# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 6 Micro-Constituents Extraction
### Extraction of Polyphenols and Their Metabolites  
The extraction of polyphenols and their metabolites from milk matrix deserves a separate discussion. As previously stated, when dairy animals consume polyphenol-rich plants (e.g., grasses, herbs and other vegetation), some polyphenols or their metabolites can be transferred into milk. However, this transfer is relatively low because polyphenols undergo significant metabolism in the digestive system of ruminants. As a consequence, their analysis requires accurate and sensitive protocols able to provide the most reliable results, starting from sample preparation and extraction. Polyphenols often bind with milk proteins through hydrogen bonding, hydrophobic interactions and ionic bonding. Hence, their effective extraction from milk requires separating them from proteins, fats and other matrix components. One recent method proposed a preliminary milk deproteinization through a one-step homogenous liquid-liquid extraction (HLLE), that uses acetonitrile as a denaturant agent and a salt solution as a separation phase [137]. The canonical precipitation of protein with solvent has been extensively studied to find the best combination of solvent (non-toxic and at a lower cost) that ensures complete precipitation, through the support of vortex, centrifugation or ultrasound treatment. Acetonitrile has been found to be the best option while the vortexing time is not influent [138]. As regards milk defatting, in general, it is carried out after defrosting at 4 \({}^{\circ}\)C and then centrifuging at 3000 rpm for 30 min [139]. Once proteins and fats have been removed from the matrix, polyphenols can undergo extraction. Conventional extraction techniques (e.g., solvent extraction) are not always efficient for milk, requiring more complex protocols like solid phase extraction (SPE) or liquid-liquid extraction with multiple steps. Common SPE sorbents include divinyl benzene (DVB), C18 (octadecyl-silica) and porous polystyrene/divinylbenzene (PS/DVB). These sorbents provide a high recovery of polyphenols, and the extraction efficiency does not vary according to the number of extractions [140]. Solvents used for polyphenols elution are acetonitrile/water 1:1 [141] or methanol [142]. Liquid-liquid extraction (LLE), instead, is carried out by using polar solvents assisted by salts (sodium chloride or ammonium chloride) [143]. For example, [PERSON] et al. [143] used ethyl acetate (710 \(\mu\)L) and acetonitrile (510 \(\mu\)L), with the addition of 0.1 g of sodium chloride for a miniaturized and more efficient version of LLE, i.e., dispersive liquid-liquid microextraction (DLLME) of gallic acid, epicatechin, epicatechin gallate, epigallocatechin gallate, daidzein, caffeic acid, quercetin, genistein, naringenin, hesperetin and kaempfer from human milk. Also, due to their sensitivity to light, pH and temperature, which make them prone to degradation during sample preparation and storage, careful handling, including refrigeration, protection from light, and sometimes pH adjustments, are often necessary to preserve compounds' integrity. Mildly acidic conditions (pH 3-5) increase the solubility of many polyphenols, minimizing interference from other milk components [144; 145]. \begin{table}
\begin{tabular}{p{56.9 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Extraction Method** & **Advantages** & **Drawbacks** & **Micro-Constituents** & **Sed Solvents** & **Refs.** \\ \hline \multirow{8}{*}{Solvent Extraction} & & & Vitamins & Water/acid for proteins denaturation & [125] \\ \cline{3-6}  & \multirow{4}{*}{Standard methods and high yield} & & & Minerals & Nitric Acid 65\% & [127; 128] \\ \cline{3-6}  & & & & & \\ \cline{3-6}  & & & & & \\ \cline{3-6}  & & & & & \\ \cline{3-6}  & & & & & \\ \cline{3-6}  & & & & & \\ \hline Nucleotides & Water & & & & \\ \cline{3-6}  & & & & & \\ \hline Polyphenols & Acetonitrile/water & [141] & & & \\ \hline \hline \end{tabular}
\end{table}