Determination of endogenous and synthetic glucocorticoids in human urine by gas chromatography–mass spectrometry following microwave-assisted derivatization

Abstract

A complete screening and confirmation analytical method for the direct determination of six endogenous (cortisol, cortisone, deoxycorticosterone, tetrahydrocortisol, tetrahydrocortisone, tetrahydro- S) and 17 synthetic (amcinonide, betamethasone, desoximethasone, dexamethasone, fludrocortisone, flumethasone, flunisolide, flucinolone acetonide, flucinonide, fluprednisolone, flurandrenolide, fluorometholone, 6-methylprednisolone, prednisolone, prednisone, triamcinolone, triamcinolone acetonide) glucocorticoids in human urine by gas chromatography with mass spectrometric detection (GC–MS) is presented. The analytical technique comprises a pre-treatment procedure and the instrumental analysis of the trimethylsilyl (TMS) derivatives, performed by GC–MS (quadrupole) with electron impact (EI) ionization. The derivatization yields obtained by two different derivatizing mixtures, namely N-methyl- N(trimethylsilyl)trifluoroacetamide (MTSFA):NH 4I:dithioerythritol (DTE) 1000:2:4 (usually indicated as TMSiodine); and N-trimethylsilylimidazole (TMSim): N, O-bis(trimethylsilyl)acetamide (BSA):trimethylchlorosilane (TMCS) 3:3:2, both under direct thermal heating and with microwave (MW) irradiation, were evaluated, also as a function of the temperature, of the MW power and of the incubation time. The highest yields of the derivatization process were obtained, for most of the compounds here considered, by a two-step procedure: a microwave-assisted derivatization stage (40 min in a microwave oven at 900 W emitted power), followed by a traditional heat transfer derivatization (1.5 h in a thermostated bath at 70 °C) with the derivatization mixture TMSim:BSA:TMCS 3:3:2. In these operating conditions, diagnostic EI–MS spectra of all considered glucocorticoids were obtained. Limits of detection (LOD) of synthetic glucocorticoids in urine ranged from 3 to 25 μg/l. The effectiveness of the method for the determination of glucocorticoids in urine was evaluated on spiked urine samples and on real samples obtained from patients under pharmacological treatment with synthetic glucocorticoids. Apart from the clinical monitoring of glucocorticoids in urine, the method can be applied as a complete screening + confirmation analytical protocol in antidoping tests for the detection of illicit administration of glucocorticoids by the athletes.