**2.6 Analytical methods for determination of fatty acids in human milk by gas chromatography: flame ionization detector (GC-FID)**

## *2.6.1 Different extraction methods for total lipids (TL)*

The fatty acid composition of human milk has been extensively studied over the last 25 years and almost all of the studies are obtained after lipid extraction.

Different methods of fat extraction have been proposed to determine the fat content in human milk by traditional method: crematocrit [77], esterified FA [78], Gerber method (butyrometer) [79], and gravimetric [80].

Recent methods have been proposed in the direct quantification of FA in human milk by gas chromatography [81]. However, the gravimetric method is considered the gold standard for extracting the total lipid (TL) content in human milk, and Folch et al. [80] is one of the most recommended methods for it. This methodology extracts non polar, polar and neutral lipid using mixture of cold solvents for extraction and not compromising the chemical structure of the lipids.

#### *2.6.2 Quantification of fatty acids in human milk by GC-FID*

Prior to chromatographic analysis, a derivation step of the TL is required, converting the different lipid classes in fatty acids methyl esters (FAMEs). This step is necessary to enable the volatilization of the compounds of interest, and to allow the determination by GC; there are several methods for this purpose [82].

Normally, the quantitative determination of FA in the human milk is data generally normalized as g of FA per 100 g of FA or expressed as percentage of weight (area normalization) FA relative to all FA exposed in a chromatogram. In the normalization methods, all the FA of the sample must be considered and, in the case of omission of a component, the other components are affected. On the other hand, the results presented by the normalization present difficulties of interpretation and, therefore, in nutritional values of the human milk [81]. The main drawback about normalization methods is that the data set does give information on the amount of FA (in mass) per volume or mass in human milk.

Recent studies express the composition of FA in mass concentrations of FA by mass of liquid human milk, and direct quantification of FA in human milk by gas chromatography has been proposed [81].

The determination qualitative and quantitative of FAMEs by GC-FID is among the most commonplace analyses in lipid matrices. Quantification of FAMEs by GC-FID has been effectively performed whereas detection with GC tandem mass spectrometry (CG-MS) has been employed mainly for qualitative analysis of FA. Both detectors FID and MS, for chromatographic analysis, the derivation step of the lipids classes is required to conversion of TAG, DAG, MAG, phospholipids (transesterification process), FFA (esterification process) in FAMEs [83].

The American Oil Chemists Society (AOCS) and the Association of Analytical Chemists (AOAC) recommend parameters for accurate quantification of FA. Both sources indicate the use of internal standard (IS, methyl tricosanoate - 23:0) and

capillary columns. The IS are used to minimize the experimental errors, control extraction, transesterification and esterification, undesired saponification. The IS cannot be part of the composition of lipid sample or whole sample [83].

#### *2.6.2.1 Relative response factor in the FID and methodology*

The FID has become one of the most popular measuring devices employed in GC and it is the most sensitive detector for hydrocarbons, being the FA merely carboxylic acids with long chains of hydrocarbons.

As the FAMEs respond differentially in FID because of the combustion of carbon compounds that produces ions due to the chain size, presence of FA-substituted functional groups (carboxylics group, double bond) in a hydrocarbon, it reduces the combustion efficiency, and therefore, relative response factors in the FID depends on the effective carbons number. Thus, it is necessary to use correction factors for the FAMEs in relation to the IS. The applied factors are the experimental (empirical correction factor) and the theoretical correction factor (FCT), theoretically determined from the number of effective carbons. It is also important the conversion factor from FAME to FA (FCEA) [83, 84]. In this chapter, methodologies employing IS (23:0) and correction factors FCT, and FCEA for the FID response are described below [83].

The follow column and chromatography condition has been used with efficiency in separation process of the methyl esters (FAMEs) in total lipid of milk. The FAMEs are prepared by transesterification and esterification of total lipids. It is injected and separated into a CG-FID. The column Select FAME (part number CP-7420) fused silica capillary column 100 × 0.25 mm, and 0.25 μm of 100% cyanopropylpolysiloxane (high polarity) was employed. The carrier gas (H2) flow rates are 1.2 mL min<sup>−</sup><sup>1</sup> ; auxiliary gas (N2) 30 mL min<sup>−</sup><sup>1</sup> ; H2 and synthetic air 35 and 350 mL min<sup>−</sup><sup>1</sup> , respectively. The volumes of the sample injection are 1.0 μL, split of 1:80. The injection temperature: 200°C, detector temperature: 240°C. The column temperature-programmed: 165°C for 7.00 min, the heating ramp of 4°C min<sup>−</sup><sup>1</sup> until 185°C (4.70 min.) after that another programming heating of 6°C min<sup>−</sup><sup>1</sup> until 235°C (5.00 min.). The FAMEs are identified by comparison of their retention times, determined by computer software analysis, with those of individual purified standards or secondary standards. The quantifications of FAMEs are performed with internal standard (IS 23:0) and the corrections factors for the FID response are utilized for the determination of concentrations [84]. The composition of fatty acids (FA) in the total lipids of samples is calculated in mg g<sup>−</sup><sup>1</sup> of total lipids (TL) using the Eq. (1) [83, 84].

$$\mathbf{M}\mathbf{x} = \begin{array}{c} \mathcal{A}\_{\mathcal{X}} \mathcal{M}\_{\rho} \mathcal{F}\_{\mathcal{C}\mathcal{T}} \\ \mathcal{A}\_{\mathcal{F}} \mathcal{M}\_{\mathcal{A}} \mathcal{F}\_{\mathcal{C}\mathcal{A}\mathcal{S}} \end{array} \tag{1}$$

**101**

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

**2.7 Analytical techniques for analysis of lipids by electrospray ionization (ESI)** 

In order to determine TAG in food, initially, is essential to extract the lipids contained on it. Folch [80] and Bligh and Dyer [85] methods are extensively employed for the extraction of milk lipids. The addition of antioxidant, such as BHT is recommended prior to extraction to avoid lipid oxidation [86]. The internal standards for

Preceding the LC–MS analysis, the extraction solvents (chloroform/methanol) must to be removed by evaporation, and the lipids are reconstituted with solvent compatible with the mobile phase of LC. Moreover, a pre-sample of SPE or TLC columns previous to LC–MS analysis may facilitate the lipid species identification

Sample direct infusion into the mass spectrometer was one of the first techniques employed in TAG analysis [88]. Its main advantage is the rapidity. However, despite the progresses in the last 15 years, there are three main problems associated with the technique: (1) ion suppression, (2) isotopic interference and (3) differentiation of isomers. Consequently, a chromatographic separation is crucial to avoid ions suppression and to differentiate the isomeric species, being the direct infusion

Nowadays, milk TAG are characterized in three levels: (1) carbon chain size, (2) level of composition in FA and (3) level of FA position; providing information, respectively, on the composition of milk fat TAG, FA composition of a lipid species

TAG is defined in this chapter as a series of species with the equal total number of acylated carbons (CN) and the equal number of double bonds (DB), regardless of its constitution in FA. So each TAG group has a unique chemical formula and a precise mass. Ammonium salt is added to the mobile phase so the TAG are detected as ammoniated adducts (to become more stable and avoid the formation of different adducts with the same molecule) in the positive ionization mode ESI+. The most abundant TAG groups contain 26–54 acyl CN and 1–8 DB, with molecular mass

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

*2.7.1 Lipid extraction for analysis by liquid chromatography-mass spectrometry* 

each lipid class are added to the matrix prior to extraction [87].

technique rarely employed for characterize lipid in milk sample.

and regiospecific distribution of FA on TAG molecules.

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

**and other techniques**

due to improved resolution.

*2.7.2 Direct infusion analysis in MS*

*2.7.3 TAG determination by LC-MS*

*2.7.4 Identification of the carbon chain size*

*2.7.5 Composition of FA contained in the TAG*

ranging from 500 to 1000.

*(LC-MS)*

Mx is the concentration of FA "x" in mg g<sup>−</sup><sup>1</sup> of TL, Ax is the FA "x" peak area, Ap is the IS (FAME 23:0) peak area, Mp is the IS mass added to the sample in mg, MA is the sample mass in grams, FCT is the theoretical correction factor of the FID and FCEA is the conversion factor from FAMEs to FA.

It is concluded that to determine the FA composition by GC-FID with high accuracy, and to express the composition of fatty acids in mass of FA per sample (by volume or mass) it is necessary to apply the correct derivation technique, internal standard and flame ionization detector relative response factors using the effective carbon number and conversion from FAMEs to FA.

*Biochemistry and Health Benefits of Fatty Acids*

ylic acids with long chains of hydrocarbons.

described below [83].

rates are 1.2 mL min<sup>−</sup><sup>1</sup>

using the Eq. (1) [83, 84].

350 mL min<sup>−</sup><sup>1</sup>

capillary columns. The IS are used to minimize the experimental errors, control extraction, transesterification and esterification, undesired saponification. The IS

The FID has become one of the most popular measuring devices employed in GC and it is the most sensitive detector for hydrocarbons, being the FA merely carbox-

As the FAMEs respond differentially in FID because of the combustion of carbon compounds that produces ions due to the chain size, presence of FA-substituted functional groups (carboxylics group, double bond) in a hydrocarbon, it reduces the combustion efficiency, and therefore, relative response factors in the FID depends on the effective carbons number. Thus, it is necessary to use correction factors for the FAMEs in relation to the IS. The applied factors are the experimental (empirical correction factor) and the theoretical correction factor (FCT), theoretically determined from the number of effective carbons. It is also important the conversion factor from FAME to FA (FCEA) [83, 84]. In this chapter, methodologies employing IS (23:0) and correction factors FCT, and FCEA for the FID response are

The follow column and chromatography condition has been used with efficiency in separation process of the methyl esters (FAMEs) in total lipid of milk. The FAMEs are prepared by transesterification and esterification of total lipids. It is injected and separated into a CG-FID. The column Select FAME (part number CP-7420) fused silica capillary column 100 × 0.25 mm, and 0.25 μm of 100% cyanopropylpolysiloxane (high polarity) was employed. The carrier gas (H2) flow

; auxiliary gas (N2) 30 mL min<sup>−</sup><sup>1</sup>

1:80. The injection temperature: 200°C, detector temperature: 240°C. The column temperature-programmed: 165°C for 7.00 min, the heating ramp of 4°C min<sup>−</sup><sup>1</sup> until 185°C (4.70 min.) after that another programming heating of 6°C min<sup>−</sup><sup>1</sup>

is the IS (FAME 23:0) peak area, Mp is the IS mass added to the sample in mg, MA is the sample mass in grams, FCT is the theoretical correction factor of the FID and FCEA

It is concluded that to determine the FA composition by GC-FID with high accuracy, and to express the composition of fatty acids in mass of FA per sample (by volume or mass) it is necessary to apply the correct derivation technique, internal standard and flame ionization detector relative response factors using the effective

235°C (5.00 min.). The FAMEs are identified by comparison of their retention times, determined by computer software analysis, with those of individual purified standards or secondary standards. The quantifications of FAMEs are performed with internal standard (IS 23:0) and the corrections factors for the FID response are utilized for the determination of concentrations [84]. The composition of fatty

acids (FA) in the total lipids of samples is calculated in mg g<sup>−</sup><sup>1</sup>

Mx is the concentration of FA "x" in mg g<sup>−</sup><sup>1</sup>

carbon number and conversion from FAMEs to FA.

is the conversion factor from FAMEs to FA.

, respectively. The volumes of the sample injection are 1.0 μL, split of

; H2 and synthetic air 35 and

of total lipids (TL)

of TL, Ax is the FA "x" peak area, Ap

until

(1)

cannot be part of the composition of lipid sample or whole sample [83].

*2.6.2.1 Relative response factor in the FID and methodology*

**100**
