Improving detection capabilities of doping agents by identification of new phase I and phase II metabolites by LC-MS/MS

Metabolic studies of doping agents have been traditionally performed by using gas chromatography coupled to mass spectrometry (GC-MS). In the last years, liquid chromatography coupled to mass spectrometry (LC-MS) has demonstrated to offer new possibilities to perform metabolic studies. The objective of this thesis was to study the metabolism (phase I and phase II) of different doping agents by LC-MS/MS in order to improve the detection capabilities of the studied substances. For mesocarb, a thermolabile compound, the parent drug 19 metabolites were detected in urine including mono-, di- and tri-hydroxylated metabolites excreted free or conjugated with glucuronic acid and sulphate. For toremifene, an anti-estrogenic drug with poor electron ionization properties, the parent drug and 20 metabolites were detected in urine. The structure of most of the metabolites was proposed. Anabolic androgenic steroids (AAS) metabolites conjugated with sulphate were investigated to improve the retrospectivity of the detection of these compounds. A study of hydrolysis and MS/MS behaviour of sulphate metabolites of AAS was performed, and sulphate conjugated metabolites of boldenone, methyltestosterone and metandienone were studied. Boldenone sulphate and epiboldenone sulphate were identified as boldenone metabolites in humans. They can be used as markers of exogenous boldenone administration. For methyltestosterone, three new sulphate metabolites were detected and structures were proposed. One of them, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate was detected in urine up to 21 days after methyltestosterone administration, improving three times the retrospectivity of the detection with respect to other previously reported long-term metabolites. Several new sulphate metabolites were detected after metandienone administration. One of them was characterized as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one conjugated with sulphate, and it was detected up to 26 days after administration, improving the retrospectivity of the detection with respect to other long-term metabolites described. The usefulness of LC-MS/MS for the detection and characterization of metabolites of doping agents has been demonstrated, especially for the study of new phase II metabolites and for metabolic studies of compounds where GC-MS shows relevant limitations.Els estudis metabòlics de substàncies dopants han estat tradicionalment realitzats mitjançant l’ús de cromatografia de gasos acoblada a espectrometria de masses (GC-MS). En els últims anys, s’ha demostrat la utilitat de la cromatografia líquida acoblada a espectrometria de masses (LC-MS) per realitzar estudis de metabolisme. L’objectiu d’aquesta tesi va ser estudiar el metabolisme (fase I i fase II) de diferents substàncies dopants mitjançant LC-MS/MS per tal de millorar la capacitat de detecció dels compostos estudiats. Per a mesocarb, compost termolàbil, es van detectar en orina el compost inalterat i 19 metabòlits incloent metabòlits mono-, di- i tri-hidroxilats excretats lliures o conjugats amb àcid glucurònic i sulfat. Per a toremifè, un fàrmac anti-estrogènic amb espectre de masses d’impacte electrònic amb pocs ions diganòstic, es van detectar el compost inalterat i 20 metabòlits en orina. Es va proposar l’estructura de la major part de metabòlits detectats. Per tal de millorar la retrospectivitat de la detecció dels esteroides anabolitzants androgènics (AAS) es van estudiar els metabòlits conjugats amb sulfat. Es va realitzar un estudi de la hidròlisi i del comportament espectromètric dels metabòlits conjugats amb sulfat dels AAS. Es van estudiar els metabòlits conjugats amb sulfat de boldenona, metiltestosterona i metandienona. Es van identificar boldenona sulfat i epiboldenona sulfat com a metabòlits de boldenona en humans. Aquests metabòlits poden ser usats com a marcadors de l’administració exògena de boldenona. Per a metiltestosterona, es van detectar i proposar les estructures de tres nous metabòlits conjugats amb sulfat. Un d’ells, el 17β-metil-5α-androstà-3α,17α-diol 3α-sulfat, va ser detectat en orina fins a 21 dies després de l’administració de metiltestosterona. Es van detectar diversos metabòlits de metandienona conjugats amb sulfat no descrits prèviament. Un d’ells, identificat com a 18-nor-17β-hidroximetil-17α-metilandrost-1,4,13-trien-3-ona conjugat amb sulfat, va ser detectat fins 26 dies després de l’administració. Tant per a metiltestosterone com per a metandienone, els metabòlits conjugats amb sulfat permeten millorar la retrospectivitat de la detecció respecte a altres marcadors descrits anteriorment. S’ha demostrat la utilitat del LC-MS/MS per a la detecció i caracterització de metabòlits de substàncies dopants, especialment per a l’estudi de nous metabòlits de fase II i per a estudis de metabolisme de compostos que mostren limitacions en GC-MS

LC-MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17β-hydroxymethyl-17α-methyl-5β-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with β-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.Grants by Ministerio de Economía y Competitividad (Gobierno de España) (Project number DEP2012-35612) and Generalitat deCatalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) are gratefully acknowledge