Rationale Exhaled breath (EB) has been demonstrated to be a promising alternative matrix in sports drug testing due to its non‐invasive and non‐intrusive nature compared with urine and blood collection protocols. In this study, a pilot‐test system was employed to create drug‐containing aerosols simulating EB in support of the analytical characterization of EB sampling procedures, and the used analytical method was extended to include a broad spectrum of prohibited substances. Methods Artificial and authentic EB samples were collected using sampling devices containing an electret filter, and doping agents were detected by means of liquid chromatography and tandem mass spectrometry with unispray ionization. The analytical approach was characterized with regard to specificity, limits of detection, carry‐over, recovery and matrix effects, and the potential applicability to routine doping controls was shown using authentic EB samples collected after single oral dose applications of glucocorticoids and stimulants. Results The analytical method was found to be specific for a total of 49 model substances relevant in sports drug testing, with detection limits ranging from 1 to 500 pg per cartridge. Both ion suppression (−62%) and ion enhancement (+301%) effects were observed, and all model compounds applied to EB sampling devices were still detected after 28 days of storage at room temperature. Authentic EB samples collected after the oral administration of 10 mg of prednisolone resulted in prednisolone findings in specimens obtained from 3 out of 6 participants up to 2 h. In octodrine, dimethylamylamine (DMAA) and isopropylnorsynephrine post‐administration EB samples, the drugs were detected over a period of 50, 48, and 8 h, respectively. Conclusions With the analytical approach developed within this study, the identification of a broad spectrum of prohibited doping agents in EB samples was accomplished. Application studies and stability tests provided information to characterize EB as a potential matrix in sports drug testing.

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Currently, primarily urine, whole blood and serum samples are analyzed for dopingrelevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features, e.g. a minimally invasive sampling procedure. In order to cope with the increased application of DBS, a comprehensive initial testing procedure (ITP) was developed, optimized and validated, comprising a total of 233 substances representing all groups on the World Anti-Doping Agency's (WADA's) Prohibited List. The sample preparation was conducted by employing a fully automated system using an efficient flowthrough extraction of a 4 mm diameter spot followed by LC-HRMS/MS analysis. The procedure was successfully validated in terms of selectivity, limit of detection, reproducibility, carryover and robustness with respect to an alternative manual sample preparation, an alternative dried blood collection device and the sample extract stability, and was thus found to meet the required criteria of the relevant guidelines published by WADA for routine application. As a proof-of-concept, DBS samples were analyzed after the administration of the glucocorticoids prednisone and dexamethasone, as well as the stimulant pseudoephedrine and the beta-blocker propranolol. All substances were detected in post-administration samples for at least 4 h and up to 24 h after intake, depending on the collection time period, using the developed testing procedure. In particular, for substances that are only banned in-competition, data obtained from DBS samples can be useful for the interpretation of adverse analytical findings. In conclusion, the developed ITP accounts for the anticipated increasing relevance of DBS in anti-doping analysis in the future and provides a foundation for optimized approaches for specific substance classes.

Rationale Exhaled breath (EB) was found to be a promising matrix in the field of sports drug testing due to the non‐invasive and non‐intrusive sampling procedure, but significant inter‐individual variations regarding detected drug concentrations have been observed in previous studies. To investigate whether the detectability of doping agents in EB is affected by sex or tobacco smoking, two administration studies were conducted with male and female smokers and nonsmokers concerning the elimination of the beta blocker propranolol and the stimulant pseudoephedrine into EB. Methods Following the administration of 40 mg propranolol or 30 mg pseudoephedrine, a total of 19 participants, including female and male nonsmokers as well as female and male smokers, collected EB and dried blood spot (DBS) samples over a period of 24 h. Respective analyte concentrations were determined using liquid chromatography and high‐resolution tandem mass spectrometry, and semi‐quantitative assays were characterized with regard to selectivity, limit of detection and identification, precision, linearity, and carryover. Results Both propranolol and pseudoephedrine were identified in post‐administration EB samples from female and male nonsmokers as well as female and male smokers, and the maximum detected drug levels ranged from 9 to 2847 pg/cartridge for propranolol and from 26 to 4805 pg/cartridge for pseudoephedrine. The corresponding DBS levels were in a range of 4–30 ng/mL for propranolol and 55–186 ng/mL for pseudoephedrine. Conclusions Neither the consumption of cigarettes nor the sex appears to represent a decisive criterion as to the detectability of propranolol or pseudoephedrine in EB, but inter‐individual variations regarding the detected drug levels were observed among all studied population groups.