonCViengchareunSMartinerieLLombèsMUrinary steroidomic profiles by LC-MS/MS to monitor classic 21-hydroxylase deficiencyJ Steroid Biochem Mol Biol20201981:CAS:528:DC%2BB3cXisFOrsw%3D%3D10.1016/j.jsbmb.2019.10555331778802 McCannSJGillingwaterSKeevilBGMeasurement of urinary free cortisol using liquid chromatography-tandem mass spectrometry: comparison with the urine adapted ACS:180 serum cortisol chemiluminescent immunoassay and development of a new reference rangeAnn Clin Biochem2005421121181:CAS:528:DC%2BD2MXktFGgsbo%3D10.1258/000456305349277515829119 MarcosJRenauNCasalsGSeguraJVenturaRPozoOJInvestigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometryAnal Chim Acta2014812921041:CAS:528:DC%2BC2cXhvV2htb0%3D10.1016/j.aca.2013.12.03024491769 KosickaKSiemiątkowskaAPałkaDSzpera-GoździewiczABręborowiczGHGłówkaFKDetailed analysis of cortisol, cortisone and their tetrahydro- and allo-tetrahydrometabolites in human urine by LC–MS/MSJ Pharm Biomed Anal20171401741811:CAS:528:DC%2BC2sXltVamsbs%3D10.1016/j.jpba.2017.03.03928359965 ChiesaLNobileMPanseriSVigoDPavlovicRArioliFSuitability of bovine bile compared to urine for detection of free, sulfate and glucuronate boldenone, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone by LC-MS/MSFood Chem20151884734801:CAS:528:DC%2BC2MXnsFGmt7w%3D10.1016/j.foodchem.2015.04.13126041220 BornsteinSRAllolioBArltWBarthelADon-WauchopeAHammerGDHusebyeESMerkeDPMuradMHStratakisCATorpyDJDiagnosis and treatment of primary adrenal insufficiency: an Endocrine Society clinical practice guidelineJ Clin Endocrinol Metab20161013643891:CAS:528:DC%2BC28XmvVSru74%3D10.1210/jc.2015-171026760044 PavlovicRCannizzoFTPanseriSBiolattiBTruticNBiondiPAChiesaLTetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC–ESI-mass spectrometryJ Steroid Biochem Mol Biol201313530351:CAS:528:DC%2BC3sXktVOltbg%3D10.1016/j.jsbmb.2012.12.01523291109 LemkeTLWilliamsDAFoyeWORocheVFZitoSWFoye’s principles of medicinal chemistry20137PhiladelphiaLippincott Williams & Wilkins McDonaldJGMatthewSAuchusRJSteroid profiling by gas chromatography–mass spectrometry and high performance liquid ch J Newell-Price (4249_CR6) 2006; 367 LM Chiesa (4249_CR31) 2016; 61 J Marcos (4249_CR17) 2014; 812 L Chiesa (4249_CR26) 2015; 188 SR Bornstein (4249_CR44) 2016; 101 MM Kushnir (4249_CR24) 2011; 44 A Cuzzola (4249_CR19) 2014; 94 L Wood (4249_CR9) 2008; 45 M Fidani (4249_CR34) 2013; 78 4249_CR37 4249_CR36 4249_CR35 C-L Lin (4249_CR27) 1997; 82 F Ceccato (4249_CR28) 2014; 171 L Chiesa (4249_CR40) 2016; 33 A Gaudl (4249_CR33) 2016; 1464 BK Matuszewski (4249_CR38) 2003; 75 R Pavlovic (4249_CR4) 2013; 135 M Dasenaki (4249_CR25) 2019; 52 BM-W Fong (4249_CR29) 2010; 396 M Araujo-Castro (4249_CR3) 2021; 72 N de Kock (4249_CR39) 2018; 8 TL Lemke (4249_CR1) 2013 SJ McCann (4249_CR16) 2005; 42 K Kosicka (4249_CR21) 2017; 140 LK Nieman (4249_CR43) 2015; 100 S Christakoudi (4249_CR13) 2012; 77 A Cuzzola (4249_CR20) 2009; 23 C Kamrath (4249_CR2) 2019; 150 C Shackleton (4249_CR5) 2010; 121 U Turpeinen (4249_CR23) 2003; 63 A Luo (4249_CR11) 2019; 146 4249_CR42 4249_CR41 M Duclos (4249_CR45) 2010; 39 MM Kushnir (4249_CR14) 2010; 29 K Deventer (4249_CR18) 2003; 17 LK Nieman (4249_CR7) 2008; 93 JG McDonald (4249_CR12) 2011; 2 A Raffaelli (4249_CR15) 2006; 830 Lv de Vries (4249_CR8) 2017; 127 K Agrawal (4249_CR32) 2021; 198 X Zhai (4249_CR22) 2015; 107 M Protti (4249_CR47) 2020; 12 A Oßwald (4249_CR10) 2019; 190 R Pavlovic (4249_CR30) 2012; 35 E Pussard (4249_CR46) 2020; 198
References_xml	– reference: de VriesLvde JongWHATouwDJBergerSPNavisGKemaIPBakkerSJLTwenty-four hour urinary cortisol excretion and the metabolic syndrome in prednisolone-treated renal transplant recipientsSteroids201712731391:CAS:528:DC%2BC2sXhsV2qsrvO10.1016/j.steroids.2017.09.00128893559 – reference: ZhaiXChenFZhuCLuYA simple LC–MS/MS method for the determination of cortisol, cortisone and tetrahydro-metabolites in human urine: assay development, validation and application in depression patientsJ Pharm Biomed Anal20151074504551:CAS:528:DC%2BC2MXitVGjtLg%3D10.1016/j.jpba.2015.01.04125668797 – reference: FongBM-WTamSLeungKS-YImproved liquid chromatography–tandem mass spectrometry method in clinical utility for the diagnosis of Cushing’s syndromeAnal Bioanal Chem20103967837901:CAS:528:DC%2BD1MXhtlynt7bP10.1007/s00216-009-3247-119898997 – reference: ChristakoudiSCowanDATaylorNFSteroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC–MS and GC–MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ringSteroids2012773823931:CAS:528:DC%2BC38Xjs1yhtr4%3D10.1016/j.steroids.2011.12.01822210448 – reference: RaffaelliASabaAVignaliEMarcocciCSalvadoriPDirect determination of the ratio of tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone in urine by LC–MS–MSJ Chromatogr20068302782851:CAS:528:DC%2BD28XksVCksQ%3D%3D10.1016/j.jchromb.2005.11.011 – reference: NiemanLKBillerBMKFindlingJWNewell-PriceJSavageMOStewartPMMontoriVMThe diagnosis of Cushing’s syndrome: an endocrine society clinical practice guidelineJ Clin Endocr200893152615401:CAS:528:DC%2BD1cXlvFWhu78%3D10.1210/jc.2008-0125183345802386281 – reference: LuoAel GierariETMNallyLMSturmerLRDoddDShiR-ZClinical utility of an ultrasensitive urinary free cortisol assay by tandem mass spectrometrySteroids201914665691:CAS:528:DC%2BC1MXmvFSgtLw%3D10.1016/j.steroids.2019.03.01430951757 – reference: CuzzolaAPetriAMazziniFSalvadoriPApplication of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoidsRCM200923297529821:CAS:528:DC%2BD1MXhtVCnsr7F10.1002/rcm.421419681065 – reference: PavlovicRCannizzoFTPanseriSBiolattiBTruticNBiondiPAChiesaLTetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC–ESI-mass spectrometryJ Steroid Biochem Mol Biol201313530351:CAS:528:DC%2BC3sXktVOltbg%3D10.1016/j.jsbmb.2012.12.01523291109 – reference: LinC-LWuT-JMachacekDAJiangN-SKaoPCUrinary free cortisol and cortisone determined by high performance liquid chromatography in the diagnosis of Cushing’s syndrome1J Clin Endocrinol Metab1997821511551:CAS:528:DyaK2sXkt1Wqsw%3D%3D10.1210/jcem.82.1.36878989250 – reference: FidaniMGamberiniMCPompaGMungiguerraFCasatiAArioliFPresence of endogenous prednisolone in human urineSteroids2013781211261:CAS:528:DC%2BC3sXht1yisrg%3D10.1016/j.steroids.2012.10.02023182764 – reference: NiemanLKBillerBMKFindlingJWMuradMHNewell-PriceJSavageMOTabarinATreatment of Cushing’s syndrome: an Endocrine Society clinical practice guidelineJ Clin Endocrinol Metab2015100280728311:CAS:528:DC%2BC2MXhsVGks7bE10.1210/jc.2015-1818262227574525003 – reference: KamrathCHartmannMFWudySAQuantitative targeted GC-MS-based urinary steroid metabolome analysis for treatment monitoring of adolescents and young adults with autoimmune primary adrenal insufficiencySteroids20191501:CAS:528:DC%2BC1MXht1OjsbrK10.1016/j.steroids.2019.10842631228484 – reference: ChiesaLMNobileMBiolattiBPavlovicRPanseriSCannizzoFTArioliFDetection of selected corticosteroids and anabolic steroids in calf milk replacers by liquid chromatography-electrospray ionisation - tandem mass spectrometryFood Control2016611962031:CAS:528:DC%2BC2MXhs1Gjsr7O10.1016/j.foodcont.2015.09.028 – reference: KushnirMMRockwoodALBergquistJLiquid chromatography-tandem mass spectrometry applications in endocrinologyMass Spec Rev2010294805021:CAS:528:DC%2BC3cXntVamtrs%3D10.1002/mas.20264 – reference: Eurachem Guide. 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Snippet	The objective of the current research was to develop a liquid chromatography-MS n (LC-MS n ) methodology for the determination of free cortisol and its 15... Abstract The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its... The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15... The objective of the current research was to develop a liquid chromatography-MSⁿ (LC-MSⁿ) methodology for the determination of free cortisol and its 15...
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SubjectTerms	Analytical Chemistry analytical methods Biochemistry Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Corticoids Corticosteroids Cortisol Cortisone Cushing syndrome detection limit Drug abuse Food Science Hormones Human wastes humans Hypothalamus Laboratory Medicine Linearity Liquid chromatography liquids Metabolism Metabolites Monitoring/Environmental Analysis Negative feedback Negative ions Pituitary Prednisolone quantitative analysis Research Paper Spectrometry Urine
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Title	Quantification of cortisol and its metabolites in human urine by LC-MSn: applications in clinical diagnosis and anti-doping control
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