Determination Of Urinary 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.

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

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.

Improved liquid chromatography–tandem mass spectrometry method in clinical utility for the diagnosis of Cushing’s syndrome

Determination of urinary free cortisol is one of the first lines in screening for the diagnosis of Cushing's syndrome where its measurement is mostly done by immunoassay. Although easy to perform, immunoassays suffer from the problem of assay interferences and are unable to measure cortisone levels. To enhance such techniques for clinical diagnosis, an improved liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous determination of urinary free cortisol and cortisone. The leftover urine samples from immunoassay were collected and subjected to facile solid-phase extraction cleanup. In the analysis of 130 urine samples from patients, 65 (50%) were found to have elevated urinary free cortisol (UFC) by immunoassay; but only 13 (10.8%) were found to have elevated UFC by this improved LC-MS/MS method. Nine out of the 13 patients, which showed elevated UFC by LC-MS/MS, were surgically confirmed to have Cushing's syndrome/disease. By setting a two times upper limit as a cut-off, the immunoassay gave a positive predictive value of 43.5%, whilst by using the improved method, a positive predictive value of 90% was obtained. Although several tests have been used extensively in first line screening for the diagnosis of Cushing's syndrome, none has ever shown with full capability of distinguishing all cases of Cushing's syndrome from normal and/or obese individuals. This method has shown superior analytical advantages over existing immunoassay type in terms of sensitivity, specificity and capability to diagnose Cushing's syndrome. Comparison between existing spectrometric methods, the reported developed method shown here, provides a simpler sample preparation procedure and meets with the high throughput demand of clinical laboratories.

Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays

Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed. The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturer's instructions. When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were <5% in each quality-control sample. The comparison of the UFC results achieved by assaying the study samples with GC-MS and LC-MS/MS indicated that the agreement between the two methods was excellent (LC-MS/MS = 1.0036GC-MS - 0.0841; r2 = 0.9937). The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.

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

Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations

Background In immunoassay kits for determination of urinary free cortisol (UFC) concentrations, the results vary markedly from kit to kit, so we compared in this study the reaction specificity among 4 commercially available immunoassay kits to determine the applicability of these assays in routine determination of UFC concentrations. Method Using 4 commercially available kits, cross-reaction was investigated. In addition, urine samples were fractionated by HPLC to investigate endogenous immunoreactive cortisol responses. HPLC fractions were subjected to gas chromatography-mass spectrometry (GCMS) to identify substances causing inter-kit assay discrepancies. Results Among the 4 kits, cortisol Kit “TFB” (Immunotech; IOT-RIA method) showed the lowest cross-reaction (2.5%) for prednisolone. Furthermore, on HPLC, 87.8% of the reaction of the entire fraction was seen in the fractions corresponding to the elution position of standard cortisol with the IOT-RIA method; this was the highest percentage among the 4 kits. GCMS revealed that the substance that showed a cross-reaction with the other 3 kits was 5α-tetrahydrocortisol (5α-THF) glucuronide. Conclusions The IOT-RIA method was found to be the most specific for UFC. The other 3 commercially available kits showed cross-reaction with a conjugate of 5α-THF, found to be one of the causes of inter-kit assay discrepancies.

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.

Simple and sensitive high-performance liquid chromatography method for simultaneous determination of urinary free cortisol and 6β-hydroxycortisol in routine practice: For CYP 3A4 activity evaluation in basal conditions and after grapefruit juice intake

Cytochrome P450 3A4 activity displays a wide variability. The urinary 6β-hydroxycortisol to cortisol ratio, as a non-invasive assay, can be useful for its pretherapeutic characterization. We developed an HPLC–UV method preceded by liquid–liquid extraction for assessment of this ratio in clinical practice. Urine was collected on second void morning-spot sample. Percentage recoveries were high and reproducible. The 6β-hydroxycortisol to cortisol ratio ranged from 1.6 to 9.9 in 12 Caucasian healthy volunteers. It was reduced by 30 to 70% after ingestion of white grapefruit juice, a CYP3A4 inhibitor. Our method, simple, sensitive and accurate, could be helpful for determination of CYP 3A4 activity before oral chemotherapy, or for the monitoring of the use of grapefruit juice as a pharmacological modulator.

Determination of urinary free cortisol by liquid chromatography‐tandem mass spectrometry

Measurement of urinary free cortisol is clinically important in the diagnosis of Cushing's syndrome. While liquid chromatography (LC) with UV detection provides much better specificity than immunologic methods, certain drugs cause interference. Detection by mass spectrometry (MS) is a potentially superior method. Our analysis utilizes 1 mL urine spiked with 6‐α‐methylprednisolone as internal standard. The samples were extracted with dichlormethane and the extract was washed, evaporated to dryness and analyzed by LC‐MS/MS operating in the negative mode after separation on a reversed‐phase C18 column. The calibration curves for analysis of urinary cortisol exhibited consistent linearity and reproducibility in the range of 10–400 nmol/L. Inter‐assay CVs were 4.0–7.6% at mean concentrations of 21–153 nmol/L. The detection limit was 1 nmol/L (signal‐to‐noise ratio=3). The mean recovery of cortisol added to urine ranged from 67% to 87% and that of the internal standard from 71% to 76%. The regression equation for the LC‐MS/MS (x) and HPLC (y) methods was: y=1.095x+8.0 (r=0.996; n=111). Drugs known to interfere with UV detection did not cause problems here. The sensitivity and specificity of the MS/MS method for urinary free cortisol offer advantages over HPLC with UV detection by eliminating drug interference. The higher equipment costs in comparison with HPLC methods using UV detection are balanced by higher throughput, thanks to shorter chromatographic run times.

Determination of urinary free cortisol by liquid chromatography-tandem mass spectrometry

Determination of urinary free cortisol by liquid chromatography-tandem mass spectrometry

Urinary Free Cortisol Determinations: What They Measure

Urinary Free Cortisol Determinations: What They Measure

Gas chromatography-mass spectrometry measurement of 6beta-OH-cortisol/cortisol ratio in human urine: a specific marker of enzymatic induction.

The urinary 6beta-OH-cortisol/cortisol ratio is a specific, non-invasive marker for evaluating inductive or inhibitory effects on cytochrome P450 3A activity. We propose a new quantitative gas chromatography-mass spectrometry with isotope dilution (GC-ID-MS) method for the simultaneous determination of urinary free cortisol (UFC) and 6beta-OH-cortisol (6beta-OHC). The method utilizes the following: (a) addition of internal standard (2H2 cortisol) to 1 ml of urine; (b) loading on to an Extrelut column and elution with dichloromethane; (c) derivatization to dimethoxime tri-(trimethyl-silyl)ether (MOX-TMS); (d) separation and identification by GC-ID-MS. The detection limit for cortisol was 22 pg injected (signal-to-noise ratio 10:1) and for 6beta-OH-cortisol 123 pg injected (signal-to-noise ratio 10:1). The intra-assay and the inter-assay imprecision were 4.69% and 7.4% for 6beta-OHC and 2.44% and 3.53% for cortisol, respectively. We used this method to analyze 57 morning urine samples of healthy volunteers and patients under different conditions. We found that chronic alcoholics had a significantly higher ratio of 6beta-OHC/UFC compared to controls (p<0.0001), whereas adults undergoing methadone therapy and patients with acute alcohol intoxication exhibited a significantly lower urinary 6beta-OHC/UFC ratio (p<0.05 and p<0.01, respectively). The proposed method allows a rapid and accurate assessment of the 6beta-OHC/UFC ratio.

Simultaneous determination of urinary free cortisol and 6β-hydroxycortisol by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry and its application for estimating hepatic CYP3A induction

A reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (HPLC–APCI-MS–MS) assay was developed to simultaneously determine monkey urinary free cortisol (C) and 6β-hydroxycortisol (6β-OHC) in 8 min. Urine sample (0.5 ml) containing fludrocortisone acetate (F-C) as the internal standard was extracted with ethyl acetate for 5 min with an extraction efficiency of 90% and 75% for C and 6β-OHC, respectively. A Perkin-Elmer Sciex API 3000 triple quadruple instrument was used for mass spectrometric detection and the column eluent was directed to a heated nebulizer probe. The assay was linear over the range 0.25–10 μ M for each analyte. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range for both analytes was less than 10%. Accuracy determined at three concentrations (0.8, 2.0 and 8.0 μ M) ranged between 95.5 and 108%. The method described herein is suitable for the rapid and efficient measurement of 6β-OHC/C ratio in Rhesus monkey urine following administration of known hepatic CYP3A inducers and can be used to estimate potential CYP3A induction by drug candidates in the process of early drug development.

SUBCLINICAL CUSHING'S SYNDROME

Classic Cushing's syndrome is a rare disease with an estimated incidence of 1 case per 100,000 persons. With routine use of imaging techniques such as ultrasound and CT, adrenal masses are being detected with increased frequency. A substantial percentage of these incidentalomas are hormonally active, with 5% to 20% of the tumors producing glucocorticoids. Autonomous glucocorticoid production without specific signs and symptoms of Cushing's syndrome is termed subclinical Cushing's syndrome. With an estimated prevalence of 79 cases per 100,000 persons, subclinical Cushing's syndrome is much more common than classic Cushing's syndrome. Depending on the amounts of glucocorticoids secreted by the tumor, the clinical spectrum ranges from slightly attenuated diurnal cortisol rhythm to complete atrophy of the contralateral adrenal gland with lasting adrenal insufficiency after unilateral adrenalectomy. Patients with subclinical Cushing's syndrome lack the classical stigmata of hypercortisolism but have a high prevalence of obesity, hypertension, and type 2 diabetes. All patients with incidentally detected adrenal masses scheduled for surgery must undergo testing for subclinical Cushing's syndrome to avoid postoperative adrenal crisis. The best screening test to uncover autonomous cortisol secretion is the short dexamethasone suppression test. Because the adrenal origin of a pathologic cortisol secretion is anticipated, the author prefers a higher dexamethasone dose (3 mg instead of 1 mg) to reduce false-positive results. A suppressed serum cortisol level of less than 3 micrograms/dL (80 nmol/L) after dexamethasone excludes significant cortisol secretion by the tumor. A serum cortisol level greater than 3 micrograms/dL requires further investigation, including confirmation by high-dose dexamethasone (8 mg) suppression testing, a CRH test, and analysis of diurnal rhythm. Determination of urinary free cortisol is less useful because increased values are a late finding usually associated with emerging clinical signs of Cushing's syndrome. Patients with suppressed plasma ACTH in response to CRH generally have adrenal insufficiency after surgery and require adequate perioperative and postoperative substitution therapy. Whether patients with subclinical Cushing's syndrome should undergo adrenalectomy is a matter of debate. The author performs surgery in young patients (< 50 years), in patients with suppressed plasma ACTH, and in patients with a recent history of weight gain, substantial obesity, arterial hypertension, diabetes mellitus, and osteopenia. In completely asymptomatic patients with normal plasma ACTH concentrations and in patients older than 75 years, the author recommends a nonsurgical approach. A large prospective randomized study is necessary to evaluate the benefits of surgery versus conservative treatment in patients with subclinical Cushing's syndrome.

Paradoxical response to dexamethasone in the diagnosis of primary pigmented nodular adrenocortical disease.

Primary pigmented nodular adrenocortical disease causes the Cushing syndrome in children and young adults and is most frequently associated with the Carney complex. To evaluate diagnostic tests for primary pigmented nodular adrenocortical disease. Retrospective cohort study. Tertiary care center. 21 patients with primary pigmented nodular adrenocortical disease. The control groups consisted of 9 patients with macronodular adrenocortical disease and 15 patients with primary unilateral adrenocortical disease (single adenomas). Clinical characteristics, radiologic imaging, and a 6-day Liddle test with determination of urinary free cortisol and 17-hydroxycorticosteroid excretion. Adrenal imaging and other tests were of limited value for the diagnosis of primary pigmented nodular adrenocortical disease. The Liddle test, however, distinguished patients with this disorder from those with other primary adrenocortical lesions. An increase of 50% or more in urinary free cortisol levels on day 6 of the Liddle test identified 9 of 13 patients (69.2% [95% CI, 46.6% to 91.8%]) with primary pigmented nodular adrenocortical disease, excluded all patients with macronodular adrenocortical disease, and was present in only 3 of the 15 patients with single adrenocortical adenomas (20% [CI, 0% to 40.2%]). An increase in urinary free cortisol excretion of 100% or more on day 6 of the Liddle test identified only patients with primary pigmented nodular adrenocortical disease. Patients with primary pigmented nodular adrenocortical disease responded to dexamethasone with a paradoxical increase in glucocorticoid excretion during the Liddle test. This feature distinguishes such patients from those who have the Cushing syndrome caused by other primary adrenal disorders and may lead to timely detection of the Carney complex (a potentially fatal disorder) in asymptomatic patients.

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?

Overnight urinary free cortisol determination: a screening test for the diagnosis of Cushing's syndrome.

The collection of urine over 24 h to measure free cortisol (UFC) is used to diagnose Cushing's syndrome. However, a complete collection of urine is not easy to achieve and the sampling is frequently inaccurate, so a 24 h collection for the determination of UFC excretion is used as a confirmatory rather than a screening test for Cushing's syndrome. Our objective was to evaluate a more convenient urine collection for screening patients suspected of Cushing's syndrome. We have studied the possibility of using night-time (from 2000 h to 0800 h) UFC excretion as a screening test for Cushing's syndrome ('overnight UFC'). Thirty patients with proven Cushing's syndrome were studied before treatment (21 cases of Cushing's disease, two cases of ectopic ACTH secretion, seven of adrenal adenoma). The results were compared to those from 150 control obese subjects. Overnight UFC, and creatininuria (UC), were evaluated at least once in the patients and in all subjects. The 24 h-UFC and UC were determined at least once in the patients and in 56 control subjects. The overnight UFC/UC ratio clearly separated all but one patient of the two groups: 194 +/- 386 vs 5.7 +/- 3.4 nmol/mumol (P < 0.0005) (ranges 16.2-2024 vs 0.6-17.4, Cushing's syndrome vs controls, respectively). The only patient with Cushing's syndrome who had an overnight UFC/UC ratio that overlapped with that of controls suffered from renal failure. The 24 h UFC/UC ratio of the patients differed from the ratio of controls: 184 +/- 365 vs 8.4 +/- 4.1 nmol/mumol (P < 0.0005) (ranges 14-1639 vs 1.5-21.2, respectively). Four patients with Cushing's syndrome had 24 h UFC/UC ratios lower than the upper control ratio. The overnight urinary sampling is a simple procedure compared to the 24 h urine collection performed in out-patients. For a sensitivity set at 100%, the specificities were 97% for the overnight UFC/UC ratio and 87% for the 24 h-UFC/UC ratio. This therefore appears to provide a good screening method for Cushing's syndrome.

Specific Determination of Urinary Free Cortisol by Solid-Phase Microparticle Extraction Capillary Electrophoresis with Fused Silica Capillaries

Measurement of 24-h urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for adrenal malfunctions, especially for Cushing syndrome (1)(2). The existing methodologies include immunoassays (3)(4)(5) and HPLC, with and without solid-phase extraction and derivatization (6)(7). Immunoassays for UFC are precise but overestimate the concentration because of antibodies’ cross-reactivity with various metabolites and with synthetic corticoids having a chemical configuration similar to that of cortisol (8). HPLCs, on the other hand, although very specific, require large volumes of mobile phase, as well as sample pretreatment. Capillary electrophoresis (CE) overcomes many of these problems through the use of an open-tubular format, thus avoiding interaction of analytes with the solid-phase resin. Earlier, we reported the feasibility of rapid UFC detection in solid-phase extraction CE with a neutral capillary (9). Using a neutral capillary, we could demonstrate a detection limit of 552 nmol/L for UFC in human urine. However, neutral capillaries have several disadvantages: Not only are they expensive, they also are unstable and lack a uniform capillary coating. This makes the neutral capillary unattractive for the development of a routine clinical laboratory assay. In the present study, we developed a solid-phase microparticle extraction (SPME) coupled micellar electrokinetic capillary chromatography (MEKC) with use of a fused-silica capillary as a …