Time-controlled online SPE: strategic approach for N-acylethanolamines quantification in complex matrices

Quantifying and monitoring the level of N-acylethanolamines (NAEs) in biological fluids is becoming increasingly important to better understand their role in health and disease. The complexity of biological matrices, however, poses significant challenges for accurate quantification, with traditional pretreatment methods often proving insufficient in certain cases. This study introduces a novel approach utilizing Online Solid Phase Extraction (SPE) to quantify NAEs in equine plasma, specifically Palmitoyl Ethanolamide (PEA) and Oleoyl Ethanolamide (OEA). In the original analytical method here developed and validated, established toluene liquid-liquid extraction was used to isolate the lipid-like fraction, followed by an innovative Time-Controlled Online SPE coupled to HPLC-MS/MS. This strategic temporal approach allows target analytes to rapidly elute to the analytical column while retaining lipidic interferents, preventing matrix contamination and ensuring selective analysis. The method validation demonstrated excellent linearity, while providing high recovery and suitable matrix effects. The limits of detection (LOD) and quantification (LOQ) were determined as 0.27 ng/mL and 0.83 ng/mL for PEA; 0.04 ng/mL and 0.11 ng/mL for OEA. The innovative approach here presented allowed for reliable NAE quantification in equine plasma and provided unprecedented data on the endogenous levels of PEA and OEA in this species. This method has potential applications for the analysis of other complex biological matrices with high levels of interferents that share chemical similarities with the target compounds. This novel approach significantly advances the understanding of the endocannabinoid system in equines and serves as a valuable tool for future research in this domain.