Analytical challenges and opportunities in the extraction, separation, and identification of food phospholipids

Phospholipids (PLs) are minor but functionally important food lipids whose amphiphilic structure and strong interactions with the complex food matrix make their extraction and analysis challenging. This review summarizes a matrix-aware synthesis of current strategies for PLs extraction, purification, separation, and identification across major food sources, including eggs, dairy products, plant oils, marine oils, and microbial oils. While traditional solvent-based systems are the "gold standard," new, emerging "greener" technologies such as supercritical CO₂, ultrasound, microwave, enzyme-assisted, pressurized liquid extraction, and deep eutectic solvents show promise in terms of selectivity, efficiency, molecular integrity, and environmental sustainability. Due to the lack of universal standard methodology, this review emphasizes a tiered, objective-driven analytical framework in which extraction is adapted to the matrix origin, followed by cartridge solid-phase extraction for PLs enrichment, and progressively deeper analytical resolution. The most effective analytical approach combines orthogonal tools, including TLC for qualitative screening, UHPLC coupled with universal detectors (CAD/ELSD) for robust class-level quantification, LC-MS(/MS) for molecular-species identification and oxidation/lysophospholipid monitoring, ³¹P NMR for independent class-level validation, and GC-FAME for fatty-acyl profiling. The substantial variability among published results is attributed to non-standardized extraction methods, different detector responses, and matrix-dependent effects. Accordingly, a flexible, matrix-aware analytical workflow is proposed highlighting future priorities in standardization, automation, and validation of greener technologies and analytical strategies to achieve reproducible, comparable, and sustainable PLs analytical and production processes.