Simultaneous detection of testosterone, nandrolone, and boldenone esters in dried blood spots for doping control in sports by liquid chromatography–tandem mass spectrometry

Abstract Testosterone, nandrolone, and boldenone, which are listed as doping substances on the World Anti‐Doping Agency Prohibited List, are mostly available commercially in esterified forms. Isotope ratio mass spectrometry (IRMS) represents a key tool for identifying these substances, as they are hydrolyzed and discharged in the urine as pseudo‐endogenous substances. However, IRMS, which comprises a complicated process, cannot achieve the direct detection of steroid esters in blood samples. These substances can be detected using dried blood spots (DBSs), reducing the impact of esterase hydrolysis. Here, a simultaneous liquid chromatography–tandem mass spectrometry method for detecting 28 steroid (13 testosterone, nine nandrolone, and six boldenone) esters was developed using three DBS types of samples, including a cellulose paper and polymer. The substances were first derivatized with methyloxime to increase their sensitivities (the limits of detection were <0.1–0.4, <0.1–0.9, and <0.1–0.9 ng/mL for the testosterone, nandrolone, and boldenone esters, respectively). Further, the DBS absorbents were verified since the effect of interferences depended on it. Next, a study involving seven participants was conducted to detect intramuscularly administered testosterone enanthate (100 mg). Polymer and cellulose papers were used to collect blood from their upper arms and fingertips, respectively, and testosterone enanthate was identified and detectable at both blood‐collection sites for up to 144 and 216 h, respectively. Furthermore, testosterone enanthate was detectable in the DBS samples stored under refrigeration after 6 months, indicating the stable nature of DBS.