The Analysis of Testosterone Esters in the Dried Blood Spot by Liquid Chromatographic Q Exactive High Resolution Mass Spectrometry

Objective: To establish an unequivocally method to analyze testosterone esters from dried blood spot and thus to discriminate exogenous with endogenous testosterone. Method: 20 microliter dried blood sample was redissolved in water and then the protein was precipitated. A liquid-liquid extraction was performed and the organic phase was collected. The residue was reacted with methoxylamine, and then the Q Exactive high resolution mass spectrometer was used to analyze the sample after reconstitution.Results: The detection limit was 0.1 to 0.3 ng/mL for testosterone acetate, testosterone propionate, testosterone benzoate,testosterone phenylpropionate, testosterone cypionate, testosterone enanthate, testosterone decanoate and testosterone undecanoate.The extraction recovery rate was 50.0% to 86.0%. The oxime products of testosterone esters were baseline separated by liquid chromatographic conditions, and the E and Z isomers of oxime were tentatively identified which meet the requirements of(World Anti-Doping Agency, WADA). In human trail, six male adults were intramuscularly injected 0.25 gram testosterone undecanoate,and the detection window was 7 to 14 days. Conclusion: the new method is fast, high sensitivity and minimally invasive. It is a direct method to detect testosterone preparations, which has passed the qualification of ISO-17025 accreditation, and it will be implemented in 2022 Beijing Winter Olympic Games and Winter Paralympic Games.