MSACL 2019 EU Abstract

Development and Validation of a Gas-Chromatography Mass Spectrometry Method for the Analysis of Testosterone Esters in Biological Matrices

Michele Iannone (1), Francesco Botrè (1, 2), Claudia Lattanzi (1), Xavier de la Torre (1).
(1) Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, Rome, Italy, (2) Dipartimento di Medicina Sperimentale, "Sapienza" University of Rome, Viale Regina Elena 324, Rome, Italy

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Michele Iannone (Presenter) Laboratorio Antidoping FMSI, Rome, Italy

Presenter Bio: After my master degree in Industrial Pharmacy (summa cum laude) in 2015 at “Sapienza” University of Rome, with an experimental thesis entitled “Metabolic modulators and doping: effects on the urinary steroid profile” carried out at “Laboratorio Antidoping” of Rome, I obtained a PhD in Pharmaceutical Science (summa cum laude) in 2018 at the same University and laboratory, with a thesis entitled “Potential confounding factors in the evaluation of the steroidal module of the Athlete Biological Passport”.
During my career I had the opportunity to carry out part of my scientific activity abroad: I worked for six months (May-October 2015) at University of Barcellona (UB), faculty of Pharmacy, Department of Nutrition and Bromatology; later on, I spent six months (July-December 2018) at DoCoLab at University of Ghent (Ugent), Department of Biological chemistry, Microbiology and Immunology.
My research interests are focused on the evaluation of confounding factors that could influence the in vitro and in vivo metabolism of anabolic androgenic steroids, on the development of new analytical method to improve the strategy followed to detect the pseudo-endogenous steroid doping, and on the developed of analytical methods to study the possible correlations between endocrine and metabolic diseases and steroid metabolism and excretion.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Conflict mitigation IN PROCESS.

Introduction
The detection of doping by testosterone (T) and its precursors is currently based on the evaluation of the so-called “urinary steroid profile”. Abnormal alterations of one or more of its parameters might indicate the misuse of T and/or related steroids and trigger subsequent confirmation analysis, by gas-chromatography coupled to isotopic ratio mass spectrometry (GC-c-IRMS), to assess the difference between the 13C/12C ratio of synthetic and endogenous T and its precursors/metabolites
In recent years, some Authors have reported the occurrence of T preparations displaying a carbon isotopic ratio very similar to the range reported for the endogenous urinary steroids. For this reason, the detection of intact T esters (in blood, plasma or serum) would be an unequivocal proof of the intake of exogenous T
Objective
The aim of the present work was to develop and validate a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the detection of fourteen T esters (acetate, propionate, valerate, isocaproate, caproate, benzoate, hexahydrobenzoate, enanthate, cypionate, octanoate, decanoate, phenylpropionate, undecanoate, laurate) in plasma and/or serum. For indeed, the presence in blood/plasma/serum of an intact synthetic T ester represents an unequivocal proof of the exogenous origin of the steroid.
Methods
After liquid-liquid extraction at pH 9 (carbonate buffer 20% w/V) with n-hexane/ethylacetate (70/30 V/V) all plasma/serum samples were analyzed by GC-MS/MS with electronic impact (EI) source and multiple reaction monitoring (MRM) acquisition mode. T esters were revealed as trimethylsilyl derivates.
Results
Qualitative validation of the method was carried out for all the T esters mentioned above. The folllowing parameters were considered: specificity, selectivity and limit of detection (LOD). The method was shown to be specific and selective for twelve out of fourteen T esters, with LODs between 0.1 and 0.2 ng/mL.
Quantitative validation was also carried out, for the five T esters (propionate, enanthate, cypionate, phenylpropionate, undecanoate) that are present in most of the pharmaceutical preparations reportedly abused by athletes. Considered parameters were the following: specificity, selectivity, LOD and limit of quantification (LOQ), recovery, linearity, repeatability and accuracy. All T esters could be determined with a combined uncertainty smaller than 20% and the calculated LODs and LOQs appeared to be adequate for the detection of the compounds over the range of concentrations corresponding to a potential alteration of the parameters of the urinary steroid profile.
Conclusions
The method here presented is specific and selective for twelve T esters and fully satisfies the WADA identification criteria for the identification of analytes for doping control purposes. The presented method can also be used to confirm the composition of seized pharmaceutical preparation by responsible authorities.