Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Buisson, Corinne; Mongongu, Cynthia; Frelat, C.; Jean-Baptiste, M.; Céaurriz, J. de

doi:10.1016/j.steroids.2008.11.001

Cited by 19 publications

(25 citation statements)

References 10 publications

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“…Two laboratories reported data using collected samples, which were investigated for CIR and which gave a significant difference in δ values between athletes from different countries but which showed that the Δ values were not influenced [19,76]. Investigations on the misuse of 4-androstene-3,11,17-trione, a nutritional supplement [77], and a comprehensive method to detect cortisone misuse, again using oxidation of target analytes, were published [78]. Another research focus was on EpiT and its metabolites 5aEpiD and 5bEpiD, which provided data for another RefPop (n=74), again showing differences between the genders in their CIR [79], and the first extensive results on the 2 H/ 1 H ratio of endogenous urinary steroids were reported [80].…”

Section: Irms Methods Developed and Used In Sports Drug Testingmentioning

confidence: 99%

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

Piper¹,

Emery²,

Saugy³

2011

Anal Bioanal Chem

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According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.For more than 15 years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available.All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urine.

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Section: Irms Methods Developed and Used In Sports Drug Testingmentioning

confidence: 99%

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

Piper¹,

Emery²,

Saugy³

2011

Anal Bioanal Chem

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“…As already known, corticosteroids undergo an intramolecular elimination of the side chain during the vaporization process in a gas chromatographic analysis To completely convert prednisone and prednisolone to Δ1‐adrenosterone, and cortisone and cortisol to adrenosterone before oxidation at the injection port, other recently proposed methods for the prednisone and prednisolone detection in GC‐C‐IRMS have added an oxidation step in the pre‐instrumental procedure. The production of the corresponding 17‐ketosteroids compounds has been described for cortisol metabolites as well as for prednisone/prednisolone as a profitable action to reduce the potential isotopic fractionation caused by spontaneous chemical modification occurring during the vaporization process, independently of the injection mode selected. Instead, in our operating conditions, no structural modification was intentionally conducted on the examined analytes during the preparation protocol.…”

Section: Resultsmentioning

confidence: 99%

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

Iannella

Botrè

Colamonici

et al. 2019

Drug Testing and Analysis

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Prednisone and prednisolone are two anti‐inflammatory steroidal drugs listed by the World Anti‐Doping Agency (WADA) within the class of glucocorticoids, which are prohibited “in competition” and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC‐C‐IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ13C value reference range (−28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC‐C‐IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro‐11‐deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC‐C‐IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.

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“…In the field of human drug testing analysis, acetylation of steroids for CIR determination is preferred because only two carbons are introduced on each alcohol function . Alternative reaction such as oxidation has also been proposed for the analysis of corticosteroids by GC‐C‐IRMS . The major advantage of such derivatization is the absence of addition of external carbon on the molecule.…”

Section: Discussionmentioning

confidence: 99%

Implementation of AICAR analysis by GC‐C‐IRMS for anti‐doping purposes

Buisson

Frelat

Mongongu

et al. 2017

Drug Testing and Analysis

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AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World AntiDoping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances.

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Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Cited by 19 publications

References 10 publications

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC‐C‐IRMS

Implementation of AICAR analysis by GC‐C‐IRMS for anti‐doping purposes

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