Multi-class screening method for the analysis of doping agents in dried blood spot (DBS) samples

In the last years, the use of Dried Blood Spots (DBS) as alternative sampling technique is reaching an increasing interest in the antidoping field. The collection of DBS is performed from small volumes of capillary blood lied on adsorbent support (e.g.cellulose) and let to dry. Compared to the conventional urine or blood collection, this technique presents several advantages as the sampling is less invasive, rapid and does not required specialized personnel to be performed. It also facilitates transport and storage, reducing the costs. Conversely, the reduced sample volume available could represent a limitation and requires the use of highly sensitive instrumentation. This work presents a multi-class screening method for 100 compounds belonging to different groups of the World Anti Doping Agency prohibited list, including anabolic agents, beta-2 agonists, hormone and metabolic modulators, and diuretics, among others. DBS samples for method development were obtained depositing 20 µL of venous blood and letting them to dry on cellulose cards. The whole DBS spot was then punched out and extracted with organic solvents prior to the analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The methodology was validated for qualitative purposes. Different parameters were evaluated. The vast majority of the compounds could be reliably detected at sub-ng/mL level. Satisfactory results were obtained in terms of recovery, precision and robustness. Also, matrix effects were negligible for most compounds, as expected considering the low volume of sample analyzed. As a final validation step, DBS samples collected after administration of boldenone, oxandrolone and tamoxifen to healthy volunteers were analyzed. The method showed good performance and robust results, making it fit-for- purpose for its application in sports drug testing.