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

Abstract

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6).

Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra‐articular administration

“…First of all, the MS behavior of THA was studied in order to find characteristic ionization and collision induced dissociation (CID) patterns that could be used to detect unknown THA metabolites in urine. In electrospray positive mode, THA was ionized at the conjugated 3‐keto function forming the [M + H] + ion and, in negative mode, forming the adduct [M + HCOO] − , as described for other GCs …”

“…Urine and plasma samples from patients obtained in the clinical study were analyzed using MRM methods described in Tables and , respectively. THA and TA and six metabolites previously described (Figure ) were measured in urine samples (Table ). In plasma samples, THA, TA, and CORT were measured (Table ).…”

“…However, this general criterion is not suitable for all GCs for several reasons: the number of administration routes, differences in both metabolism and excretion rates, as well as differences in pharmacological activity and therapeutic doses. As a consequence, great research activity has been developed to find strategies to distinguish allowed from forbidden administration for some compounds of the group . For triamcinolone acetonide (TA), a drug related to THA, a previous study showed that a reporting level of 30 ng/mL was not able to detect forbidden IM administration.…”

“…Therefore, the urinary metabolites described for TA are also expected for THA. TA and six metabolites have been reported as the main compounds present in urine after TA administration (Figure ). However, the possibility of the occurrence of specific metabolites of THA has never been tested.…”

“…Chemical structures of the compounds under study: A, THA; B, TA and its metabolites detected in previous studies…”