*2.7.5 Composition of FA contained in the TAG*

Aiming the determination of the FA contained in the TAG, the diacylglycerols (DAG+) are formed after neutral loss of one of the three FA chains. For each TAG molecule, three DAG+ ions correspond to the loss of each of the three FA, so the FA composition of any TAG molecule can easily be deduced by the mass difference (for example, neutral loss of 245, 273 and 301 corresponding to the loss of FA 14:0, 16:0 and 18:0, respectively). As expected, each TAG group may contain different

FA compositions (referred as isomeric species), being the liquid chromatography with reverse phase (RP-LC) combined to a C18 column, non-aqueous mobile phase (generally acetonitrile and isopropanol) and a surface gradient (up to 150 min) the greatest recognized method to separate species of TAG isomers from the same group or even ECN [89].

### *2.7.6 Determination of FA position in the TAG*

Determining the position of FA in the TAG (sn-1, sn-2 or sn-3) is more challenging. Two protocols are commonly employed to achieve it, both based on the partial hydrolysis of TAG to cleave FA at sn-1 and sn-3 with Grignard reagent [90] or pancreatic lipase [91], being the last one, the method approved by the AOCS [92]. It is followed by MAG TLC isolation and lastly the GC determination of the FA that was at the sn-2 position after transesterification of the MAG-sn-2-FA. Both methods may reveal the percentage distribution of the different FA at sn-2 position and also for a particular FA, the percentage at sn-2 position compared to sn-1/ sn-3 position. In other words, this approach generates information on the overall percentage of FA in sn-2, the FA composition of a mixture and not for each individual TAG species.

It is essential to mention that the precision of these two methods depends on the complete conversion of TAG and DAG into MAG, which needs to be carefully monitored. This is a rather time-consuming procedure, so a simplified protocol combining lipase digestion with direct LC–MS and quantification of MAG is urgently needed.

## **2.8 Determination of TAG, phospholipids and sterols from human milk by HPLC analysis**

The first approaches for the analysis of TAG in lipid-rich matrices have been based on GC coupled with FID and MS. However, in recent years, to simplify sample preparation, *i.e*., to avoid time-consuming preliminary treatments, TAG analysis have been carried out by high performance liquid chromatography (HPLC) [93, 94]. TAG analysis by HPLC can be performed by normal phase (NP) and reversed phase (RP). Among it, RP with non-aqueous (NA) mobile phases is the most extensively used mode. In NARP-HPLC the separation is based on the equivalent carbon numbers (ECNs); TAG with the same ECN can be separated based on the position, configuration of the double bounds, the length and unsaturation of FA. In NP separation mode it has been used silver ions impregnated columns, and this mode is named as silver ion (Ag)-HPLC. In (Ag)-HPLC the separation is based on specific silver ion/double bond interactions and the retention times depends on the position, on the cis/trans double bonds configuration and increases with the unsaturation number of the chains [93, 95].

Several detection systems have been used for TAG analysis, such as ultra-violet (UV) detector, evaporative light scattering detector (ELSD) and refractive index detector (RID) which are suitable for quantitative analysis by means of reference, but it does not permit structural information. Thus, HPLC coupled to MS is the currently preferred TAG analysis technique, being electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) the favorite ionization sources [93, 96].

For phospholipids analysis, HPLC is the most commonly used chromatographic technique. PLs are primary determined by NP-HPLC, but RP-HPLC and, in the last years, hydrophilic interaction liquid chromatography (HILIC) has also been

**103**

**3. Conclusions**

newborns.

1589/2017).

None.

**Acknowledgements**

**Conflict of interest**

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis*

used. In NP and HILIC modes the separation is based on the different polarity of the "headgroups" and in RP mode the separation is based on the features of the chain lengths, number of unsaturation and geometry of acyl chains [97, 98]. Among the detection systems used for phospholipids determination, low-wavelength UV detectors, ELSD, RID and most recently, charged aerosol detector (CAD), ELSD is probably the most extensively reported for phospholipids class analysis in the food matrices. The determination of phospholipids by HPLC coupled to MS has been also increased in the last years, being ESI and matrix-assisted laser desorption ionization (MALDI) the preferred ionization sources. Despite the advantages of MALDI, ESI is more used due to the difficulty in coupling HPLC to MALDI-MS [99–101].

Finally, for sterols analysis the most conventional used chromatographic techniques are GC coupled to FID or MS detection systems and HPLC coupled to UV detection systems. However, the high temperatures achieved during GC methods can cause degrade some sterols and HPLC-UV methods have relatively poor sensitivity and selectivity towards sterol molecules [102–104]. Thus, during the last decade, based on the accurate identifications and good selectivity and sensitivity of MS detectors, the use of HPLC coupled to MS for sterols analysis gained ground. Because sterols are highly lipophilic and have few polar groups, APCI is the most widely used ionization technique, although conventional ESI methods have also been applied. In the same way, RP-HPLC is the preferred analysis mode, and the analyte interactions with the stationary phase increase with increasing molecular sizes and decreasing number of double bonds in sterol molecules [104, 105].

The human milk fat contains 98% of neutral lipids (TAG, DAG and MAG) and the fatty acid composition of these constituents is directly related to the nutritional and physico-chemical properties of human milk fat. Especially, the most significant fatty acids in human milk are LCPUFA, including EPA, DHA and AA, essential for proper growth and development. Analytical techniques such as gas chromatography with flame ionization detector (GC-FID) can be employed to evaluate the fatty acid composition of human milk fat. Prior the chromatographic analysis, the lipids derivatization is essential to allow volatilization of the interest compounds. Moreover, TAG analysis can be carried out by mass spectrometry and high performance liquid chromatography (HPLC), in order to determine the FA contained in the TAG, as well as TAG's FA position. Conclusively, breastfeeding is an incomparable ideal food for the healthy growth and development of infants and offers numerous short- and long-term health benefits for breastfed

The authors are grateful to Laboratory of Immunogenetics of the State University of Maringa (LIG-UEM) for funding support (Process number

*DOI: http://dx.doi.org/10.5772/intechopen.80429*

#### *Lipids and Fatty Acids in Human Milk: Benefits and Analysis DOI: http://dx.doi.org/10.5772/intechopen.80429*

used. In NP and HILIC modes the separation is based on the different polarity of the "headgroups" and in RP mode the separation is based on the features of the chain lengths, number of unsaturation and geometry of acyl chains [97, 98]. Among the detection systems used for phospholipids determination, low-wavelength UV detectors, ELSD, RID and most recently, charged aerosol detector (CAD), ELSD is probably the most extensively reported for phospholipids class analysis in the food matrices. The determination of phospholipids by HPLC coupled to MS has been also increased in the last years, being ESI and matrix-assisted laser desorption ionization (MALDI) the preferred ionization sources. Despite the advantages of MALDI, ESI is more used due to the difficulty in coupling HPLC to MALDI-MS [99–101].

Finally, for sterols analysis the most conventional used chromatographic techniques are GC coupled to FID or MS detection systems and HPLC coupled to UV detection systems. However, the high temperatures achieved during GC methods can cause degrade some sterols and HPLC-UV methods have relatively poor sensitivity and selectivity towards sterol molecules [102–104]. Thus, during the last decade, based on the accurate identifications and good selectivity and sensitivity of MS detectors, the use of HPLC coupled to MS for sterols analysis gained ground. Because sterols are highly lipophilic and have few polar groups, APCI is the most widely used ionization technique, although conventional ESI methods have also been applied. In the same way, RP-HPLC is the preferred analysis mode, and the analyte interactions with the stationary phase increase with increasing molecular sizes and decreasing number of double bonds in sterol molecules [104, 105].
