Several pathological conditions can be related to the alteration of the urinary levels of cortisol (F) and itsmetabolites. The determination of each of them in the free and free plus conjugated form can provide adeeper insight into the impaired activity of the cortisol metabolism enzymes, thus improving the diag-nosis 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, cor-tisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) wasthus developed and validated. Deconjugation of glucocorticoids was carried out by enzymatic hydroly-sis. Analytes were extracted by solid phase extraction, separated by liquid chromatography and analyzedvia electro-spray ionization (negative ion mode) triple-quadrupole mass spectrometry in the selectedreaction monitoring mode using a stable isotope-labeled internal standard.Baseline separation for all compounds, in particular the two stereoisomers A-THF and THF, wasobtained. Matrix effects, not reported so far, were observed and minimized for the determination ofurinary free E and THE. Validated range was 0.5–1000 ng/mL for A-THF and THF, 5–800 ng/mL for E andTHE and 1–1000 ng/mL for F, with R2values greater than 0.9981. The LOD and LOQ of the describedmethod ranged from 0.1 to 3.0 ng/mL, while the extraction recoveries resulted close to 100% for all theglucocorticoids determined. Precision and accuracy were well within ±10%. As suggested by the resultsobtained in the preliminary study on polycystic ovary syndrome (PCOS) urine samples, the method canbe used to support clinical diagnosis of pathologies related to cortisol metabolism. In fact, levels of freeand total glucocorticoids in control subjects were in agreement with previously reported data, as well asfree and total A-THF/THF ratio in PCOS patients. Conversely, in the latter free F/E and A-THF + THF/THEratios were lower than in control subjects (P < 0.01), suggesting a possible alteration of 11-HSD1 and11-HSD2 activity, to be further investigated.

A comprehensive study for the validation of a LC/MS/MS method for the determination of free and total forms of urinary cortisol and its metabolites

CUZZOLA, ANGELA ROSA;PETRI, ANTONELLA
2014

Abstract

Several pathological conditions can be related to the alteration of the urinary levels of cortisol (F) and itsmetabolites. The determination of each of them in the free and free plus conjugated form can provide adeeper insight into the impaired activity of the cortisol metabolism enzymes, thus improving the diag-nosis 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, cor-tisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) wasthus developed and validated. Deconjugation of glucocorticoids was carried out by enzymatic hydroly-sis. Analytes were extracted by solid phase extraction, separated by liquid chromatography and analyzedvia electro-spray ionization (negative ion mode) triple-quadrupole mass spectrometry in the selectedreaction monitoring mode using a stable isotope-labeled internal standard.Baseline separation for all compounds, in particular the two stereoisomers A-THF and THF, wasobtained. Matrix effects, not reported so far, were observed and minimized for the determination ofurinary free E and THE. Validated range was 0.5–1000 ng/mL for A-THF and THF, 5–800 ng/mL for E andTHE and 1–1000 ng/mL for F, with R2values greater than 0.9981. The LOD and LOQ of the describedmethod ranged from 0.1 to 3.0 ng/mL, while the extraction recoveries resulted close to 100% for all theglucocorticoids determined. Precision and accuracy were well within ±10%. As suggested by the resultsobtained in the preliminary study on polycystic ovary syndrome (PCOS) urine samples, the method canbe used to support clinical diagnosis of pathologies related to cortisol metabolism. In fact, levels of freeand total glucocorticoids in control subjects were in agreement with previously reported data, as well asfree and total A-THF/THF ratio in PCOS patients. Conversely, in the latter free F/E and A-THF + THF/THEratios were lower than in control subjects (P < 0.01), suggesting a possible alteration of 11-HSD1 and11-HSD2 activity, to be further investigated.
Cuzzola, ANGELA ROSA; Mazzini, Francesco; Petri, Antonella
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/599467
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