Comparability of different analytical workflows for steroid esters detection in doping control field

In this study different microsampling devices (Whatman DMPK-C card, Whatman 903 Protein Saver card, PerkinElmer-226 card, Tasso-M20, and Mitra® tips), extraction (methanol, ethylacetate, pentane, methanol/acetonitrile, methanol/isopropanol, methanol/water, methanol/isopropanol/tert-butylmethyl ether, and methanol/isopropanol/cyclohexane) and derivatization (Girard's reagent P, Girard's reagent T or methoxyamine) protocols were evaluated to select optimal conditions for the analysis of 27 steroid esters in dried blood spot. Samples were analyzed using LC-MS/MS. RESULTS: showed: (i) methanol provided the highest recoveries. However, when used with the Tasso-M20 device, chromatographic issues and interfering peaks were observed with Girard's Reagent P and T. These problems were partially solved using mixed organic solvents or methoxyamine for derivatization, but lower recoveries were registered. (ii) Girard's Reagent P and T provided the highest sensitivity, but boldenone esters were detected mainly as underivatized compounds, whereas methoxyamine facilitated their derivatization. (iii) High extraction efficiency (>50 %) and minimal matrix effects (< 25 %) were obtained when Mitra tip was used as device. In contrast, cellulose-based devices showed significant matrix effects (>25 %). For initial screening procedure, methanol and Girard's Reagent P or T (1.0 mg/mL in acetic acid/methanol/water 2:6:2, v:v:v, 30 min, 65 °C) are recommended (limits of detection: 0.5-1.0 ng/mL for boldenone esters; 0.05-0.50 ng/mL for the other esters). For confirmation, a compound-specific approach is suggested: methanol and methoxyamine (100 mM for 30 min, 70 °C) for boldenone esters, testosterone cypionate, enanthate and decanoate (limits of identification: 0.25-2.00 ng/mL), while Girard's Reagent P or T remains optimal for the other esters (limits of identification: 0.2-1.0 ng/mL).