Simultaneous identification of prednisolone and its ten metabolites in human urine by high performance liquid chromatography‐tandem mass spectrometry

Abstract

The use of prednisolone and prednisone is prohibited by the World Anti-Doping Agency (WADA) due to their performance-enhancing effect. The purpose of the present work was to explore the possibility of identification and detection of various metabolites of prednisolone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in excretion study samples. Ten metabolites of prednisolone could be identified namely prednisone (11-oxo metabolite) [M-1], 6-β-OH-prednisolone [M-2], 20-β-OH-prednisolone [M-3], 20-α-OH-prednisolone [M-4], 20-α-OH-prednisone [M-5], 20-β-OH-prednisone [M-6], 2 tetrahydro epimers of 20-β-OH-prednisolone [M-7], 2 tetrahydro epimers of 20-α-OH-prednisolone [M-8], 2 tetrahydro epimers of 20-β-OH-prednisone [M-9], and 2 tetrahydro epimers of 20-α-OH-prednisone [M-10]. Prednisolone was administered in 10-, 20-, and 40-mg dosage to healthy volunteers to study detection of various metabolites. The parent, M-1, M-2, and M-3 could be detected up to 72 h while rest of the metabolites were detectable up to 24 h after drug administration. The detection of newer metabolites of the drug can further be used for confirmatory purposes.

Detection and characterization of prednisolone metabolites in human urine by LC-MS/MS

“…Derivatization of these compounds is even more complicated than other PRED metabolites because of presence of extra hydroxyl group, and this fact could explain why these compounds were not detected in previous studies using GC‐MS . These metabolites were also unreported by previous LC‐MS/MS methods . This fact can be due to a co‐elution with 6 β OH‐PRED, which is circumvented by the UHPLC‐MS/MS method used in this present work.…”

“…This knowledge helped us to propose reliable structures for seven of the other nine detected metabolites. Among those seven, only M‐XVIII has been previously reported as a PRED metabolite …”

“…Prednisone is both a prodrug and a metabolite of the active drug PRED . Some metabolites were detected in late 1950s using paper chromatography (Florisil), and the number of identified and characterized metabolites has increased since the use of chromatography coupled to mass spectrometry . The previously reported metabolites of PRED are prednisone, 20 α ‐dihydro‐PRED and 20 β ‐dihydro‐PRED, 6 β ‐hydroxy‐PRED (6 β OH‐PRED), 20 α ‐dihydro‐prednisone and 20 β ‐dihydro‐prednisone, several isomers of 4,5‐dihydroprednisolone and 4,5‐dihydroprednisone, 3,4,5‐tetrahydroprednisolone, 3,4,5‐tetrahydroprednisone and the 20 α isomers and 20 β isomers of a 3,4,5,20‐hexahydroprednisone …”

Urinary excretion profile of prednisone and prednisolone after different administration routes

“…Prednisolone is, instead, the active compound, and it is available for both systemic and topical use. The phase I metabolic profile of both compounds was already described by previous investigators . Twenty metabolites were isolated in urine after oral administration of prednisolone; the phase I metabolic reactions included oxidation of the hydroxyl group in C11, reduction of ketone in C20, hydroxylation in C6, or a combination of some of them, being prednisone, prednisolone and 20β‐dihydro‐prednisolone the most abundant compounds detected, followed by 20α‐dihydro‐prednisolone, 20α‐dihydro‐prednisone and 20β‐dihydro‐prednisone …”

Hormone Use and Abuse

“…Several groups have described protocols and screening methods for the analysis of endogenous GC urinary metabolites using liquid chromatography-mass spectrometry (Mazzarino et al, 2008;Brooker et al, 2012;Ahi et al, 2012;Musenga and Cowan, 2013). Some methods allows for the analysis of a large number of analytes from most of the classes in the WADA Prohibited List, including anabolic agents, b2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids, and b-blockers, and do so while meeting the WADA sensitivity requirements (Musenga and Cowan, 2013).…”