A Multitarget Screening Method for the Detection of Small Peptides in Dried Blood Spots for Doping Control Analysis

Over the past few years, dried blood spots (DBS) have been approved as a valid matrix for drug testing in sport. They undoubtedly offer advantages but also pose analytical challenges. For example, various DBS supports are nowadays available (polymeric and cellulose based) and an extraction method suitable for one support is not necessarily transferable to a different one. Herein, we present a qualitative screening method for the detection of a representative selection of small peptides and their metabolites in both cellulose and polymeric DBS. The analytes were extracted using an extraction solvent containing formic acid 1% in water/acetonitrile/methanol (70/15/15), followed by a second extraction with acetate buffer. To remove interferences and increase sensitivity, the combined extracts were further purified using solid-phase extraction (mixed-mode, weak cation exchange). Analysis was performed using ultrahigh-performance liquid chromatography combined with high-resolution mass spectrometry (orbitrap Q-Exactive). The analysis time was 7.5 min, and the acquisition was performed in full-scan mode, with the addition of some product ion scan acquisitions to increase selectivity or sensitivity for a few compounds that were particularly challenging. The method permits the analysis of small peptides on both polymeric and cellulose DBS samples with the same procedure for either matrix. Validation was performed following the World Anti-Doping Agency regulations, and the method proved satisfactory in terms of selectivity and sensitivity (limits of detection in the low ng/mL range) and applicable to the analysis of sport samples for the detection of small peptides.