Mass Spectrometry-Based Anti-Doping Analysis: A Critical Review of Emerging Analytical and Sample Pretreatment Strategies.

Illicit use of doping substances has raised significant ethical and health concerns in sports. Therefore, continuous doping control is essential for maintaining fairness and athlete's safety. Accurate and rapid detection of prohibited substances at their lowest levels in complex biological matrices, such as blood, urine, and plasma, is crucial for reliable anti-doping analysis. This critical review focuses on the applications of mass spectrometry (MS) for the detection of doping agents across diverse biological matrices. Special emphasis is given to advanced mass spectrometry platforms, particularly LC-QQQ-MS, LC-QTOF-MS, Orbitrap-MS/MS, and IRMS which are widely used for the routine screening, confirmation, and identification of emerging doping agents. Microsampling approaches such as dried blood spots (DBS) and volumetric absorptive microsampling (VAMS) provide superior alternatives to traditional sampling. Furthermore, comprehensive understanding of sample pretreatment and analytical techniques is essential. In this regard, novel microextraction techniques, including solid-phase microextraction (SPME), liquid-phase microextraction (LPME), supramolecular solvents (SUPRAS), and deep eutectic solvents (DES), have improved multi-analyte sample pretreatment strategies and facilitated advanced automation. This review also offers valuable guidance in selecting appropriate chromatography coupled mass spectrometry‑based analytical methods with sensitive detection, sample pretreatment techniques, and storage conditions during sports drug testing.