Extraction Of Cortisol Research Articles

A-046 Quantification of Corticosteroids and Androgens in Serum, utilizing Waters MassTrak™ Steroid Serum Sets 2 & Sets 3 for Clinical Research

Abstract Background Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting. The availability of lyophilized calibrators and QCs reduces sample preparation time, aids in method harmonization, and assists with metrological traceability in accordance with ISO15189:2022, when used alongside an analytically selective chromatographic method. Methods A quantitative clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals & QCs and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17- OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol and 21-deoxycortisol from human serum samples. Chromatographic separation was performed on an Waters ACQUITY™ UPLC™ I Class Plus (FL-I) System, using an ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer. Results Accuracy (±6%) and precision (±10%) have been confirmed through comparison to External Quality Assurance (EQA) LC-MS/MS schemes, panels and QC material for all seven steroid hormones. All analytes were assessed at the low, medium and high concentrations for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Deming and Linear regression analysis were performed. No statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3. The clinical research method was shown to be linear for all analytes over the calibration ranges specified, furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r2) > 0.999 for all analytes. Analytical sensitivity using Signal:Noise (S/N) of the low calibrator (Calibrator 1) of each set, was >10:1 for each analyte across several analytical runs. Conclusions This evaluation has demonstrated that the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17- OHP, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum using Waters I-Class Xevo TQ-S micro system. For Research Use Only. Not for Use in Diagnostic Procedures.

Salting-Out Assisted Liquid-Liquid Extraction for UPLC-MS/MS Determination of Thyroxine and Steroid Hormones in Human Serum and Fish Plasma

Measuring the level of steroid and thyroxine hormones is key to understanding organism health conditions. Liquid chromatography coupled with tandem mass spectrometry has become the method of choice for such hormone analyses in clinical laboratories. Detection of hormones at low levels typically requires a time-consuming sample preparation, such as liquid-liquid extraction followed by solvent evaporation and re-solubilization of the sample extract. Instead, we applied salting-out assisted liquid-liquid extraction (SALLE) for the extraction of thyroxine, testosterone, cortisone, and cortisol from human serum and fish plasma samples. SALLE allowed direct injection of sample extracts. Sodium chloride and ammonium sulfate were evaluated as salting-out reagents together with four different organic solvents. High extraction recovery and reduced matrix interference were achieved by using ammonium sulfate together with 10% methanol in acetonitrile. Limits of quantification were in the range of 0.1–0.2 ng/mL and signal responses were linear (R2 > 0.997) up to at least 100 ng/mL for all hormones. The method was applied for hormone measurements in fish plasma. In conclusion, SALLE combines the simplicity of crude protein precipitation with the high analyte enrichment of a liquid-liquid extraction. Here we have presented it as a novel sample preparation method for clinical laboratories where mass spectrometry is utilized in the field of endocrinology.

Impact of hair type, hair sample weight, external hair exposures, and race on cumulative hair cortisol

The biomarker cortisol assesses the impact of biopsychosocial stressors that activate the stress response system. Hair has emerged as a valid and non-invasive means of gauging cumulative cortisol deposited over month-long periods of time. Established protocols for the extraction of hair cortisol are being validated and refined in humans, yet methodological information about hair characteristics on cortisol remains limited. In addition to external hair exposures (e.g. dye, time spent outside), we examined hair categorization or type (e.g. kinky, straight) by extending a hair typing methodology for scientific use that is currently popular among hair care professionals. We then examined the interaction between hair type and race on cortisol levels with a hair questionnaire. Three studies were pooled to investigate how sample weight, hair type, race, heat exposures, and hair treatments impacted cumulative hair cortisol concentrations. Study 1 consisted of Adult Kenyan Medical Workers (N = 44); Study 2 Mexican and Mexican Americans (N = 106); and Study 3 American Youth (N = 107). We found significantly higher cortisol in 5 mg of hair when compared to larger sample weights, and higher cortisol in those who spent more time outdoors. Cortisol concentrations differed between racial groups and varied by hair type; moreover, there were directional differences in cumulative cortisol from straighter to curlier hair types which depended on racial group. In addition to demonstrating the impact of relatively novel control factors like hair sample weight, outdoor exposure, and hair type, the present study illustrates the importance of disentangling hair type and race to understand variability in cumulative hair cortisol. These influences should be included in future studies that measure hair cortisol.

Deep eutectic solvent extraction of cortisol and cortisone from human saliva followed by LC-UV analysis

A novel extraction method was developed for the determination of cortisol and cortisone. In this study, we prepared a hydrophobic deep eutectic solvent (DES) by mixing trioctylmethylammonium chloride and pentafluorophenol as a hydrogen bond acceptor and a hydrogen bond donor, respectively, for use as the extraction solvent. The extraction of cortisol and cortisone was performed by adding a small volume of the DES to the aqueous sample. After centrifugation, the resulting sedimented DES phase was injected into a reversed-phase liquid chroamtography column, and the analytes were detected with an ultraviolet detector at 254 nm. Under the optimized extraction conditions, the enrichment factors of cortisol and cortisone were 9.3 and 8.5, respectively. Furthermore, the linear dynamic ranges were established over a concentration range of 10–200 pmol mL−1 (r2 > 0.9992), and the limits of detection of cortisol and cortisone were found to be 2.1 and 1.8 pmol mL−1, respectively. The applicability of this method was evaluated by analyzing the cortisol and cortisone contents of human saliva samples.

A Modified Protocol for Cortisol Analysis in Zebrafish (Danio rerio), Individual Embryos, and Larvae.

A modified protocol for the extraction and analysis of cortisol in individual zebrafish, Danio rerio, embryo, and larva samples has been developed and evaluated. Recovery efficiency of the method was high, specifically calculated at 93.8% ± 6.5%. Dilution tests showed high parallelism, while increasing the number of individuals used in each extraction sample resulted in a linear, although slightly underestimated, increase of cortisol yield. Results of cortisol content from 0, 3, and 5 days postfertilization (dpf) fish using the proposed protocol were within the range of most published studies analyzing cortisol in pooled samples of 10-30 individuals. Moreover, 5 dpf larvae had significantly higher cortisol levels than embryos, a pattern commonly observed in literature. Finally, application of an osmotic stress in 5 dpf larvae led to a statistically significant increase in cortisol content.

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 ± 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 °C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 ± 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 °C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

Ionic liquid dispersive liquid-liquid microextraction combined with LC-UV-Vis for the fast and simultaneous determination of cortisone and cortisol in human saliva samples

Ionic liquid dispersive liquid–liquid microextraction (IL-DLLME) was used for the first time for the extraction and preconcentration of cortisone and cortisol from human saliva samples. This approach allows the determination of both compounds by liquid chromatography (LC) with ultra-violet/visible (UV/Vis) detection at physiological levels. The IL 1-butyl-3-methylimidazolium hexafluorophosphate [C4MIM][PF6] and methanol (MeOH) were used as extraction and dispersive solvents, respectively. The parameters that affect extraction (ionic strength, pH, amount of IL, volume of dispersive solvent, vortex and centrifugation time) were optimized. Limits of detection were 0.11 (cortisone) and 0.16 μg L−1 (cortisol) and enrichment factors of 5.0 (cortisol) and 6.3 (cortisone) were achieved. Recoveries from 83.3 ± 1.6 to 115.8 ± 3.0% were obtained for the extraction of the target analytes in spiked human saliva samples. Intraday and interday precision, expressed as relative standard deviation, was below 4.2 and 7.8% for cortisone and cortisol, respectively. The method was successfully applied to the determination of cortisone and cortisol in saliva from healthy volunteers (n = 9) in the morning and in the evening. The method is simple, faster, and more environmentally friendly than the current ones.

Assessment of hair cortisol in euthyroid, hypothyroid, and subclinical hypothyroid subjects.

Hypothyroidism is associated with an increase in serum cortisol level while the long-term activity of hypothalamic-pituitary-adrenal (HPA) axis in hypothyroid, and subclinical hypothyroid (SCH) subjects has not been studied. This study aimed to assess the hair cortisol levels as a long-term activity of HPA axis in hypothyroid, SCH and a group of healthy adult subjects. Also, it aimed to examine the correlation of hair cortisol levels with hypothalamic-pituitary-thyroid (HPT) axis and anthropometric measures. We prospectively evaluated a group of normal, SCH and hypothyroid subjects. Serum TSH, FT4, and FT3 were measured as a component of the HPT axis. Hair samples were collected, prepared, followed by extraction of hair cortisol and measurement in pg/mg of hair. Hair cortisol levels were compared in normal, SCH and hypothyroid groups and correlated with HPT axis and anthropometric data. A total of 65 healthy volunteers were analyzed, and the mean hair cortisol level was reported to be 17.38 pg/mg of hair. Hair cortisol level was slightly higher in the SCH subjects, 18.19 pg/mg of hair; however the difference was not significant. Compared to the euthyroid subject, a significantly higher hair cortisol level was recorded in the hypothyroid subjects, 24.17 pg/mg hair, p < .05. Hair cortisol was significantly and positively associated with each of the serum TSH, age, weight and BMI (p < .05). Overt hypothyroidism but not SCH is significantly associated with higher hair cortisol levels compared to normal subjects, and a significant relation between hair cortisol with HPT axis was found. Also, weight and BMI were positively correlated with hair cortisol level.

A novel method for assessing chronic cortisol concentrations in dogs using the nail as a source

Cortisol, a glucocorticoid secreted in response to stress, is used to assess adrenal function and mental health in clinical settings. Current methods assess cortisol sources that reflect short-term secretion that can vary with current stress state. Here, we present a novel method for the extraction and quantification of cortisol from the dog nail using solid phase extraction coupled to enzyme-linked immunosorbent assay. Validation experiments demonstrated accuracy (r = 0.836, P < 0.001) precision (15.1% coefficients of variation), and repeatability (14.4% coefficients of variation) with this method. Furthermore, nail cortisol concentrations were positively correlated to an established hair cortisol method (r = 0.736, P < 0.001). Nail cortisol concentrations did not differ with dog sex, breed, age, or weights; however, sample size limitations may preclude statistical significance. Nail cortisol may provide information on cortisol secretion integrated over the time corresponding to nail growth and may be useful as a tool for diagnosing stress and adrenal disorders in dogs.

Technical approaches to a cortisol assay in hair for retrospective stress evaluation

The traditional assessment of the intensity of stress effects includes measurements of the content of the stress hormone cortisol in biological fluids, such as blood and less frequently, saliva or urine. However, various factors that influence the biosynthesis and/or metabolism of cortisol and the importance of measurement of cortisol over a large period of time make group studies and interpretation of data difficult using this biological material. Moreover, detection of the level of cortisol at the time of sampling of blood or saliva does not reflect the total stress load to which the patient was subjected in the period preceding the study. Recently, a quantitative assay of cortisol in hair was considered as an approach to the evaluation of the long-term (months) period of system stress load. Here, we adapted an assay method for cortisol in human hair. The method is based on methanol extraction of cortisol from a hair sample, methanol evaporation, and dilution of the extracted substances in phosphate-buffered saline followed by the enzyme-linked immune-sorbent cortisol assay. We also applied this approach to assessment of the corticosterone level in rodent hair, which may be used for comparative/translational studies.

Extraction and Analysis of Cortisol from Human and Monkey Hair

Extraction and Analysis of Cortisol from Human and Monkey Hair

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT "point samples" obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months. In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair's protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Evaluation of Two Techniques for Extraction of Cortisol from Human Saliva

The aim of the study was to compare two kinds of extraction of cortisol from human saliva. The first was made with dichloromethane and the second by solid-phase extraction. Saliva was sampled in the evening into plastic tubes then cortisol was extracted and analyzed by LC with detection at 240 nm. The limit of detection was 1 ng mL−1 for extraction with dichloromethane and 3 ng mL−1 for SPE extraction. Both methods of extraction were applied to the analysis of saliva samples without any interference peaks.

Measurement of cortisol in small quantities of saliva.

The determination of cortisol in saliva has become popular for human research on stress reactions (1)(2)(3)(4)(5). Depending on the sensitivity and reliability of the assays used, the required sample volume varies between 0.025 and 2 mL of saliva (6)(7)(8). Infants and toddlers, however, often produce only small amounts of saliva and are usually sampled by swabbing the mouth with cotton dental rolls (5) or commercial cotton swabs (Salivette; Sarstedt Inc.) (9), or by pipettes or alternative devices that aspirate saliva from the floor of the mouth (10)(11)(12)(13). Cotton rolls must either be centrifuged to obtain saliva (9) or be placed in the barrel of a syringe (needleless), from which the saliva is expressed into a vial by compression of the plunger (5). With these procedures, saliva remaining in the swabs is thus lost for analysis. When we tested seven different types of cotton rolls, we found that, depending on the individual type, 135–450 μL of saliva could not be centrifuged from the rolls. Oral stimulants (such as presweetened Kool-Aid crystals) can increase saliva production, but they affect the concentration of cortisol (14). Finally, in the case of Salivettes, the material covering the cotton swab is hard and makes sampling unpleasant. In this report, we present a new method that uses soft cotton swabs without hard covering material and solvent extraction of cortisol from saliva in the cotton. Saliva was collected from volunteers in the laboratory and from infants and toddlers participating in studies on cortisol and behavior. Volunteers and the parents of the infants gave informed consent. These studies had been approved by the Medical Ethical Committee of the University Medical Center Utrecht. After collection, either direct or with use of cotton …

Clean-up and re-use of kieselguhr (Extrelut) for liquid–liquid extraction of urinary cortisol

Cortisol was isolated from human urine using kieselguhr (Extrelut)-filled columns. After use, Extrelut was cleaned-up once with distilled water and twice with ethanol. Before re-use, the cleaned-up kieselguhr was dried for 24 h by a warm air stream. The comparison of cortisol recovery from human urine and HPLC chromatograms of urinary extracts show that Extrelut can be repeatedly used for liquid–liquid extraction of urinary cortisol.

Transuteroplacental metabolism of cortisol and cortisone during mid- and late gestation in the baboon.

We measured uterine extraction (i.e. metabolism) and transuteroplacental interconversion of cortisol (F) and cortisone (E) to determine whether metabolism across the uterus changes during pregnancy and contributes to the MCR of these corticosteroids. On day 100 (n = 4) or 170 (n = 3) of pregnancy (term = day 184), baboons (Papio anubis; 14-18 kg) were sedated with ketamine, and a constant infusion (0.38 ml/min) of 8-12 microCi [3H]F and 9-15 microCi [14C]E in 80 ml 0.9% NaCl-1% ethanol was initiated (time zero) via a maternal antecubital vein. At 60 min, animals were laparotomized, and at 70, 80, and 90 min, blood samples were obtained from right and left uterine veins and from a maternal saphenous vein. At 95 min, a transverse incision was made in the uterus, the fetus was isolated, and blood samples were obtained from the umbilical vein and artery. The cord was then clamped, and the fetus was delivered. Radio-labeled F and E were extracted from serum and purified by sequential paper chromatography, and metabolic parameters were calculated. Endogenous F and E levels were determined by RIA. In the mother, the percent conversions of E to F at midgestation (mean +/- SE; 72 +/- 4) and late gestation (65 +/- 3) were similar and exceeded (P less than 0.01) respective values for oxidation of F to E (51 +/- 7 and 46 +/- 7, respectively), indicating that maternal corticosteroid metabolism favors F formation and is unchanged during the second half of gestation. In contrast, corticosteroid metabolism across the uterus and placenta (transuteroplacental) was altered during pregnancy. At midgestation, transuteroplacental conversion of E to F (37 +/- 9) exceeded (P less than 0.05) the reverse reaction (18 +/- 3), whereas oxidation of F to E at term (28 +/- 4) was 7-fold greater (P less than 0.05) than reduction of E to F (4 +/- 1). At midgestation, essentially all of the F and E in umbilical vein was derived from maternal F. This contrasts with that observed in near-term baboons in which only 41 +/- 9% of the F and 64 +/- 8% of the E entering the fetal circulation was of maternal origin. As a result of uterine, placental, and fetal metabolism, 30% of the F and 15% of the E in maternal circulation were extracted by the uterus at both mid- and late gestation. We conclude that transuteroplacental corticosteroid metabolism changes from reduction at midgestation to oxidation at term.(ABSTRACT TRUNCATED AT 400 WORDS)

High-performance liquid chromatographic (HPLC) separation and quantitation of endogenous glucocorticoids after solid-phase extraction from plasma.

This study describes a method for the extraction and simultaneous measurement of cortisone, cortisol and corticosterone using dexamethasone as an internal standard. Solid-phase extraction of plasma steroids with C18 columns allows the samples to be extracted, washed and concentrated in a single step with minimal sample handling and without the use of large volumes of organic solvents. HPLC separation of the steroids is accomplished within 10 min and the individual steroid peaks are quantitated by UV detection at 239 nm. This assay was examined for linearity, extraction efficiency, precision and potential interference by commonly used drugs. Plasma values of glucocorticoids are reported for samples obtained from human subjects as well as from rats. HPLC was also compared to RIA for the determination of plasma levels of corticosterone in the rat. Solid-phase extraction and assay by HPLC provides a rapid and specific method for the simultaneous determination of plasma glucocorticoids.

Solid-state extraction of cortisol from plasma or serum for liquid chromatography.

Solid-state extraction of plasma or serum offers significant advantages over traditional liquid-liquid methods in cortisol determinations by liquid chromatography (HPLC). Sample preparation is facilitated and HPLC column contamination is minimized. Extract yield from the solid-state columns is enhanced by sample pretreatment with methanol. Cortisol values by solid-state extraction/HPLC correlate well (r = 0.92) with those from a commercial radioimmunoassay procedure.

Protein-Bound Corticosteroid in Human Serum Is Selectively Transported into Rat Brain and Liver in Vivo*

It is generally regarded that only free corticosteroid is available for entry into tissues in vivo, although some studies have suggested that albumin-bound corticosteroid is available for liver uptake. However, recent studies suggest that owing to favorable kinetic relationships among tissue capillary transit times and hormone dissociation rates from plasma proteins, free plus albumin-bound hormone may be available to peripheral tissues. Moreover, globulin-bound hormone may enter the liver under normal conditions and be available to peripheral tissues under pathological circumstances. The present studies measure the free and noncorticosteroid-binding globulin (non-CBG)-bound corticosteroid fractions in vitro and compare these measurements to the fraction of corticosteroid in human sera that is available for entry into rat brain and rat liver in vivo. Corticosterone was used as the model corticosteroid, since this compound binds to CBG with the same affinity as does cortisol, and the brain extraction of corticosterone is more readily measured in vivo than is the brain extraction of cortisol. Serum was obtained from 51 human subjects and included samples from newborns, pregnant mothers, normal subjects, and patients with either cirrhosis or renal failure. Serum levels of CBG varied more than 6-fold, and both the free and the non-CBG-bound fractions were generally inversely related to the CBG concentration. The fraction of corticosterone available for entry into the brain was much greater than the free fraction, but was not significantly different from the non-CBG-bound moiety. Moreover, the rate of corticosterone dissociation from CBG (t 1/2 = 27 +/- 1 sec at 22 C) was not increased in any of the serum samples studied. The fraction of corticosterone available for uptake by the liver was up to 3-fold greater than that of the non-CBG fraction and had no relationship with the serum concentration of CBG. These studies indicate that albumin-bound, but not globulin-bound, corticosteroid is available for entry into a peripheral tissue such as the brain. However, globulin-bound corticosteroid is readily transported into the liver. It is suggested that the routine measurements of the non-CBG-bound corticosteroid provide a more accurate index of the corticosteroid available to peripheral tissues in vivo than does the measurement of free corticosteroid.

Candidate definitive method for the determination of cortisol in human serum.

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-[14C]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 m/z 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-1 serum.