Using complementary mass spectrometric approaches for the determination of methylprednisolone metabolites in human urine.
BACKGROUND
The metabolism of methylprednisolone is revisited in order to find new metabolites that could be important for distinguishing between different routes of administration. Recently developed liquid chromatography/tandem mass spectrometry (LC/MS/MS) strategies for the detection of corticosteroid metabolites have been applied to the study of methylprednisolone metabolism.


METHODS
The structures of these metabolites were studied using two complementary mass spectrometric techniques: LC/MS/MS in product ion scan mode with electrospray ionization and gas chromatography/mass spectrometry (GC/MS) in full scan mode with electron ionization. Metabolites were also isolated by semipreparative liquid chromatography fractionation. Each fraction was divided into two aliquots; one was studied by LC/MS/MS and the other by GC/MS after methoxyamine-trimethylsilyl derivatization.


RESULTS
The combination of all the structural information allowed us to propose a comprehensive picture of methylprednisolone metabolism in humans. Overall, 15 metabolites including five previously unreported compounds have been detected. Specifically, 16β,17α,21-trihydroxy-6α-methylpregna-1,4-diene-3,11,20-trione, 17α,20β,21-trihydroxy-6α-methylpregna-1,4-diene-3, 11-dione, 11β,17α,21-trihydroxy-6α-hydroxymethylpregna-1,4-diene-3,20-dione, 11β,17α,20ξ,21-tetrahydroxy-6α-hydroxymethylpregna-1,4-diene-3-one, and 17α,21-dihydroxy-6α-hydroxymethylpregna-1,4-diene-3,11,20-trione are proposed as feasible structures for the novel metabolites. In addition to the expected biotransformations: reduction of the C20 carbonyl, oxidation of the C11 hydroxy group, and further 6β-hydroxylation, we propose that hydroxylation of the 6α-methyl group can also take place.


CONCLUSIONS
New metabolites have been identified in urine samples collected after oral administration of 40 mg of methylprednisolone. All identified metabolites were found in all samples collected up to 36 h after oral administration. However, after topical administration of 5 g of methylprednisolone aceponate, neither the parent compound nor any of the metabolites were detected.
Bioanalysis Research Group, IMIM, Institut de Recerca Hospital del Mar, Doctor Aiguader 88, 08003, Barcelona, Spain.
Oscar J
OJ
Pozo
Josep
J
Marcos
Xavier
X
Matabosch
Rosa
R
Ventura
Jordi
J
Segura
D000284:Administration, Oral; D000287:Administration, Topical; D002853:Chromatography, Liquid / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005938:Glucocorticoids / Q000008:administration & dosage / Q000378:metabolism* / Q000652:urine*; D006801:Humans; D008297:Male; D008775:Methylprednisolone / Q000008:administration & dosage / Q000378:metabolism* / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods
D005938:Glucocorticoids; D008775:Methylprednisolone