# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
###### Abstract  
In recent years, there has been a growing interest in identifying both the macro- and micro-constituents of milk, as their functional and health benefits have been recognized. This review provides a comprehensive overview of all milk constituents, including fats, proteins, oligosaccharides and polysaccharides, vitamins, minerals and polyphenols, along with their extraction protocols and the analytical methods employed for their separation and detection over the last ten years. The principles, strengths and limitations of these methods are discussed, highlighting their importance in advancing milk research. Traditional methods such as spectrophotometry, and advanced technologies, like mass spectrometry and omics approaches, are described to collectively enable a comprehensive understanding of milk's nutritional and functional components. Election criteria for the analytical platforms suitable for target analytes are provided.  
m 2025 2  
The analysis of milk involves unique challenges due to its complexity, with components spanning a wide range of molecular sizes, polarities and concentrations. Furthermore, the presence of a complex matrix of proteins, fats and carbohydrates can interfere with the detection and quantification of trace components [9]. Key issues include matrix effects during ionization in mass spectrometry, interferences in spectroscopic measurements and the need for rigorous sample preparation protocols to isolate target analytes [10]. To this regard, pre-concentration and purification techniques such as solid phase extraction (SPE) can help remove interfering matrix components before analysis, while the use of validated and reproducible protocols tailored to specific metabolite classes allows for target analysis. Modern analytical methods must overcome these challenges while providing high sensitivity, specificity and reproducibility. Recent advancements in analytical chemistry have introduced techniques like ambient ionization mass spectrometry (MS), which enables direct and automated analysis of milk with minimal sample preparation [11]. Moreover, omics approaches, including proteomics, lipidomics and metabolomics, are increasingly being integrated with advanced analytics to comprehensively study milk's constituents and their interactions [12; 13; 14]. After a brief description of the main classes of macro- and micro-constituents of milk, this review focuses on the analytical protocols employed in the last ten years for their extraction and analysis, highlighting their principles, strengths and limitations. Relevant papers related to the topics under review were searched in internationally available databases, i.e., Scopus, Google Scholar, Web of Science, Science Direct and PubMed. As keywords, three different ones were chosen, namely milk micro-constituents' analysis, milk macro-constituents' analysis and milk metabolomics. After an initial screening of titles, abstracts and keywords, eligible articles were downloaded and carefully reviewed to evaluate the relevance of their methodology, results and conclusions. Publications without available full-text and no significant contribution to the analytical field were excluded.