A sustainable nano-liquid chromatography method for the determination of phenolic acids in coffee and its by-products

• Lab-made nano-LC for separation of 10 phenolic acids in coffee and by-products. • Baseline separation achieved in 35 min using C18 core-shell particles. • Large volume injection with on-column focusing boosts sensitivity. • Sustainable extraction of phenolic acids using Natural Deep Eutectic Solvents. • Workflow sustainability assessed with AGREEprep and BAGI greenness metrics. This study presents a method based on lab-made nano-liquid chromatography (nano-LC) for the simultaneous separation of ten phenolic acids (PAs) in coffee powder and spent coffee ground, using both ultraviolet and MS detection. Preliminary experiments were performed with a 100 µm I.D. capillary column packed in-house to select suitable stationary phases and optimize chromatographic conditions. Baseline separation of all analytes was achieved using a capillary column packed with C18 core–shell particles (5 µm particle size, 100 Å) within 35 minutes under step-gradient elution. Method sensitivity was enhanced through on-column focusing by investigating the effects of the dilution solvent type and injection volume. PAs were extracted from both coffee matrices using different green solvents and four natural deep eutectic solvents (NaDES) via vortex-assisted liquid phase microextraction (VA-LPME). The analytical method was fully validated, showing intra- and inter-day repeatability (RSD%) below 4.5% and 6.0% for retention time and peak area, respectively. LOD and LOQ values ranged from 0.1 to 6.6 µg/mL and 0.4 to 18.8 µg/mL, while linearity over the concentration range of interest for all analytes was estimated with R 2 ≥ 0.966. The developed nano-LC-UV method was extended to MS detection using a liquid–junction ESI interface to confirm the presence of PAs in all coffee samples previously identified by UV detection via the fortification method. Finally, to ensure compliance with Green Analytical Chemistry principles, the procedure was evaluated using greenness and practicability metrics, evaluated by AGREEprep (score, 0.66) and BAGI (score, 57.5), respectively, which highlighted the methodology’s low environmental footprint and high practical applicability within the framework of miniaturized analytical chemistry.