Screening of In Vitro Synthesised Metabolites of 4,9,11-Trien-3-One Steroids by Liquid Chromatography-Mass Spectrometry

Abstract

The aim of the work was to develop a flexible in vitro synthesis procedure, which can be applied in order to study and predict the metabolic patterns of new derivatives of anabolic androgenic steroids (AAS) with respect to most prominent target compounds for doping control purposes. Microsomal and S9 fraction of human liver preparations were used as a source of metabolising enzymes and the co-substrates of the synthesis mixture were selected to favour phase-I metabolic reactions and glucuronidation as phase-II conjugation reactions. Model compounds within the study were 4,9,11-trien-3-one steroids, structural derivatives of gestrinone and trenbolone, which both are included in the list of prohibited compounds in sports by the World Anti-Doping Agency (WADA). The correlation between in vitro metabolism of human microsomes and in vivo excretion studies in human was compared with gestrinone and subsequently, the applicability of the in vitro model for prediction of AAS metabolic pathways for new doping agents was evaluated. All the AAS examined within this study were successfully metabolised using the developed in vitro model, hydroxylation, reduction and glucuronide conjugation being the most prominent reaction pathways. Hydroxylated and glucuronide-conjugated metabolites of in vivo experiment with gestrinone were the same metabolites formed in the enzyme-driven process, thus showing good in vitro-in vivo correlation. Liquid chromatographic-mass spectrometric and tandem mass spectrometric methods were developed, relying on the positive polarity of electrospray ionisation, which also allowed the direct detection of intact glucuronide-conjugated AAS metabolites. Due to charge delocalisation and high proton affinity, the developed method was proven effective in the analysis of AAS metabolites bearing extensive conjugated double bond systems in their structures.

Urinary detection of conjugated and unconjugated anabolic steroids by dilute‐and‐shoot liquid chromatography‐high resolution mass spectrometry

“…Therefore the corresponding glucuronide conjugates of the parent compound or the metabolites in the excretion urines were confirmed with the corresponding glucuronides synthesized by an earlier-described methodology by Kuuranne et al [42] Studying excretion urines of stanozolol, two other isomeric peaks, alongside 3STANG, were observed in the chromatograms, considering two diagnostic ions: the protonated ([M + H] + ) and the deprotonated ([M-H] À ) molecules. Therefore the corresponding glucuronide conjugates of the parent compound or the metabolites in the excretion urines were confirmed with the corresponding glucuronides synthesized by an earlier-described methodology by Kuuranne et al [42] Studying excretion urines of stanozolol, two other isomeric peaks, alongside 3STANG, were observed in the chromatograms, considering two diagnostic ions: the protonated ([M + H] + ) and the deprotonated ([M-H] À ) molecules.…”

Doping control analysis of emerging drugs in human plasma – identification of GW501516, S‐107, JTV‐519, and S‐40503

“…The lack of detailed pharmacokinetic information complicates the incorporation of new compounds into urine‐based analytical methods since the metabolic fate of orally or subcutaneously administered drugs is difficult to predict. In vitro metabolism models are an excellent approach that simulate metabolic reactions in the human body, and have been utilized for a variety of drugs relevant for doping control analyses 44, 45. A complementary approach enabling the detection of the intact analytes under nearly authentic conditions in plasma specimens adds another tool to the current options available to sports drug testing laboratories until details on the pharmacokinetics of new and potentially performance‐enhancing substances are known.…”

A review of designer anabolic steroids in equine sports

“…To model the liver, which is the principle organ involved in detoxification and metabolism of exogenous substances, preparations involving liver metabolic enzymes are amongst the most commonly used in vitro methods . Equine liver microsomes and S9 fraction are most commonly used to model equine metabolism due their ease of use and commercial availability.…”

“…These enzymes are typically supplemented with several biological co‐factors, including nicotinamide adenine dinucleotide (NADH), or nicotinamide adenine dinucleotide phosphate (NADPH) to promote the metabolic reactions. Most systems use an excess of these reagents, although systems have been developed which employ co‐factor regeneration, in which an NADH/NADPH‐generating reaction is coupled to the NADH/NADPH‐dependant metabolic reactions. Typically, the reaction of glucose‐6‐phosphate with a glucose‐6‐phosphate dehydrogenase (G6PDH) enzyme is used, which regenerates NADH/NADPH as a by‐product of oxidation.…”