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.

Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber

“…The limits of detection (LODs) using nanoLC/MS/MS were found to be 7.8 fg/μL, 7.2 fg/μL and 9 fg/μL for progesterone, testosterone and hydrocortisone, respectively. These LODs are an improvement compared to that reported for the analysis of urinary steroids using conventional LC where the LOD was 50 fg/μL . This improvement in sensitivity over conventional LC is of particular importance where the sample quantity is limited.…”

“…Steroid hormone analyses using LC/MS/MS techniques have been conducted in various species and tissues . Although there has been great utility of nanoLC/MS/MS for peptide analysis due the increased sensitivity of this method, there has been limited use of nanoLC/MS/MS for steroids.…”

Quantitative Profiling of Cortisol Metabolites in Human Urine by High-Resolution Accurate-Mass Ms

“…Deuterium isotope effect caused [ 2 H 6 ]-A-THF to elute 0.5 min earlier than the unlabeled THF and A-THF (Supplementary Figure 4A). Previous studies reported a partial separation of THF and A-THF by using chromatographic condition with 0.1% acetic acid or formic acid (FA) [9,25]. However, we observed considerable ion suppression and lost 40-80% ion signals of cortisol metabolites when the concentration of FA increased from 0.01 to 0.05%, and 0.1% during optimization of chromatographic conditions.…”

“…LOD and LOQ values of cortisol metabolites were summarized in Table 1. Sensitivities were achieved for cortisol and cortisone in the urine with LOD as low as 0.05 and 0.02 pg/μl urine, respectively, 10-75-times more sensitive than the methods in previous reports [9,25]. LOD and LOQ estimates were higher for A-THF/THF, 6-hydroxycortisol, dihydrocortisol, THE and dihydrocortisone, likely because these compounds were labile under ESI and underwent in-source fragmentation to lose H 2 O molecules in the full MS scan, which caused a drop of ion signals measured at MS 2 and MS 3 scan stages.…”

Urinary Exosomes and Their Cargo: Potential Biomarkers for Mineralocorticoid Arterial Hypertension?

“…Human blood, saliva, and urine are biofluids that constitute a source of non-invasive, convenient and easy to access biomarkers that can be collected many times over long periods of time. Spot urine and 24-h urine is the focus for the identification of novel peptide, steroidal, or exosomal biomarkers with a potential role in diagnosis and classification of diseases related to renal system ( 65 , 66 ).…”