Determination Of Free Cortisol Research Articles

Urinary Free Cortisol Determination and Interferences Studies Using Liquid Chromatography Coupled to Tandem Mass Spectrometry after On-Line Solid Phase Extraction Based on TurboflowTM Chromatography.

(1) A 24 h urinary free cortisol (UFF) is one of the first-line exams recommended for the diagnosis of Cushing's syndrome. In a hospital hormonology department, this activity can exceed several hundred dosages per week. The UFF is generally determined via an immunoassay with an automate using a chemiluminescence or electrochemiluminescence detection system. To increase the cortisol concentration in the analyzed sample, the automated analysis is preceded by urine extraction, which does not prevent there from being some interferences due to other steroids with close structures. (2) This paper describes the development of on-line solid phase extraction coupled to liquid chromatography and mass spectrometry for the analysis of urinary free cortisol. The on-line extraction was based on the TurboflowTM chromatography coupled to the analytical column by two valves, easily available for the laboratories. (3) The choice of the Accucore Polar Premium® analytical column made it possible to avoid analytical interferences with exogenous or endogenous molecules having the same SRM transition (363 → 121) as cortisol. (4) The method was fully validated in the range of clinically relevant concentrations from the lower limit of quantification (LLOQ) to 411.75 nmol·L-1.

Analysis of microsamples by miniaturized magnetic-based pipette tip microextraction: determination of free cortisol in serum and urine from very low birth weight preterm newborns.

Miniaturized magnetic-based pipette tip microextraction is presented as a sample preparation approach for microsamples. It involves quick dispersion of a diminutive amount of a magnetic sorbent material in a low-volume sample (10 μL) to entrap the target analytes. Next, the dispersion is aspirated using a (semi)automatic pipette through a pipette tip with a small cubic neodymium magnet inside, which retrieves the magnetic sorbent containing the analytes. After discarding the rest of the sample, the sorbent is properly rinsed by aspirating/dispensing deionized water, and then, the analytes are eluted by aspirating/dispensing an appropriate solvent. This approach was employed for the determination of free cortisol in serum and urine from very low birth weight preterm newborns, a vulnerable patient group who present low availability for sampling biological fluids. A magnetic immunosorbent made of a cortisol antibody was employed for the selective extraction, followed by liquid chromatography-tandem mass spectrometry. Good analytical features were obtained, such as limits of detection and quantification of 0.08 and 0.27 ng mL-1, respectively, linearity up to 50 ng mL-1 (R2 > 0.999), RSD values under 15% and relative recoveries between 91 and 111%. The cross-reactivity with other glucocorticoids (i.e., cortisone and prednisolone) was evaluated to show the selectivity of the extraction. Finally, the method applicability was demonstrated towards the determination of free cortisol in the serum and urine samples from low birth weight preterm newborns.

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-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

Urinary free cortisol is a reliable index of adrenal cortisol production in patients with liver cirrhosis.

The measurement of total and free cortisol has been studied as a clinical index of adrenal cortisol production in patients with liver cirrhosis. Correlations between free plasma and salivary cortisol have previously been reported in stable cirrhotic patients. Urinary free cortisol constitutes an index of adrenal cortisol production; however, it has never been used in assessing adrenal function in patients with liver cirrhosis. The aim of this observational study was to determine associations between urinary free cortisol, serum total, salivary, measured and calculated plasma free cortisol levels in cirrhotics, determining which of them can be used as an indirect index of free cortisol levels. Moreover, we investigated the potential use of 24 h urinary free cortisol as a prognostic factor for mortality. Seventy-eight outpatients with liver cirrhosis were included. Serum, salivary and urinary free cortisol were measured using the electrochemiluminenscence immunoassay. Plasma free cortisol determination was conducted using a single quadrupole mass spectrometer. The quantification of free cortisol was achieved by determining the signal response on negative ESI-MS mode. Twenty-four hour urinary free cortisol levels correlated with free cortisol determined by mass spectrometer, total cortisol and calculated free cortisol levels. Patients with low levels of urinary free cortisol presented a significantly higher mortality rate compared to those with high levels. The factors associated with death risk were determined by Cox regression. In the multivariate analysis, two models were applied; in the first model, CP score, PVT and urinary free cortisol were found to be significantly related to patients' survival, whereas in the second, MELD score, ascites and urinary free cortisol were independently related to survival. This study suggests that 24 h urinary free cortisol could be considered as a potential index of adrenal cortisol production in patients with liver cirrhosis and it potentially detects patients with a high mortality risk.

Simultaneous Determination of Free Cortisol, Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography–Tandem Mass Spectrometry

A fast, efficient and low-cost high performance liquid chromatography–tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH2Cl2, followed by reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C18 column with a mobile phase consisting of 0.05%v/v formic acid in water—acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34 ng mL−1 (cortisol) to 1.37 ng mL−1 (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96 years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.

Investigation for Paediatric Cushing's Syndrome Using Twenty-Four-Hour Urinary Free Cortisol Determination

Objective: Paediatric Cushing's syndrome (CS) remains a challenge to diagnose and exclude. We assessed the accuracy of 24-hour urinary free cortisol (UFC) determination in children referred for suspected CS. Design: We conducted a retrospective study of paediatric patients referred to our centre with suspected CS between 1982 and 2014. Patients: Of 66 subjects (mean age 12.9 years; range 4.4-16.9), there were 47 cases of CS (29 males), which included Cushing's disease (CD; 39 patients, 25 males), primary pigmented nodular adrenocortical disease (8 patients, 4 males) and 19 ‘controls' (6 males) in whom the diagnosis of CS was excluded. Measurements: The subjects had between one and five 24-hour UFC collections analysed by radioimmunoassay, chemiluminescent immunoassay or liquid chromatography-mass spectrometry. The data were normalised, corrected for body surface area (m²) and assessed using receiver operating characteristic analysis and an independent two-tailed t test. Results: The diagnostic accuracy of 24-hour UFC for CS was excellent (area under the curve 0.98, 95% CI 0.946-1.00, sensitivity 89%, specificity 100%). Conclusions: Twenty-four-hour UFC is a reliable and practical investigation with high diagnostic accuracy for paediatric CS. However, further investigations may be required if the UFC is normal but there is a high diagnostic suspicion of CS.

Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to liquid chromatography-tandem mass spectrometry (TFC-HPLC-MS/MS)

Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders. Here we present a new method, designed for routine laboratory use, which enables quick determination of these analytes with minor sample workup.

Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography–tandem mass spectrometry (TFC–HPLC–MS/MS)

Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders. Here, we present a new method, designed for routine laboratory use, which enables quick determination of these analytes with minor sample workup. Turbulent flow chromatography shortens sample preparation, and connection to a fused-core particle-packed column (rugged amide-embedded C18 phase) permits a rapid and effective separation of the analytes, as well as additional separation from other related and isobaric compounds present in urine. Urinary isobaric compounds were successfully identified. The method requires only 100μl of urine supernatant per sample. The total time between injections is 9.5min. The solvents used for both turbulent and analytical chromatography are water and methanol, and the relatively low flows needed during the method resulted in an extended life of the columns. Linearity showed a R (2) > 0.994. Limit of detection and limit of quantification are 0.5 and 1.0ng/ml for cortisone and 1.0 and 2.0ng/ml for cortisol. Recoveries ranged from 99.7 to 109.1% for cortisone and from 98.7 to 102.9% for cortisol. Accuracy values (relative errors) for intra- and inter-assay experiments were always below 8%, whereas precision (percent CV) ranged from 3.7 to 10.7%. No matrix effects were detected during the validation process. The reproducibility for each analyte's retention time was excellent, with a coefficient of variation always below 0.2%. The final validation step included the study of urine samples from healthy children and from children previously diagnosed with corticoidal disorders. The high selectivity achieved enables quick data handling.

Simultaneous determination of urinary cortisol, cortisone and corticosterone in parachutists, depressed patients and healthy controls in view of biomedical and pharmacokinetic studies

A rapid and sensitive reversed-phase liquid chromatographic method (RP-LC) with UV detection has been developed for the determination of free cortisol, cortisone and corticosterone in human urine. The assay was performed after a solid-phase extraction procedure (SPE) with dexamethasone as the internal standard. Chromatographic separation was carried out on a Nucleosil 100 C(18) analytical column using a mixture of acetonitrile and water (30 : 70, v/v) as a mobile phase at a flow-rate of 1 mL min(-1). Spectrophotometric detection was performed at 240 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The absolute recoveries of glucocorticoids were above 94.6%. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 2 ng mL(-1), respectively, for all analytes. Linearity was confirmed in the range of 2-300 ng mL(-1) with a correlation coefficient greater than 0.9997 for all steroid hormones. The proposed method was sensitive, robust and specific allowing reliable quantification of steroid hormones. This method was successfully applied for determination of three endogenous glucocorticoid levels in human urine. The studies were performed on 20 sedentary healthy volunteers in comparison to two socially diversified groups, namely 10 parachutists before and after jump and 10 patients with depression. Pharmacokinetic studies performed on these groups indicated that urinary free cortisol and cortisol-to-cortisone ratios can be treated as biomarkers of stress and depressive disorders.

Application of nanofiber‐packed SPE for determination of salivary‐free cortisol using fluorescence precolumn derivatization and HPLC detection

Salivary cortisol has emerged as an easy-to-collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary-free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high-throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro-column was designed. The LOD of cortisol was 0.01 microg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 microg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 microg/L. The nanofiber-packed SPE overcame the low extraction recovery and bad clean-up effect of the conventional methods, and increased the sensitivity and selectivity of the method.

The diagnosis of Cushing’s syndrome

Spontaneous Cushing's syndrome is well known but unusual clinical disorder. Many of the clinical features (central weight gain, glucose intolerance, hypertension, muscle weakness) are seen in other common conditions. Recognition of patients with multiple features, features unusual for their age (i.e. early onset osteoporosis or hypertension), patients with features more specific to Cushing's syndrome (i.e. easy bruising, facial plethora, and violaceous striae), and patients with incidental adrenal mass or polycystic ovary syndrome should prompt an evaluation for cortisol excess. Late-night salivary cortisol, 1mg overnight dexamethasone suppression testing, or 24h urine free cortisol determination have excellent diagnostic characteristics and should be obtain in patients with suspected Cushing' syndrome. If this initial testing is abnormal, further evaluation should be directed by an endocrinologist experienced in the diagnosis and differential diagnosis of Cushing' syndrome.

Determination of cortisol in human blood sera by a new Ag(III) complex–luminol chemiluminescent system

A new chemiluminescence (CL) system based on the reaction of Ag(III) complex with luminol is, for the first time, reported in this work. Incorporated with a flow injection analyses (FIA), the new CL system has been applied for the determination of free cortisol in human sera. The system is based on the CL reaction of luminol with Ag(III) in alkaline solutions, while cortisol can dramatically enhance CL intensities. Under optimum conditions, CL intensities are proportional to concentration of cortisol in the range of 0.05–7.5 nM. The limit of detection is 2.0 × 10 −11 M (3σ), with a relative standard deviation ( n = 11) of 1.9% for 3.5 × 10 −9 M cortisol. Eight human blood serum samples were all handled by solid-phase extraction (SPE) clean-up and enrichment before detection. This detection system is highly sensitive and convenient and may find wide applications. Based on the chemiluminescent spectra, a possible reaction mechanism is also suggested.

Interference in urinary free cortisol determination by components of the NuvaRing® contraceptive device

Interference in urinary free cortisol determination by components of the NuvaRing® contraceptive device

Thin-Layer Chromatographic Competitive Protein-Binding Assay for Cortisol and Cortisone, and its Application to Urine Samples from Healthy Men Undergoing Water Diuresis

Specific and rapid determination of free cortisol (compound F) and cortisone (compound E) in human urine has been achieved by concentration of the urine samples, liquid–liquid extraction of the concentrated samples, thin-layer chromatography of ethanolic extracts on aluminium foil-backed silica gel 60 TLC plates, location of the steroids under UV light, elution of cortisol and cortisone from sections of the plates with phosphate buffer, and measurement by competitive protein-binding assay. Chicken serum was used as the source of corticosteroid binding globulin, because it binds cortisol and cortisone with similar high affinity. The method combining thin-layer chromatography and competitive protein-binding assay is easy to perform, specific, sensitive, and quite rapid. Free cortisol and cortisone were measured in the urine of male individuals who abstained from water intake for 2 h or who ingested 1 L of water. The results show, for the first time, that short-term water diuresis markedly increases urinary free cortisone excretion, supporting our previous hypothesis that its excretion is positively correlated with urine volume, i.e. with the volume of 24-h urine samples.

Actualité des investigations biologiques pour le diagnostic du syndrome de Cushing

The demonstration of cortisol overproduction and the determination of its cause are still challenging in spite of the progress in imaging studies performances and in techniques for cortisol overproduction demonstration. The latter is established with the 24-hour urinary free cortisol determination, the best screening test when the threshold is settled at 180 μg/24 h. But, since cortisol overproduction is sometimes periodic in true Cushing's disease, two to three determinations might be necessary for the diagnosis. If 24-hour urine collection cannot be adequately obtained, measurement of salivary cortisol is a good alternative. Overnight dexamethasone test is a good screening test for the exclusion of Cushing's syndrome. Though, false negative and false positive results are frequent. Then, in many cases, the standard dexamethasone test is useful. When cortisol overproduction is established, ACTH determination and high dose dexamethasone test usually lead to the etiological diagnosis.

Urinary Free Cortisol Determinations: What They Measure

Urinary Free Cortisol Determinations: What They Measure

Rapid and specific determination of free cortisol in guinea pig urine and faeces by thin-layer chromatography-competitive protein-binding assay

The present work describes a specific, rapid and sensitive determination of free cortisol in the urine and faeces of guinea pigs. This method offers some advantages over previous methods including the use of dexamethasone as a marker for cortisol localization on TLC sheets, the use of buffer instead of organic solvents to recover cortisol from silica gel 60 and the rapidity of the competitive protein-binding assay (1–2 h).

Lack of Specificity of Urinary Free Cortisol Determinations: Why Does It Continue?

Lack of Specificity of Urinary Free Cortisol Determinations: Why Does It Continue?

Lack of Specificity of Urinary Free Cortisol Determinations: Why Does It Continue?

Lack of Specificity of Urinary Free Cortisol Determinations: Why Does It Continue?

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C 18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.