Simultaneous determination of urinary cortisol, cortisone and corticosterone in parachutists, depressed patients and healthy controls in view of biomedical and pharmacokinetic studies (original) (raw)
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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.
Journal of Pharmaceutical and Biomedical Analysis, 2014
Several pathological conditions can be related to the alteration of the urinary levels of cortisol (F) and its metabolites. The determination of each of them in the free and free plus conjugated form can provide a deeper insight into the impaired activity of the cortisol metabolism enzymes, thus improving the diagnosis protocol currently based only on the determination of total amount of urinary cortisol metabolites. In that view, an LC-MS/MS method for the determination of the free and total amount of urinary F, cortisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) was thus developed and validated. Deconjugation of glucocorticoids was carried out by enzymatic hydrolysis. Analytes were extracted by solid phase extraction, separated by liquid chromatography and analyzed via electro-spray ionization (negative ion mode) triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable isotope-labeled internal standard. Baseline separation for all compounds, in particular the two stereoisomers A-THF and THF, was obtained. Matrix effects, not reported so far, were observed and minimized for the determination of urinary free E and THE. Validated range was 0.5-1000 ng/mL for A-THF and THF, 5-800 ng/mL for E and THE and 1-1000 ng/mL for F, with R2 values greater than 0.9981. The LOD and LOQ of the described method ranged from 0.1 to 3.0 ng/mL, while the extraction recoveries resulted close to 100% for all the glucocorticoids determined. Precision and accuracy were well within ±10%. As suggested by the results obtained in the preliminary study on polycystic ovary syndrome (PCOS) urine samples, the method can be used to support clinical diagnosis of pathologies related to cortisol metabolism. In fact, levels of free and total glucocorticoids in control subjects were in agreement with previously reported data, as well as free and total A-THF/THF ratio in PCOS patients. Conversely, in the latter free F/E and A-THF + THF/THE ratios were lower than in control subjects (P<0.01), suggesting a possible alteration of 11b-HSD1 and 11b-HSD2 activity, to be further investigated.
Cortisol assays and diagnostic laboratory procedures in human biological fluids
Clinical Biochemistry, 2009
The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.
Determination of urinary free cortisol by HPLC
2000
We here report a reversed-phase HPLC method for the determination of free cortisol in human urine, using methylprednisolone as the internal standard. Before chromatography, samples were extracted with a C18 solid-phase extraction column and the steroids were separated on a LiChrospher 100 C18 column with a mobile phase of methanol/acetonitrile/water (43/3/54 by vol). Linearity, precision, and accuracy of the method
Annals of Clinical Biochemistry, 2019
Background The analysis of steroids in biological matrices is challenging. One can apply immunoassay as well as gas and liquid chromatography with various types of detection, depending on the available equipment and the experience of the analyst. The question is how the methods are interchangeable between themselves. Doubts were reported having compared immunoassays and chromatography-mass spectrometry, but there are scarce data on chromatographic methods with detection types other than mass spectrometry. Methods Here, we present the detailed comparison of two liquid chromatographic methods for the determination of free urinary cortisol and cortisone: one with fluorescence detection (high-performance liquid chromatography [HPLC-FLD]) and the other with tandem mass spectrometry (HPLC-MS/MS). The comparison was made with 199 human urine samples. The data analysis included Passing–Bablok and Deming regression, Bland–Altman test, Wilcoxon test, mountain plot and Lin’s concordance correl...
Multi-matrix assay of cortisol, cortisone and corticosterone using a combined MEPS-HPLC procedure
Journal of Pharmaceutical and Biomedical Analysis, 2014
The development and validation of a bioanalytical assay for the simultaneous determination of cortisol, cortisone and corticosterone levels in several matrices, such as saliva, plasma, blood and urine samples have been described. The method is based on a rapid test which combines a microextraction by packed sorbent procedure and liquid chromatography-diode array technique. Chromatographic separation of the analytes (cortisol, cortisone and corticosterone) and the internal standard (methylprednisolone) was achieved in less than 10 min on a reversed-phase pentafluorophenyl column using a mobile phase composed of phosphate buffer and acetonitrile. The assay was performed after an innovative microextraction procedure by means of C8 sorbent which guaranteed good clean-up of the matrices and satisfactory extraction yield of the analytes. Moreover, the method gave linear results over a range of 5-100 ng mL −1 and showed good selectivity and precision. This method was successfully applied for quantifying corticosteroids in specific matrices derived from some healthy volunteers in comparison to two socially diversified groups, namely former heroin addicts undergoing opioid replacement therapy and poly-drug abusers.
Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2005
A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a solid-phase extraction (SPE) column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post-exercise versus pre exercise condition in cyclists (F: 136 ± 93 nmol/l versus 67 ± 50 nmol/l (p < 0.001); E: 797 ± 400 nmol/l versus 408 ± 252 nmol/l (p < 0.001)). The F/E ratio was 0.18 ± 0.11 versus 0.16 ± 0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11 ± 0.07) and cyclist individuals at rest (p < 0.05). . was mistakenly published in uncorrected form in Volume 824, Issues 1-2, September 2005. It can be viewed by visiting the following link: http://dx.
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.