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

Area of Science:

• * Analytical Chemistry
• * Biochemistry
• * Forensic Science

Background:

• * Dried blood spots (DBS) are increasingly used for drug testing in sports, offering convenience but presenting analytical challenges due to varied support materials.
• * Existing extraction methods are often specific to either cellulose or polymeric DBS, limiting their universal application.
• * The detection of small peptides and their metabolites in sports doping control requires sensitive and selective analytical techniques.

Purpose of the Study:

• * To develop and validate a single, qualitative screening method for detecting small peptides and their metabolites in both cellulose and polymeric DBS.
• * To address the analytical challenges posed by different DBS matrices in sports drug testing.
• * To ensure the method meets the stringent requirements set by the World Anti-Doping Agency (WADA).

Main Methods:

• * Extraction of analytes from both cellulose and polymeric DBS using a dual-step solvent extraction process (formic acid solution followed by acetate buffer).
• * Purification of combined extracts via solid-phase extraction utilizing a mixed-mode, weak cation exchange approach.
• * Analysis using ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Orbitrap Q-Exactive) with optimized acquisition modes.

Main Results:

• * A robust method capable of analyzing small peptides on both polymeric and cellulose DBS using a single procedure.
• * Satisfactory validation results according to WADA regulations, demonstrating good selectivity and sensitivity.
• * Achieved limits of detection in the low nanogram per milliliter (ng/mL) range, suitable for sports drug testing.

Conclusions:

• * The developed method provides a versatile and effective solution for detecting small peptides in DBS for anti-doping purposes.
• * This approach simplifies sample analysis by accommodating different DBS matrices with a unified protocol.
• * The method's sensitivity and selectivity make it suitable for routine application in sports drug testing programs.