Anabolic agents, such as anabolic-androgenic steroids or β2-adrenergic agonists, are designed to positively affect muscle mass, strength, and recovery, and they are the most commonly detected doping agents used for performance enhancement in sports [1]. The steroid hormone testosterone and its precursors are the most abused anabolic doping agents, according to the World Anti-Doping Agency (WADA) [2,3]. Amongst β-agonists, clenbuterol is the most effective, and it has been used in sports because it can stimulate the central nervous system and promote growth, thus, it is included in the list of prohibited substances published by the WADA. Moreover, athletes and bodybuilders often use higher doses than those used for medical reasons, resulting in uncontrollable health risks [4]. Fatalities due to long-term use of anabolic androgen steroids have been described in the previous studies [5]. Thus, the effective control and detection of anabolic agents in human matrices is critical in the field of anti-doping. Sample preparation is essential to enable the reliable detection of low amounts (µg/kg or ng/kg) of anabolic steroids and β-agonists in complex matrices.

Liquid-liquid extraction (LLE) [[6], [7], [8]] and solid-phase extraction (SPE) [[9], [10], [11], [12], [13]] are the most commonly used extraction and clean-up techniques for anabolic agent residues in samples (such as urine, serum, saliva, and dietary supplements) before chromatographic analysis. Compared to LLE, SPE requires smaller amounts of organic solvents and sample volumes and provides high preconcentration. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method, which involves dispersive SPE with C18/primary secondary amine as the sorbent, have been used for the extraction and purification of anabolic steroids and β-agonists from milk and protein supplements [14,15]. Recent studies focused on developing fast and simple techniques with low levels of sample and solvent consumption. Liquid-liquid extraction (LLME) [16] and molecularly imprinted polymer-based solid-phase extraction (SPME) fibers [17] have been employed to determine anabolic steroids in urine samples.

Magnetic solid-phase microextraction (MSPE), which is a simple alternative to these approaches, uses a magnetic sorbent that can be retrieved using an external magnet, avoiding separation via filtration and centrifugation [18]. Magnetic materials are crucial in the extraction procedure [[19], [20], [21], [22], [23], [24]]. When the size of the adsorbent material is reduced to the nanoscale, its adsorption performance is enhanced due to the increased specific surface area and number of accessible active sites. After suitable modifications, magnetic nanoparticles (MNPs) can effectively separate and/or enrich analytes in complex matrices.

Polyethylene glycol (PEG) is a polyether compound with a flexible hydrophilic long chain, and it exhibits good biocompatibility and low toxicity. PEG is an ideal agent for use in modifying the surfaces of MNPs, which provides them with hydrophilic surfaces, minimizes agglomeration, and reduces environmental hazards.

In this study, PEG-coated MNPs were synthesized, characterised, and evaluated for their potential as adsorbents for the extraction of anabolic agents from urine. The extraction performance of MNPs was improved by PEG modification, and this adsorbent displayed good reusability. The extraction mechanisms were discussed. The optimal conditions were determined after a detailed study of the MSPE experimental parameters. Method validation was conducted to assess the applicability of the MSPE-LC-MS method, including its linearity, sensitivity, precision, and recovery. A comparative study of this method with the previously reported methods used in analyzing anabolic substances in complex samples was conducted to demonstrate the simplicity and sensitivity of the proposed method.