While misuse of testosterone esters is widespread in elite and recreational 
sports, direct detection of intact testosterone esters in doping control samples 
is hampered by the rapid hydrolysis by esterases present in the blood. With 
dried blood spot (DBS) as sample matrix, continued degradation of the esters is 
avoided due to inactivation of the hydrolase enzymes in dried blood. Here, we 
have developed the method further for detection of testosterone esters in DBS 
with focus on robustness and applicability in doping control. To demonstrate the 
method's feasibility, DBS samples from men receiving two intramuscular 
injections of Sustanon® 250 (n = 9) or placebo (n = 10) were collected, 
transported, and stored prior to analysis, to mimic a doping control scenario. 
The presented nanoLC-HRMS/MS method appeared reliable and suitable for direct 
detection of four testosterone esters (testosterone decanoate, isocaproate, 
phenylpropionate, and propionate) after extraction from DBS. Sustanon® was 
detected in all subjects for at least 5 days, with detection window up to 
14 days for three of the esters. Evaluation of analyte stability showed that 
while storage at room temperature is tolerated well for a few days, testosterone 
esters are highly stable (>18 months) in DBS when stored in frozen conditions. 
Collectively, these findings demonstrate the applicability of DBS sampling in 
doping control for detection of steroid esters. The fast collection and reduced 
shipment costs of DBS compared with urine and standard blood samples, 
respectively, will allow more frequent and/or large-scale testing to increase 
detection and deterrence.