# 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  
After the chromatographic separation, several systems are used for milk micro-constituents' detection. UV-Vis detection is commonly used for vitamins and polyphenols. For vitamins analysis, the most used wavelengths are 247, 280, 320 and 450 nm [179] (Table 6) [175]. A fluorescence detector (FLD) is often used for the analysis of specific vitamins. For example, riboflavin can be detected via HPLC-FLD at 420 nm for excitation and 530 nm for emission as reported in the work of [PERSON] et al. [180]. Another example is topcherol, determined by HPLC-FLD at 290 nm for excitation and 320 nm for emission [181]. Instead, as most of BAs lack chromophores, derivatization is usually needed to reduce their polarity and improve both chromatographic behavior and UV-Vis detectability [182]. Several derivativizing reagents have been used for LC-UV determination of BAs, with dansyl chloride being the preferred one. This derivativizing agent has been used by [PERSON] et al. [183] to detect BAs in commercial millks for infants and young children. Very low concentrations of BAs were found in ready-to-use liquid milk, with concentrations and profiles related to the production processes of the different samples. A thermogenic accumulation of BAs was noticed in samples manufactured with drastic thermal treatments. As regards mass spectrometric detection coupled to liquid chromatography for the analysis of milk micro-constituents, different ionization methods are available, each suited for specific types of analytes and applications. Among these, electron ionization (EI), chemical ionization (CI), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) are widely used. However, in LC-MS applications for milk analysis, ESI is \begin{table}
\begin{tabular}{l l} \hline \hline
**Analytes** & \(\mathbf{\lambda}\)**max (nm)** \\ \hline Protocatechic acid & 259 \\ \hline (+)-Catechin & 278 \\ \hline Gentisic acid & 327 \\ \hline Vanillic acid & 261 \\ \hline Syringaldehyde & 308 \\ \hline p-Coumaric acid & 309 \\ \hline Ferulic acid & 323 \\ \hline m-Coumaric acid & 278 \\ \hline o-Coumaric acid & 276 \\ \hline Cinnamic acid & 277 \\ \hline Quercetin & 372 \\ \hline Kaempfer & 366 \\ \hline Retinol (Vitamin A) & 323 \\ \hline Vitamin B1 & 260 \\ \hline Vitamin C & 290 \\ \hline Vitamin B7 & 210 \\ \hline Vitamin B6 & 210 \\ \hline Vitamin B12 & 445 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Wavelength of maximum absorbance for polyphenols and vitamins commonly detected in milk samples. Reproduced with the permission of Elsevier [179]. particularly favored due to its compatibility with liquid phase chromatographic outputs and its ability to ionize polar and thermally labile compounds directly from solution [184]. The application of LC-MS to milk micro-constituents represents a significant advancement in food analysis, enabling the characterization of complex biochemical profiles with a level of detail that was previously unattainable. With its ability to differentiate compounds based on both mass and structural properties, MS coupled with LC remains a cornerstone technique for advancing the understanding of milk's nutritional and functional properties. Vitamins such as riboflavin and thiamine, as well as polyphenols introduced through cow feed or fortification, can be effectively analyzed using LC-MS systems. LC-MS and LC-MS/MS are increasingly used to determine vitamin levels in biological samples, offering a reliable alternative to traditional immunoassays due to their ability to simultaneously measure multiple vitamins, namely fat-soluble vitamins (A, D, E) and water-soluble B vitamins. B vitamins, though grouped by their common role as coenzymes or coenzyme precursors, are structurally different, with wide-ranging polarities and molecular weights (123-1579 Da). This chemical heterogeneity makes simultaneous analysis challenging. As a result, analyses often focus on individual B vitamins or small subsets rather than the entire group.