# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 4 Analytical Methods for Milk Macro-Constituents  
### Analysis of Fatty Acids  
Some of the more conventional techniques that have been extensively used for the analysis of milk fatty acids are gas-chromatography coupled with flame ionization detection (GC-FID) or mass spectrometric detection (GC-MS) [61]. Due to their low volatility and high polarity, both GC-FID and GC-MS methods require a preliminary derivatization step, which involves the esterification of FAs to fatty acid methyl esters (FAMEs). FAs esterification is typically conducted with hydrochloric acid (HCl), acetyl chloride (CH\({}_{3}\)COCl), sulfuric acid (H\({}_{2}\)SO\({}_{4}\)) and boron trifluoride (BF\({}_{3}\)) as derivativizing agents [62; 63]. In general, 30 m columns are preferred for the chromatographic separation, as they ensure fast analysis, with good values for limit of detection (LOD) and limit of quantification (LOQ) [64; 65]. The dynamic liner range is usually comprised between 10 \(\mu\)g/mL and 500 \(\mu\)g/mL [66], while the LOQ and LOD are around 0.07 \(\mu\)g/mL and 0.02 \(\mu\)g/mL, respectively [67]. Longer columns result in the separation of a higher number of fatty acids [68]. For example, [PERSON] et al. [69] separated saturated, monounsaturated and polyunsaturated lipids with a number of carbon atoms ranging between 8 and 22, with a 40 m column. On the other hand, [PERSON] et al. [70] used a 50 m column for the successful separation of the CLA isomers, i.e., _cis-9_, _trans-_11 CLA, _trans-_10, _cis-_12 CLA and _trans-_11 C18:1. Moreover, [PERSON] et al. [71] performed the separation of more than 50 different fatty acids, including short-chain (4:0), very long-chain (23:0), highly unsaturated fatty acids (22:5n-3), as well as various branched-chain fatty acids, 18:1 isomers and CLAs, in a 90-min run with a coating select FAME, 100% bonded cyano-propyl phase, 100 m \(\times\) 0.25 mm column (Figure 2). Compared to GC-FID, GC-MS has the advantage of providing more structural information, as the comparison with the MS spectral database allows for unambiguous identification of the compounds, while ensuring higher efficiency and better selectivity [72; 73]. Electron ionization (EI) is used with different type of MS analyzers, i.e., single quadrupole (Q), ion trap, time-of-flight (TOF). Methyl esters of straight-chain fatty acids have characteristic EI-MS spectra. In particular, [M \(-\) 43]\({}^{+}\) and [M \(-\) 31]\({}^{+}\) ions are characteristic of straight-chain saturated fatty acid methyl esters (n-SFA) (Figure 3). The MS spectra of shorter-chain fatty acids methyl esters typically show a fragment ion at \(m/z\,74\) ([CH\({}_{3}\)-O-C(OH)=CH\({}_{2}\)]\({}^{+}\)) due to \(\gamma\)-hydrogen migration and McDonald's rearrangement (Figure 3) [73]. Moreover, fragment ions due to McLafferty rearrangement allow the identification of most ester derivatives of fatty acids. With the McLafferty rearrangement, a hydrogen atom from position 4 of the aliphatic chain migrates to the carbo-methoxy group, presumably through a six-membered transition state, which is sterically favored. One result is the elimination of an olefin. If one of the hydrogen atoms on the C4 atom is substituted, then the McLafferty ion will be appreciably lower in intensity than expected [73]. \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline
**Extraction Method** & **Advantages** & **Drawbacks** & \begin{tabular}{l} **Macro-** \\ **Constituents** \\ \end{tabular} & **Used Solvents** & **Refs.** \\ \hline Solid phase & \(\bullet\) & \begin{tabular}{l} Low consumption \\ of solvents \\ High yield \\ Pre-concentrated \\ and purified \\ extract obtained \\ \end{tabular} & \(\bullet\) & \
\begin{tabular}{l} High cost \\ Pre-conditioning \\ and cleaning steps \\ necessary for SPE \\ cartridges \\ \end{tabular} & Lipids & \(
\begin{tabular}{l} 1\% formic acid in \\ methanol \\ \end{tabular} & [49] \\ \hline \hline \end{tabular}
\end{table}