Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath

Abstract

Rationale: Continuously refining and advancing the strategies and methods employed in sports drug testing is critical for efficient doping controls. Besides improving and expanding the spectrum of target analytes, alternative test matrices have warranted in-depth evaluation as they commonly allow for minimal-/non-invasive and non-intrusive sample collection. In this study, the potential of exhaled breath (EB) as doping control specimen was assessed.

Methods: EB collection devices employing a non-woven electret-based air filter unit were used to generate test specimens, simulating a potential future application in doping controls. A multi-analyte sports drug testing approach configured for a subset of 12 model compounds that represent specific classes of substances prohibited in sports (anabolic agents, hormone and metabolic modulators, stimulants, and beta-blockers) was established using unispray liquid chromatography/tandem mass spectrometry (LC/MS/MS) and applied to spiked and elimination study EB samples. The test method was characterized concerning specificity, assay imprecision, and limits of detection.

Results: The EB collection device allowed for retaining and extracting all selected model compounds from the EB aerosol. Following elution and concentration, LC/MS/MS analysis enabled detection limits between 5 and 100 pg/filter and imprecisions ranging from 3% to 20% for the 12 selected model compounds. By means of EB samples from patients and participants of administration studies, the elimination of relevant compounds and, thus, their traceability in EB for doping control purposes, was investigated. Besides stimulants such as methylhexaneamine and pseudoephedrine, also the anabolic-androgenic steroid dehydrochloromethyltestosterone, the metabolic modulator meldonium, and the beta-blocker bisoprolol was detected in exhaled breath.

Conclusions: The EB aerosol has provided a promising proof-of-concept suggesting the expansion of this testing strategy as a complement to currently utilized sports drug testing programs.

Figure 1

Exhaled breath (EB) samples tested by reversed‐phase liquid chromatography/tandem mass spectrometry for 12 model compounds including anabolic agents, hormone and metabolic modulators, stimulants, and beta‐blockers: (A) blank EB specimen containing only the internal standards D₃‐testosterone, D₃‐meldonium, (S)‐2‐aminooctane, and D₇‐propranolol. Y‐axes are normalized to the abundance of the corresponding spiked specimen shown under (B), which illustrates the results of an EB sample fortified with 500 pg of each target analyte plus ISTDs

Figure 2

Chromatograms of exhaled breath samples collected from patients and participants of elimination studies with (A) dehydrochloromethyltestosterone (DHCMT, collected 4 h post‐administration of 20 mg), (B) meldonium (collected 15 h post‐administration of 500 mg), (C) methylhexaneamine (collected 8 h post‐administration of 40 mg), (D) pseudoephedrine (collected 5 h post‐administration of 30 mg), and (E) bisoprolol (collected 3 h post‐administration of 3.75 mg)