ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2006) 12 P119

Determination of xenobiotic glucocorticoids by gas chromatography–mass spectrometry for clinical purposes

CJ Walker, DA Cowan, NF Taylor & AT Kicman

King’s College London, London, United Kingdom.

Methyl oxime–trimethylsilyl (MO-TMS) derivatisation with gas chromatography–mass spectrometric (GC-MS) analysis has been well established in the analysis of glucocorticoids and is still used in many laboratories for profiling endogenous steroids for clinical purposes. There appears to be a clinical need to extend the analysis to xenobiotic glucocorticoids, primarily to elucidate cases of patients presenting with adverse symptoms associated with glucocorticoid excess, where the pathology shows no obvious intrinsic cause but there is a history of use of non-pharmaceutically approved medications, e.g. herbal products or lotions that may have been surreptitiously prepared with steroids.

Here we present a refined method that can detect mainstream xenobiotic glucocorticoids (betamethasone, budesonide, desonide, dexamethasone, fludrocortisone, flunisolide, prednisone, 6-methylprednisolone, prednisolone, triamcinolone) in human urine. The optimised derivatisation conditions and subsequent removal of the derivatisation agents prior to GC-MS are critical.

Following extraction from urine (2 ml), methoximation was carried out using 0.05 ml methylhydroxylamine hydrochloride 8% (w/v) in pyridine and heated at 60 °C overnight. Excess pyridine was evaporated and silylation was carried out using 0.05 ml N-methyl-N-trimethylsilyltrifluoroacetamide: trimethylsilylimidazole (4:1) heated at 105 °C for 6 h. After cooling, cyclohexane (1 ml) containing 2% (v/v) dodecane and 100 ng/ml cholestane was added, the tube contents were vortex mixed and water (1 ml) was then added, vortex mixed again and centrifuged (1300 g for 5 min). The aqueous layer containing silylating reagents was discarded. The organic solvent was dried using anhydrous sodium sulphate (∼250 mg) and the solvent was evaporated under nitrogen at 60 °C for 10–12 min. The residual dodecane was transferred to an autosampler vial for GC-MS analysis (electron impact).

Operating in selected ion mode, the limits of detection were fit for the purpose (<10 ng/ml). Full scan analysis of larger concentrations yielded particularly informative spectra.

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