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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 0.5-1000ng/mL for A-THF and THF, 5-800ng/mL for E and THE and 1-1000ng/mL for F, with R(2) values greater than 0.9981. The LOD and LOQ of the described method ranged from 0.1 to 3.0ng/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 11β-HSD1 and 11β-HSD2 activity, to be further investigated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A previously reported method for the assessment of the ratio of tetrahydrocortisol (THF) + allo-tetrahydrocortisol (A-THF) to tetrahydrocortisone (THE) by HPLC-MS-MS has been significantly improved, in order to increase either ruggedness and reliability. That was achieved by the introduction of an on-line sample cleanup stage, which made use of a perfusion column as a solid phase microextraction (SPE) cartridge. The set of analytes was expanded, by introducing cortisol and cortisone, whose ratio supply additional diagnostic information. The response factors of both THF and A-THF has been checked, resulting almost identical, as well as the influence of the matrix on the calibration curves which, although different for water and urine, had similar effect on the ratios of interest. As a consequence, the calibration solutions can be prepared in pure water. The influence of several different storage procedures has also been tested, resulting in no substantial effect on the final result. Finally, the improved method has been used to run real samples from healthy volunteers, with satisfactory results., (Copyright (c) 2009 John Wiley & Sons, Ltd.)