Automated online dried blood spot sample preparation and detection of anabolic steroid esters for sports drug testing

Dried blood spots (DBSs) provide a valuable complementary sample matrix for routine doping analysis, and the full automation of analysis for DBS samples is achievable to avoid extensive and repetitive manual laboratory work. In the current study, a fully automated online DBS preparation and detection method for the screening and quantification analysis of 13 anabolic steroid esters by means of the DBSA-TLX-HRMS system was developed and validated, based on the purpose for the determination of the abuse of anabolic steroid esters in athletes. Validation of the method yielded linear (R2 > 0.99), precise, and accurate (RSD% and Re% < 20% at low, medium, and high concentration levels) results. The LOD of testosterone laurate was established at 0.5 ng/ml and at 0.2 ng/ml for other steroid esters. The extraction recovery of the target compounds from DBS ranged from 10.5% to 88.9%, and matrix effects were moderate. Furthermore, the developed and validated method was applied in the analysis of DBS samples collected after the oral administration of a single dose of 80 mg testosterone undecanoate demonstrating its applicability. Evaluation of analyte stability showed that testosterone undecanoate are more stable (8 weeks) in DBS samples of administration study when stored in frozen (-20°C) condition compared with cold storage (4°C). Collectively, these findings demonstrate the applicability of automated DBS analysis in doping control for detection of anabolic steroid esters.