Nontargeted detection of designer androgens: Underestimated role of in vitro bioassays

Abstract

Androgens, both steroidal and nonsteroidal in nature, are among the most commonly misused substances in competitive sports. Their recognized anabolic and performance enhancing effects through short-and long-term physiological adaptations make them popular. Androgens exist as natural steroids, or are chemically synthesized as anabolic androgenic steroids (AAS) or selective androgen receptor modulators (SARMs). In order to effectively detect misuse of androgens, targeted strategies are used. These targeted strategies rely heavily on mass spectrometry, and detection requires prior knowledge of the targeted structure and its metabolites. Although exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A nontargeted approach to androgen detection involves the use of cell-based in vitro bioassays. Both yeast and mammalian cell androgen bioassays demonstrate a clear ability to detect AAS and SARMS, and if paired with high resolution mass spectrometry can putatively identify novel structures. In vitro cell bioassays are successfully used to characterize designer molecules and to detect exogenous androgens in biological samples. It is important to continue to develop new and effective detection approaches to prevent misuse of designer androgens, and in vitro bioassays represent a potential solution to nontargeted detection strategies.androgen, in vitro bioassay, nontargeted detection | INTRODUCTIONAndrogens have been used as performance-enhancing drugs for many decades. 1 Since their first detection in various sporting arenas, 1 a battle has ensued between the ability to detect versus evasion of detection strategies. Currently, detection relies on methods that depend on up-to-date cataloguing of all known androgens, as well as in some cases understanding their metabolism, so that reference material of parent compounds and/or their metabolites can be synthesized. The large number of known target compounds, coupled with an increasing list of emerging targets, lends itself to a nontargeted detection approach. In vitro cell-based androgen bioassays that detect on biological activity rather than chemical identification represent a feasible solution for the nontargeted screening of sports doping samples. | ANDROGENSAccording to World Anti-Doping Agency statistics for human athletes, the most abused performance-enhancing drugs are anabolic (muscle-building) agents inclusive of androgenic molecules (2019 anti-doping testing figures, WADA, www-ama.org). Reports on adverse analytical findings show that anabolic agents are responsible

Adulteration of Brain Health (Cognitive, Mood, and Sleep Enhancement) Food Supplements by the Addition of Pharmaceutical Drugs: A Comprehensive Review of Analytical Approaches and Trends

“…To date, extant reviews have provided valuable insights into the adulteration of food supplements by the addition of pharmaceutical drugs by addressing different analytical techniques employed for detecting and identifying such fraudulent practices. However, the predominant focus has been confined to specific classes of dietary supplements, particularly those most prone to be tainted with synthetic pharmaceutical adulterants, encompassing the categories of supplements intended for enhancing sexual potency [ 57 , 58 ], promoting bodybuilding or sports performance [ 59 , 60 , 61 ], or a combination thereof, addressing aspects such as weight loss, muscle development/sports performance, and sexual performance [ 11 , 62 , 63 ]. Additionally, some studies encompassed multiple supplement groups associated with conditions such as erectile dysfunction, obesity/overweight, diabetes mellitus, and hypertension [ 64 ].…”

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay

“…Cell-based in vitro bioassays have shown potential as alternative untargeted approaches for the screening of structurally diverse (and/or designer) molecules in other doping classes involving anabolic agents. 12,24,25 These "steroid" bioassays exploit the natural biology of androgenic signaling in a cell on which the anabolic doping agents act to mediate their major effects in humans. 26,27 Similarly, we recently developed cell-and activity-based HIF bioassays exploiting the natural phenomenon of oxygen sensing in which HIF signaling, and more specifically HIF stabilization, is involved.…”

“…Cell-based in vitro bioassays have shown potential as alternative untargeted approaches for the screening of structurally diverse (and/or designer) molecules in other doping classes involving anabolic agents. ,, These “steroid” bioassays exploit the natural biology of androgenic signaling in a cell on which the anabolic doping agents act to mediate their major effects in humans. , Similarly, we recently developed cell- and activity-based HIF bioassays exploiting the natural phenomenon of oxygen sensing in which HIF signaling, and more specifically HIF stabilization, is involved . These bioassays have already shown broad specificity, capable of generating concentration–response curves for multiple recently approved and current investigational HIF stabilizers and other HIF-related drugs. , In the current study, we optimized the assay format, generating stable cell systems, and evaluated the capability of this untargeted activity-based approach to detect HIF stabilizers in biological samples within a doping control context.…”

Non‐targeted screening of drugs

“…The aim for this special issue of Drug Testing and Analysis was a multidisciplinary and technology-agnostic focus on the potential for detecting screening abnormalities without prior information as to the specific compounds present in complex matrices. The issue begins with a review by Lund et al 1 of bioassay technology for anabolic-androgenic steroid (AAS) and Selective Androgen Receptor Modulator (SARM) detection. Both yeast-and mammalian-cell androgen bioassays have demonstrated this capability and if paired with high resolution mass spectrometry (HRMS) can putatively identify novel structures in biological samples.…”