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

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. Graphical abstract

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity

“…Inter-individual differences in urine matrix may affect the MF because some cases showed avoidance of ion suppression for 6β-OHC from urine and others did not. 50,51) Although several analytes affected matrix effects based on the characteristics of each analyte and inter-individual differences in human urine specimens, a complex matrix, REs, and CVs for IS-corrected MFs were within ±15%, which satisfy the acceptance criteria of the U.S. FDA guidelines. Because the background subtraction method was applied for the quantification of endogenous analytes, the increase in background peak area after spiking with LLOQ samples might be slightly higher than the reproducibility limits.…”

From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography–mass spectrometry bioanalysis

“…Even if suppliers are manufacturing an increasing number of high-quality SILs, their commercial availability is limited to the most important compounds of any chemical family. When a high number of analytes must be quantified, the possibility to use one IS for multiple analytes should be carefully evaluated based on chromatographic retention and MS response [81,82]. For quantification purposes, the use of one IS per target compound is generally recommended when available because they are assumed to compensate for specific differences in matrix effect and extraction recovery between the calibration methodology and study samples.…”

Pitfalls in the Diagnosis and Management of Hypercortisolism (Cushing Syndrome) in Humans; A Review of the Laboratory Medicine Perspective

“…Subsequently, LC-MS/MS techniques have been developed that are robust and suitable for discriminating adrenal carcinoma from adrenal incidentaloma or adenoma [ 103 , 104 , 105 ]. Undeniably, the steroid metabolome can provide useful information in diagnosing both hypercortisolism and its cause, and the developing technology is improving feasibility and method performance [ 49 , 106 , 107 , 108 ].…”