# Methods for Determining Polycyclic Aromatic Hydrocarbons (PAHs) in Milk: A Review
## 2 Polycyclic Aromatic Hydrocarbons
### PAHs in Milk  
Choosing the suitable PAH extraction and measurement method is crucial for obtaining reliable results. Milk is a complex matrix, and lipophilic PAHs are trapped in fats, not just dispersed droplets. The fats are also trapped in protein structures. Therefore, an \"ideal\" extraction process should not only involve the transfer of PAHs from the matrix to the solvent (milk \(\rightarrow\) fat \(\rightarrow\) solvent), but should also include a fat release step. This will allow for more accurate measurements and more reliable results [22, 65].  
A challenge is the presence of numerous interfering substances in the matrices of dairy products. Casein micelles can undergo structural changes under the influence of temperature and pH, leading to the entrapment of lipids within their structures. Along with these lipids, the lipophilic PAH molecules can also become embedded, creating additional challenges during PAH extraction. Furthermore, milk contains structures such as the milk fat globule membrane (MFGM), a three-layered structure that forms fat globules. Due to their non-polarity, PAHs can also interact within these membranes. The disintegration of micelles, driven by modifications in electrostatic interactions, increases membrane rigidity and promotes the formation of protein-lipid aggregates, with protein molecules positioned on the outer layer [66, 67, 68]. Consequently, it is essential to release PAHs from these various structures during PAH extraction from milk and processed milk products. Failure to adequately address this step can result in inaccurate analytical results. These can be co-extracted with PAHs, making separating and quantifying them difficult. Current PAH  
Figure 2: Structures of PAHs and regional breakdown of special concern [40, 64].  
extraction and identification techniques still have significant limitations. Many traditional methods, such as LLE (liquid-liquid extraction) and Soxllet extraction, are time-consuming, labor-intensive, and often require large amounts of hazardous solvents. In addition, the high-fat content of dairy products, causing PAHs to accumulate in them, also contributes to the difficulty of extraction and the use of large amounts of solvents [32]. It is, therefore, advisable to develop promising new methods, such as solid-phase extraction (SPE) or solid-phase microextraction (SPME) (Figure 3). These are currently more popular and better-performing than the methods previously used [7; 22; 65].  
As part of developing \"green chemistry\", efforts are being made to modify conventional extraction methods to minimize solvent use [69]. One such approach is liquid-phase microextraction (LPME), which requires significantly less solvent than traditional liquid-liquid extraction (LLE) [70]. Various techniques can facilitate fat release, including conventional methods like saponification and unconventional methods such as ultrasound and microwave-assisted extraction [65].  
Beyond the fat release step, selecting appropriate solvents for extracting fat from milk is essential. This choice depends on the polar and non-polar nature of the matrix and PAH molecules, as well as the extraction method and chromatographic techniques employed (HPLC or GC) [71]. The following section discusses the parameters of methods used for analyzing milk and dairy products in greater detail.