Dried blood spots for doping controls—Development of a comprehensive initial testing procedure with fully automated sample preparation

Abstract

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.

Detection of Gene Doping Using Dried Blood Spots from a Mouse Model with rAAV9 Vector-Mediated Human Erythropoietin Expression as a Pilot Study

“…Therefore, automating most processes in doping testing is desirable as well. There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18].…”

“…There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18]. Hence, future research should not only focus on the target substance but also focus on research and development in order to automate the DBS pretreatment and detection processes.…”

Recent progress on the detection of animal-derived food stimulants using mass spectrometry-based techniques

“…Nanofiber-based SPE is another promising technique because of its simple operation, high speed, and low organic-solvent consumption. Currently, urine, whole blood, and serum samples are the primary matrices for detection; however, new supplementary matrices, such as DBS ( 71 ), DUS ( 72 ), and VAMS ( 71 ), have been introduced and verified successfully with improved analyte stability and low cost ( 73 ). Synthetic two-dimensional chromatography techniques, such as GC × GC and LC × LC, exhibit enhanced separation capabilities and retrospective analysis potential in the field of anti-doping.…”