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Clinical Chemistry 50: 2345-2352, 2004. First published October 14, 2004; 10.1373/clinchem.2004.033605
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(Clinical Chemistry. 2004;50:2345-2352.)
© 2004 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Quantitative, Highly Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for Detection of Synthetic Corticosteroids

Robert L. Taylor, Stefan K. Grebe and Ravinder J. Singha

1 Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, Rochester, MN.

aAddress correspondence to this author at: Hilton 730, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905. Fax 507-284-9758; e-mail singh.ravinder{at}mayo.edu.

Background: Measurements of serum or urine concentrations of synthetic glucocorticoids are useful for assessing suspected iatrogenic hypothalamic-pituitary-adrenal axis suppression and Cushing syndrome. We have developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantitative analysis of beclomethasone dipropionate, betamethasone, budesonide, dexamethasone, fludrocortisone, flunisolide, fluorometholone, fluticasone propionate, megestrol acetate, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide.

Methods: Stable isotopes of cortisol-9,11,12,12-d4 and triamcinolone-d1 acetonide-d6 were added as internal standards to calibrators, controls, and unknown samples. After acetonitrile precipitation, these samples were extracted with methylene chloride, and the extracts were washed and dried. Reconstituted extract (15 µL) was injected on a reversed-phase column and analyzed by LC-MS/MS in positive-ion mode. Assay precision, accuracy, linearity, and sample stability were determined by use of enriched samples. Clinical validation included analysis of 8 serum and 20 urine samples from patients with undetectable cortisol concentrations and analysis of different types of tablets.

Results: Functional assay sensitivity was as low as 0.6–1.6 nmol/L for all compounds except for triamcinolone (7.6 nmol/L). Interassay CVs were 3.0–20% for concentrations of 0.6–364 nmol/L for all analytes. Recoveries of all analytes (except triamcinolone in serum) were 82–138% at 19.2–693 nmol/L. All but one of the serum and urine samples from patients who were tested because of suppressed cortisol concentrations contained at least one synthetic steroid. Tablet analysis recovered 75% of the synthetic steroids in suspected drugs.

Conclusions: LC-MS/MS allows simultaneous quantitative detection of various synthetic steroids in serum, plasma, urine, and tablets. This provides a valuable tool for evaluating the clinical effects of topical and systemic synthetic corticosteroids.




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