Cortisol assays and diagnostic laboratory procedures in human biological fluids (original) (raw)
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Sample-pretreatment procedure for routine liquid chromatographic assay of serum cortisol
Talanta, 1986
A specific method for measuring concentrations of cortisol in serum, by preliminary isolation on a "minicolumn" followed by elution and determination by liquid chromatography, is described. This assay requires only 500 ~1 of serum. The limit of determination of cortisol was found to be 5 fig/l. The analytical recovery of cortisol added to serum ranged from 98 to 102%. The coefficients of variation ranged from 2.4 to 3.4% (within-day) and from 3.6 to 8.8% (day-today), depending on the cortisol concentration. The method compares well with a commonly used radioimmunological method.
Candidate definitive method for the determination of cortisol in human serum
Biological Mass Spectrometry, 1983
As a substantial part in the development of a reference material in clinical chemistry, a highly accurate and precise isotope dilution mass spectrometric method has been worked out for the determination of cortisol in human serum. The candidate definitive method consists of addition of 4-['4C]cortisol and, following equilibration, solvent extraction of cortisol and its internal standard from the serum matrix. After conversion into methoxime-trimethylsilyl derivatives, the extract is purified by gel chromatography. Measurements are made by combined capillary gas chromatography mass spectrometry. The ratio of peak heights at mlz 605 and 607 for each sample and calibration mixtures is used for quantification. To ensure maximum accuracy each set of calibration mixtures was composed from two stock solutions and closely bracketed the anticipated serum concentration. An analysis of variance, involving comparison of within-run and between-run variability, gave a total coefficient of variation (CV) of 0.38% for a serum pool containing 90.79 ng cortisol ml-' serum.
Therapeutic Drug Monitoring, 2007
Cortisol is an important adrenal steroid hormone involved in the regulation of metabolic homeostasis. A new liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) multiple reactant monitoring (MRM) procedure for the measurement of cortisol concentration in plasma ultrafiltrate, whole plasma, and urine was developed and validated. Plasma, plasma ultrafiltrate, or urine was extracted by ethyl acetate. The extract was subjected to liquid chromatography with an Inertsil ODS-3 column with an aqueous NH 4 Cl (1 mM, pH 9.0):methanol mobile phase. The presence of NH 4 Cl in the mobile phase induced the formation of [M+ 35 Cl]in the first quadrupole at m/z 397 and 409 for cortisol and 6a-methylprednisolone (internal standard), respectively. In the collision cell, the complex dissociated to the neutral parent and the chloride ion at m/z 35; the latter ion was used for quantification. The calibration curve was linear from 0.5 to 100 ng/mL. The lower limit of quantification was 0.50 ng/mL and the limit of detection was 0.25 ng/mL. For quality control samples prepared in water, the intrabatch assay precision was 5.6%, 9.6%, and 9.9% at 50, 10, and 1 ng/mL, respectively. The interbatch assay precision was 4.2%, 6.3%, and 7.5% at 50, 10, and 1 ng/mL, respectively. For measurement of endogenous cortisol in plasma and urine samples, the intra-assay and interassay precision was 10.8% and 4.8% for total plasma cortisol, 13.1% and 5.2% for free plasma cortisol, 10.9% and 13.1% for cortisol protein-binding free fraction, and 8.9% and 14.4% for urine cortisol, respectively. A simple procedure of ultrafiltration coupled with the highly sensitive LC-MS/MS quantification offered a rapid and reproducible assay for plasma free cortisol, which may be useful in the assessment of adrenal function in patients, especially critically ill patients with abnormal protein binding. It may also be useful for plasma and urinary cortisol measurements in pharmacodynamic studies of adrenocorticoid response.
Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine, 2013
Background: LC-MS/MS is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum cortisol. We have used this assay to investigate the effects of gender and exogenous steroid interference on the immunoassay measurement of serum cortisol. Methods: Zinc sulphate (40 mL) was added to 20 mL of sample. This was vortexed for 10 s followed by the addition of 100 mL of internal standard in methanol. Following mixing and centrifugation, 10 mL of sample was injected into an Acquity LC system coupled to a Quattro Premier tandem mass spectrometer. Serum samples (n ¼ 149) were analysed by LC-MS/MS and two commercial immunoassays. Results were then compared for all samples and for gender differences. A further set of serum samples (n ¼ 171) was analysed by the LC-MS/MS assay and a GC-MS assay. Results: Cortisol had a retention time of 0.98 min and the assay had an injection-to-injection time of 2.6 min per sample. Mean recovery was 99% and mean CV was 8%. The immunoassays gave comparisons of: Roche ¼ 1.23 Â LC-MS/MS À1.12 nmol/L and Abbott ¼ 0.94 Â LC-MS/MS þ 11.97. The comparison with GC-MS showed LC-MS/MS ¼ 1.11 Â GC-MS -22.90. Discussion: We have developed an LC-MS/MS assay for serum cortisol analysis that is suitable for routine clinical use and has been in use in our laboratory for 12 months. The availability of this assay will give more reliable results in patients receiving exogenous steroid therapy.
A Radioimmunoassay System for Human Serum Cortisol
Biochemistry Letters
Background: Cortisol (hydrocortisone) is the most potent glucocorticoid produced by the human adrenal cortex with about 55-69 µmol (20-25 mg) released daily, and the rate of release has a pronounced diurnal rythm. The plasma concentration is highest in the early morning around 06:00 (140-700 nmol/L), while the nadir is about midnight (~250 nmol/L). Cortisol acts through specific intracellular receptors and has effects in numerous physiologic systems, including immune function, glucosecounter regulation, vascular tone, substrate utilization and bone metabolism. For accurate detection of this low concentration of cortisol in human serum, Radioimmunoassay (RIA) is used; which is known as the most recognized sensitive microanalytical technique for the determination of the very low concentrations of awide range of substances of biological and medical interest. No other analytical method can reach the low detection limits of assayed analytes attained by RIA realted techniques.Applicability, simplicity , to operate and economy of RIA have paved the way for the last advancement in medical research during the past five decades. Materials & Methods: Highly purified Hydrocortisone-21-hemisuccinate and Bovine Serum Albumin used to prepare immunogen (cortisol-21-hemisuccinate: BSA), which is immunized into rabbits to produce cortisol polyclonal antibodies, Na 125 I (370 MBq/100µl) carrier and reductant free is the radioactive material used to prepare the tracer (Institute of Isotopes Co.,Hungary). All above materials in addition to prepared standards of cortisol used to prepare radioimmunoassay system to assess cortisol level. Objective: This work was carried to assess and development the technical as well as the economic feasibility of establishing local radioimmunoassay for human serum cortisol. Results: immunogen obtained was of 17 cortisol molecule : 1 BSA molecule ,the dilution of the antibody was chosen to be 1/8000 according to the best displacement percent (76.5%) obtained at this dilution from the the 5 th bleed of the 1 st rabbit in group A and the prepared tracer was of radiochemical yield percent (58.5%), radiochemical purity (96%)and specific activity (120 µCi/µg). Conclusion: This RIA-technique method for detection of cortisol level in human serum is simple, accurate and can be used as a decisive diagnostic tool for adrenal status.
Measuring cortisol in human psychobiological studies
Physiology & Behavior, 2007
The steroid cortisol is an extensively studied and important variable in developmental and other behavioral studies. Cortisol has been assayed by various methods using a range of substrates including blood, saliva, and urine. Cortisol in blood exists in two forms. While most is bound to carrier proteins, a small portion exists in a soluble free form. The informed choice of cortisol fraction and measurement method is critical for research. Such choices should be influenced by understanding the characteristics of the various cortisol fractions, along with their binding proteins' biological functions and relationship to the hypothalamic-pituitary-adrenal (HPA) axis. The goal of this paper is to familiarize researchers with key points for evaluating the choice of total and free cortisol in research as well reviewing various options for measuring free cortisol. These points are raised with special emphasis on their significance during pregnancy and the post-partum. Such information may prove useful in informing researcher's cortisol-related protocols and in the interpretation of cortisol data.
A Comparison of Three Widely Used Immunoassay Systems in Cortisol Measurement
Clinical laboratory, 2015
Interassay variability is one of the challenging issues of routine clinical laboratory practice. Commercial plasma cortisol immunoassays are also subject to this issue. In this study, we intended to evaluate the interchangeability of cortisol results of three widely used immunoassay systems. The cortisol values of 150 serum samples measured by three immunoassay systems, Beckman Coulter DXI 800, Roche Modular E170, and Siemens Immulite 2500, were compared. A degree of proportional biases was observed between all three methods; DXI 800 showed the worst biases with the other two systems (slope values 0.67 and 0.77 with E170 and Immulite 2500, respectively). DXI 800 showed poor agreement with other methods (CCC: 0.83 and 0.87, respectively). There was a moderate agreement between E170 and Immulite 2500 (CCC: 0.92). All three methods showed a degree of variability among themselves. DXI 800 results were not interchangeable with the other two systems.
2014
Background: The purpose of this study was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) developed for measuring free cortisol in human saliva and total cortisol concentration in diluted human serum, for its applicability in measuring cortisol concentration in pig saliva. Collection of saliva is less stressful than e.g. blood sampling, and is a non-invasive method. Findings: Saliva was collected by allowing sows to chew on cotton swabs held by forceps. Thereafter, the swabs were centrifuged to retrieve the saliva. The ELISA was performed according to instructions provided by the manufacturer. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV was below 10% and inter-assay CV below 15% for samples of high, medium and low cortisol concentrations. The mean recovery was 117% and the linearity and parallelism showed an r 2 -value of 0.994 and 0.993, respectively. For biological assessment of induced social stress, two saliva samples were collected in the morning from 6 primiparous and 21 multiparous sows. One sample was collected when the sows were individually housed in a farrowing pen and a second sample was collected when the sows were group housed. The primiparous sows had a significant higher cortisol concentration compared to the multiparous sows when group housed. Conclusion: The results obtained in this validation study indicate that the ELISA is suitable for measuring cortisol concentration in porcine saliva.
LaboratoriumsMedizin, 2000
A commercially available radioimmunoassay developed for the determination of coriisol in human plasma and serum was used to measure salivary cortisol. Clearly elevated levels for spiked saliva (3-10 fold: P/B regression: y = 1.9x + 6.4; r = 0.983) were found. We suggest that differing biochemical conditions in the standard material (plasma) and the sample matrix (saliva) were responsible for the elevated measurements. For this reason we adapted the assay by replacing the original plasma standards by in-house saliva standards. In addition the concentrations of the antibodies and the tracer were decreased and the time for incubation was increased. After adaptation, cortisol could be measured in excellent accordance with the amounts added to the saliva samples (P/B regression: y = 1.03x-0.01; r = 0.998). The 50% intercept was 8 ng/ml. We measured intra-assay variation coefficients of 7.9% (1 ng/ml) and 6.1% (10 ng/ml), respectively. Inter-assay variation coefficients were 11.2% (1 ng/ml) and 9.6% (10 ng/ml), respectively. Saliva samples of healthy volunteers measured after adaptation were in good accordance with data from in-house assays reported in the literature. There was no disturbing influence of either dental care or food intake on the reproducibility of salivary cortisol levels by this specific assay (p=0.76 and p>0.05, respectively). After adaptation, the assay is therefore a reliable tool for salivary cortisol measurement. •
Asian Journal of Pharmaceutical and Clinical Research
Objective: The objective of this study is to develop and validate a simple, sensitive, specific, and rapid assay for quantification of clinically relevant cortisol level in human plasma and urine samples.Methods: Ultra performance liquid chromatographic-tandem mass spectrometric (UPLC-MS/MS) analysis was performed on Atlantis dC18 column (2.1×100 mm, 3 μm) with a mobile phase consisting of acetonitrile and 2 mM ammonium acetate (50:50, v: v) that was delivered at a flow rate of 0.3 ml/min. Tolperisone (2 ng) was used as an internal standard (IS). Biological samples were extracted with a mixture of hexane and methyl tert-butyl ether (8:2, v: v). The eluents were monitored using electrospray ionization in the positive ion mode with transition mass to charge ratio set at 363.1 → 121.0 and 246.0 → 97.9 for cortisol and IS, respectively. The method was validated according to international guidelines.Results: Retention times of cortisol and IS were about 1.4 and 2.3, respectively. Relatio...