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

Area of Science:

• Analytical Chemistry
• Forensic Toxicology
• Pharmacokinetics

Background:

• Dried blood spot (DBS) sampling offers a minimally invasive method for analyzing various analytes, including steroid esters.
• Accurate quantification of steroid esters in DBS is crucial for applications in doping control, clinical diagnostics, and pharmacokinetic studies.
• Optimization of extraction and derivatization protocols is essential to overcome challenges associated with DBS analysis, such as matrix effects and analyte stability.

Purpose of the Study:

• To evaluate and optimize microsampling devices, extraction solvents, and derivatization reagents for the sensitive and accurate analysis of 27 steroid esters in DBS.
• To establish reliable analytical conditions for both screening and confirmation of steroid esters using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Main Methods:

• Evaluation of five microsampling devices: Whatman DMPK-C, Whatman 903, PerkinElmer-226, Tasso-M20, and Mitra® tips.
• Testing of eight extraction solvents, including methanol and various organic solvent mixtures.
• Assessment of three derivatization reagents: Girard's reagent P, Girard's reagent T, and methoxyamine, followed by LC-MS/MS analysis.

Main Results:

• Methanol demonstrated the highest recoveries, though Tasso-M20 with Girard's reagents caused chromatographic interferences.
• Mitra® tips provided high extraction efficiency (>50%) and minimal matrix effects (<25%), outperforming cellulose-based devices.
• Girard's reagents P and T offered high sensitivity, while methoxyamine improved derivatization of boldenone esters.

Conclusions:

• For initial screening, methanol with Girard's reagent P or T is recommended for steroid ester analysis in DBS.
• For confirmation, a compound-specific approach using methoxyamine for boldenone and testosterone esters, and Girard's reagents for others, is advised.
• The Mitra® tip is identified as a superior microsampling device for steroid ester analysis due to its efficiency and low matrix effects.