###### Abstract  
Despite the considerable steps taken in the last decade in the context of antineoplastic drug (AD) handling procedures, their mutagenic effect still poses a threat to healthcare personnel actively involved in compounding and administration units. Biological monitoring procedures usually require large volumes of sample and extraction solvents, or do not provide adequate sensitivity. It is here proposed a fast and automated method to evaluate the urinary levels of cyclophosphamide and bihosphamide, composed of a miniaturized solid phase extraction (\(\mu\)SPE) followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The extraction procedure, developed through design of experiments (DoE) on the ePrep One Workstation, required a total time of 9.5 min per sample, with recoveries of 77-79% and a solvent consumption lower than 1.5 mL per 1 mL of urine sample. Thanks to the UHPLC-MS/MS method, the limits of quantification (LOQ) obtained were lower than 10 pg/mL. The analytical procedure was successfully applied to 23 urine samples from compounding wards of four Italian hospitals, which resulted in contaminations between 27 and 182 pg/mL. antineoplastic drugs; biological monitoring; ultrahigh-performance liquid chromatography; tandem mass spectrometry; \(\mu\)SPEed; micro solid phase extraction Article  
28 December 2023  
23 January 2024  
26 January 2024  
229 638 2024 20  
medical products (HMPs). In 2020, the European Commission conducted a study and consultation to further amend the CMD [5; 6; 7], which resulted in the last revision (Directive 2022/431/EU) [8] in March 2022 being adopted by national laws in all EU member states by 5 April 2024. Both Directive 2022/431/EU and the resulting guidelines, \"Guidance for the safe management of hazardous medicinal products at work\" [9], published in 2023, encourage the development of monitoring methods and biological surveillance for exposed health professionals. While surface contamination sampling remains an important tool to detect incorrect working procedures and increase the awareness of hospital workers [10; 11; 12; 13; 14], biological monitoring is still the only way to truly assess occupational exposure and identify possible pathological correlations. Nowadays, ADs can still often be found in the biological fluids of exposed healthcare personnel [15]. Numerous analytical methods can be encountered in the literature for the extraction of ADs in urine samples, mainly based on solid-phase extraction (SPE) or liquid-liquid extraction (LLE), but they usually require large volumes of samples and extraction solvents, or do not provide adequate sensitivity [16; 17; 18; 19; 20; 21] due to the progressive decrease in encountered contaminations [22]. Solvents' consumption shown in previously cited studies can vary from 5 to 45 mL and, in many cases, by the use of not environmentally friendly substances such as dichloromethane and n-hexane. Thus, the development of growingly sensitive, green and fast methods of analysis via the means of innovative technologies is the only way to keep up with the decreasing levels of contamination which can be encountered after the latest safety implements, following the \"as low as reasonably achievable\" (ALARA) principle. Many miniaturized solid-phase extraction techniques (\(\upmu\)SPE) are being developed and applied to sample preparation in numerous fields of application [23; 24]. This sample treatment technique aims to improve selectivity and sensitivity through sample clean-up and pre-concentration. It generally involves miniaturized cartridge-type devices that contain packed solid particles of porous chromatographic material. The mechanism of action of SPE consists in the interaction between the solid sorbent phase and the analytes contained inside a liquid sample solution that is percolated through the SPE bed. The analytes bonded to the sorbent are then recovered thanks to the affinity of a small amount of solvent.