Carbon isotopic characterization of prednisolone and prednisone pharmaceutical formulations: Implications in antidoping analysis

Abstract

Twenty‐two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) method. All of them showed typical non‐endogenous δ13C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13C content (−17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ13C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ13C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre‐treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC‐C‐IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro‐11‐deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method used routinely for the quali‐quantitative detection of glucocorticoids. δ13C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ13C value (−29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated.

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…The carbon isotope signatures of eight pharmaceutical products (six prednisone and two prednisolone) were determined, all exhibiting δ 13 C values below −28.4‰, and proof‐of‐concept for the capability of the assay to determine the exogenous origin of urinary prednisolone and prednisone was provided by means of the analysis of selected administration study urine specimens. Another 14 (seven prednisone and seven prednisolone) preparations obtained from four different countries were also subjected to the developed carbon isotope ratio analysis, yielding similar δ 13 C values, except for one prednisolone nasal spray, which was found to present δ 13 C values of −17.8‰, 170 indicating also here both the confirmatory capability and the limitations of GC/C/IRMS.…”

Urinary excretion profile of prednisolone and prednisone after rectal administration: Significance in antidoping analysis

“…Due to its systemic effects, the rectal use of glucocorticoids, similarly to any injectable and oral administration, is prohibited by the WADA “in competition” only. The excretion profile of prednisone and prednisolone after local (eye drops, nasal spray, ointment) and oral (tablet, vial) intake was extensively investigated in the antidoping field 19–27 . In this work, we explored the rectal administration of both prednisolone and prednisone, verifying whether it should be considered as a systemic route, regardless of the type of drug formulation used.…”

“…The excretion profile of prednisone and prednisolone after local (eye drops, nasal spray, ointment) and oral (tablet, vial) intake was extensively investigated in the antidoping field. 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 In this work, we explored the rectal administration of both prednisolone and prednisone, verifying whether it should be considered as a systemic route, regardless of the type of drug formulation used. As already stated, analyses were carried out by the analytical procedure currently adopted in our laboratory to detect (the LOD for prednisone and prednisolone is 1 ng/ml) and confirm (the LOI for prednisone and prednisolone is 5 ng/ml) the presence of glucocorticoids in urine for doping control tests.…”

“…A reporting level of 30 ng/ml was initially established by the WADA to discriminate the allowed (topical) and the prohibited (systemic) administration. 18 Data reported by previous investigations carried out after administration by different routes of prednisone, prednisolone, 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 betamethasone, 28 budesonide, 29 , 30 or triamcinolone acetonide 31 , 32 have showed the need to adequately modify the WADA guidelines. New reporting levels were, therefore, established by the WADA for several glucocorticoids, including prednisone (i.e., 300 ng/ml) and prednisolone (i.e., 100 ng/ml).…”

Recent progress on the detection of animal-derived food stimulants using mass spectrometry-based techniques

“…The analysis of glucocorticoids shows promise using the GC-C-IRMS method. Iannella et al ( 70 ) successfully applied the GC-C-IRMS method to analyze 22 commercially available pharmaceutical preparations containing prednisolone or prednisone, demonstrating its excellent applicability.…”