# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 8 Mass Spectrometric Advances in Milk Metabolomic and Lipidomic  
Nowadays, the recent introduction of ambient ionization MS techniques has allowed to simplify and greatly shorten the time required for milk metabolomic and lipidomic analysis. Indeed, ambient ionization is a set of ionization techniques used in mass spectrometry that enable the analysis of samples in their natural state with minimal or no sample preparation [195]. These methods work under atmospheric pressure and are designed to ionize molecules directly from complex matrices such as biological tissues, surfaces, or liquids, allowing real-time analysis. Furthermore, great utility comes from the possibility to use smaller, less expensive and potentially portable mass spectrometers, making the technique particularly well suited to in situ or on-site experiments. In the field of milk analysis, ambient ionization is mostly used for authentication studies, to allow the precise differentiation of cow, sheep and goat milks, as well as other dairy matrices. The commonly used techniques are desorption electrospray ionization (DESI) and direct analysis in real-time (DART) coupled to high-resolution mass spectrometry. The primary differences among DESI and DART, lie in their ionization mechanisms and sample handling [196; 197; 198; 199; 200]. With DESI, a charged solvent spray is directed onto the sample surface under atmospheric pressure. The interaction between the droplets and the surface causes desorption of analytes, which are then ionized and transferred to the mass spectrometer [198; 200]. On the contrary, the mechanism of DART involves the ionization of a stream of heated, inert gas (e.g., helium or nitrogen), thus creating excited-state species [196; 197]. These species interact with the analyte in an open environment, transferring energy to produce ions. In this case, the sample is directly exposed to the ionizing gas stream.  
In a recent study, [PERSON] et al. [201] developed a DESI-MS based method to detect biomarkers from five distinct animal and plant-based milk sources with the support of chemometric tools. Their method was optimized for MS signal intensity, by choosing a suitable sample treatment. Different protocols were tested, involving milk dilution with organic solvent (methanol) or deionized water. As methanol removed most of the lipids occurring in cow milk, thus causing a loss of biomarkers for milk classification, milk  
Figure 5: Comparison of untargeted vs. targeted extraction methods for milk metabolite analysis.  
diluted with water in a 1:4 ratio was directly loaded onto the glass slide sample plate and evaporated to dryness at room temperature for DESI-MS analysis. Notable differences in spectral data were observed among cow milk, goat milk, camel milk, soy milk and oat milk. Their results showed that lipid composition can be used as a key factor to discriminate among milk from different species, with sphingolipids (SP) and glycerophospholipids (GP) being more abundant in cow milk. A recent DART-HRMS approach developed by [PERSON] et al. (2020) allowed to easy and deep access to the chemical composition of milk directly from samples diluted with an organic solvent. Ethyl acetate was used in this case. The conducted metabolomic analysis demonstrated that monoacylglycerollycerols (MAG) mainly characterize and explain the metabolic variation of grazing milk, while lowland milk is mainly characterized by ketoacid derivates, amines and organic acids.  
Hence, the recent advances in the MS field and the introduction of the ambient ionization MS techniques, have revolutionized milk metabolomic and lipidomic analysis by enabling rapid, real-time profiling with minimal sample preparation. These methods allow for precise milk authentication, species differentiation and insights into metabolic variations between grazing and lowland milk, highlighting lipids as key discriminating compounds.