Protocol for the upgraded high-throughput SPME system for biocompatible<i> in</i><i> vitro</i> extraction from small volume for metabolomics and pharmaceutical assays

This study presents an updated application of Solid Phase Microextraction (SPME) technology for high- throughput, time-course metabolomic analysis in in vitro cell culture models. SPME, a versatile and minimally invasive sample preparation technique, was integrated with 96-well plate systems to enhance metabolomic profiling while maintaining cell viability. The proposed SPME-lid system was used to evaluate employed SPME fibers with miniaturized polytetrafluoroethylene (PTFE)-based coatings. The innovative SPME-lid system enables in-incubator sampling, preserving optimal cell growth conditions and permitting repeated extractions from the same culture overtime. Experiments demonstrated the biocompatibility of the SPME coatings and their negligible influence on key cellular parameters. This approach's time-course analysis revealed shifts in metabolite levels, showcasing its potential for biochemical and drug development studies. Furthermore, the method aligns with green chemistry principles, reducing solvent and plastic consumption, and was evaluated with a 0.75 AGREEprep score for sustainability. This protocol represents a significant advancement in metabolomic methodologies, providing a scalable, ecofriendly solution for comprehensive metabolomic profiling in pharmaceutical and toxicological research.