High-performance liquid chromatography methods for the analysis of endogenous cortisol and cortisone in human urine: comparison of mass spectrometry and fluorescence detection (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.
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...
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.
Analytical and Bioanalytical Chemistry, 2014
Blood cortisol level is routinely analysed in laboratory medicine, but the immunoassays in widespread use have the disadvantage of cross-reactivity with some commonly used steroid drugs. Mass spectrometry has become a method of increasing importance for cortisol estimation. However, current methods do not offer the option of accurate mass identification. Our objective was to develop a mass spectrometry method to analyse salivary, serum total, and serum free cortisol via accurate mass identification. The analysis was performed on a Bruker micrOTOF high-resolution mass spectrometer. Sample preparation involved protein precipitation, serum ultrafiltration, and solid-phase extraction. Limit of quantification was 12.5 nmol L −1 for total cortisol, 440 pmol L −1 for serum ultrafiltrate, and 600 pmol L −1 for saliva. Average intra-assay variation was 4.7 %, and interassay variation was 6.6 %. Mass accuracy was <2.5 ppm. Serum total cortisol levels were in the range 35.6-1088 nmol L −1 , and serum free cortisol levels were in the range 0.5-12.4 nmol L −1. Salivary cortisol levels were in the range 0.7-10.4 nmol L −1. Mass accuracy was equal to or below 2.5 ppm, resulting in a mass error less than 1 mDa and thus providing high specificity. We did not observe any interference with routinely used steroidal drugs. The method is capable of specific cortisol quantification in different matrices on the basis of accurate mass identification.
Journal of Endocrinological Investigation, 2019
Purpose One of the best indicators of adrenal gland dysfunction is the level of free cortisol measured in the 24-h urine (UFC) which faithfully reflects the level of biologically active serum cortisol not subjected to circadian variations. Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a sensitive, accurate and precise method recently available in routine laboratories that could remedy interference problems of immunoassays. Methods In this study, a literature reference range for UFC measured by LC-MS-MS was verified, and UFC values measured by LC-MS-MS and immunoassay were compared. Immunometric UFC measurement was performed by ACCESS COR-TISOL assay without preliminary extraction, using Beckman Coulter UniCel DxI 600 highly automated platform. Liquid chromatography-tandem mass spectrometry UFC measurement was performed by a home-made validated method using cortisol-D 4 as internal standard with preliminary deproteinization of urinary samples by centrifugal filter and injection on reverse-phase column. Cortisol was analyzed in positive ion mode with an ESI interface. Results The reference interval from literature (11-70 μg/day) was confirmed by results obtained for healthy study group. Comparison study of the two methods highlighted a constant and proportional systematic error with a general tendency to overestimate results for the in-use method. Conclusions In conclusion, the direct immunometric method overestimates UFC results with respect to liquid chromatography-tandem mass spectrometry which represents the reference method. The literature reference range 11-70 μg/day was confirmed and can be adopted by our lab that will shift all UFC tests performed in routine to the mass spectrometry-based method, satisfying clinicians' request.
Steroids, 1996
A method for the profiling of several important 3-oxo-4-ene urinary steroids is reported. The methodology is combined gas chromatography/mass spectrometry (GC/MS) utilizing stable isotope-labeled internal standards'. The following standards were obtained or easily ). For 18-hydroxycortisol in particular, the excretions were much higher for males than fiw Jemales. We found that the larger part of urinary cortisol and cortisone is not free but is released Jrom conjugation by enzymes present in snail digestive juice. Using a pooled urine sample from an equal number ~[ male and female subjects, we found that for cortisol 29% was excreted free, 28% as glucuronide and 43% as other conjugates (probably sulfates). For cortisone 41% was free, 45% ~-glucuronide and 14% as other conjugates. Relatively little (3-8%) of the hydroxylated cortisols were excreted conjugated.
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: 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.
Clinical Chemistry and Laboratory Medicine, 2000
Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans.