# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 7 Micro-Constituents' Analysis
### Chromatographic Techniques
#### 7.4.1 LC Coupled Detectors
Both hydrophilic and lipophilic vitamins are generally analyzed through ESI(+), thus generating protonated molecular ions with predictable fragmentation patterns. For example, vitamin B6 produces a characteristic fragment ion at \(m/z\) 134 due to cleavage of its side chain. Vitamin A (retinol) exhibits fragmentation due to cleavage of the isoprenoid chain, yielding fragments at \(m/z\) 83 and \(m/z\) 93. Specific transitions are selected for multiple reaction monitoring (MRM) MS studies. In detail, fragment ions having the highest intensity (quantifier transition) are used to perform quantitative analysis, while the least intense ones are used for identification purposes (qualifier transition). Table 7 summarizes the qualifier and quantifier MRM transitions used for the detection of the main vitamins occurring in milk [185]. [PERSON] et al. [186] developed and validated an LC-MS/MS method for the quantification of 25-hydroxyvitamin D (25(OH)D\({}_{2}\)) and vitamin D\({}_{2/3}\) (ergocalciferol/cholesterol) in human milk. This study found significant variability in milk vitamin D content between individuals, with milk fat concentration being a key predictor. While vitamin D\({}_{3}\) was the most abundant form, the 25(OH)D\({}_{3}\) accounted for about two-thirds of the total biological activity, highlighting its greater physiological relevance despite lower concentrations. In another study, [PERSON] et al. [187] quantified the total B vitamins of fresh cow milk. Their results suggested that fresh cow milk contains higher levels of total B vitamins compared to pasteurized milk, allowing to state that pasteurization may reduce heat-sensitive B vitamins. An inverse relationship was observed between vitamins B5 and B2 across different animal millks. It could be noticed that the B5/B2 ratio may serve as a potential marker to distinguish milk types, as it is typically 1 for goat, >1 for cow and <1 for buffalo milk. \begin{table}
\begin{tabular}{l c c} \hline \multicolumn{3}{c}{**Qualifier Transition (\(m/z\))**} & **Quantifier Transition (\(m/z\))** \\ \hline Vitamin A & 269/83 & 269/93 \\ \hline Vitamin B1 & 265/144 & 265/122 \\ \hline Vitamin B3 & 124/78 & 124/80 \\ \hline Vitamin B5 & 220/184 & 220/202 \\ \hline Vitamin B6 & 170/134 & 170/152 \\ \hline Vitamin B8 & 245/123 & 245/227 \\ \hline \end{tabular}
\end{table} 
Table 7: Qualifier and quantifier MRM transitions used for the LC-MS/MS detection of the main vitamins occurring in milk [185; 186; 187]. Due to its sensitivity and accuracy, LC-MS/MS is the most used method also for nucleotides analysis. LC-MS/MS based methods allow the simultaneous quantitation of different nucleotides, such as cytidine 5\({}^{\prime}\)-monophosphate (CMP), uridine 5\({}^{\prime}\)-monophosphate (UMP), adenosine 5\({}^{\prime}\)-monophosphate (AMP), inosine 5\({}^{\prime}\)-monophosphate (IMP) and guanosine 5\({}^{\prime}\)-monophosphate (GMP). [PERSON] et al. [188] separated these nucleotides by using a C18 column and gradient elution with water containing 0.1% (_v/v_) formic acid and acetonitrile, while their detection was accomplished through an MS/MS system consisted of a triple quadrupole analyzer and an ion source equipped with an electrospray ionization operating in negative mode. In the context of polyphenol analysis, specific mass detectors like Ion Trap, Orbitrap, Time of Flight and Triple Quadrupole are used due to their unique advantages [184; 189]. [PERSON] et al. [190] proposed an UHPLC-QTOF-MS untargeted method to exploit the impact of different pasture-based diets on the chemical profile of Sarda sheep milk, with a particular reference to the polyphenolic composition. On the other hand, a triple quadrupole MS has been successfully used to validate methods for the determination of phenolic compounds in human milk, highlighting its robustness and sensitivity for such applications [143; 191]. Also, in a study of [PERSON] et al.