Structure and nomenclature

Modern techniques

Before  looking at the various strategies to study fatty acids you would like to learn details about the history  of their discovery, so read the next chapter. 


We owe a considerable debt to ancient investigators who, prior to about 1935, made enormous contributions to our knowledge of the fatty acid composition of natural lipids despite primitive equipments and analytical techniques. Since the first works of Chevreul and for about a century, chemists isolated lipids using only solubility properties of solvents, the formation of salts of fatty acids which were further characterized by their raw formula, and ebullition or fusion temperatures.
The period following 1935 has been marked by new and more efficient procedures for separating and studying fatty acid mixtures. These procedures include ester distillation, crystallization of urea complexes or of various metallic salts, various forms of chromatography and countercurrent distribution.

An overview of these techniques applied to fatty acids can be found 

in clicking here

The discovery in the mid-1950's of gas-liquid chromatography (GLC) has revolutionized the analysis of fatty acids and, undoubtedly, this technique is the most frequently used. Indeed, for the quantification of individual fatty acids in any acylated lipids, GLC must be adopted.
In some other studies, complementary techniques should be considered. Metabolic studies involve the knowledge of the intensity of labelling of molecular species with radioactive atoms while identification studies require the separation and quantification of hydroxylated, branched-chain, trans or conjugated fatty acids. All these investigations are more easily run with HPLC than with GLC procedures since positional and conformational isomers are more easily separated by HPLC than by GLC. Furthermore, HPLC is the method of choice for preparative scale separations of particular fatty acids for further structural or metabolic studies. In contrast to GLC which preferred flame ionization detection (FID), the choice of the detector for HPLC analysis is important and determines the adopted procedure. Several detections are possible, the most used are light scattering, UV, fluorescence and radioactivity.
In general, fatty acids are separated by HPLC as derivatized molecules but unesterified forms can also be chromatographed if acidic solvent systems are used.

For some precise purposes only the amount of fatty acids is to be known. Global methods are useful when the fatty acid profile is not in the scope of the investigation. 

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1- How to prepare fatty acids (free or bound) for further analysis ?

2- How to derivatize fatty acids before GLC ?

3- How to purify or fractionate fatty acids ?

4- How to analyze fatty acids by GLC ?

5- How to analyze fatty acids by HPLC ?

- study of normal fatty acids

- study of trans fatty acids

- study of conjugated fatty acids

- study of dicarboxylic acids

- study of cyclic fatty acids


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Fatty acids may be found in scarce amounts in free form but, in general they are combined in more complex molecules through ester or amide bonds.
The isolation of free fatty acids from biological materials is a complex task and precautions should be taken at all times to prevent or minimize the effects of hydrolyzing enzymes.

Free fatty acids

A simple procedure was described previously using silica gel column chromatography with an acidic elution of fatty acids. Furthermore, free fatty acids may be isolated during the TLC separation of acylglycerols but may also be collected during the separation by HPLC of neutral lipids. They may be either methylated yielding fatty acid methyl esters (FAME) or reacted with various UV absorbing or fluorescent tags.

When fatty acids (medium and long-chain) are in aqueous media they may be accurately extracted using a small C18 bonded phase column (SPE) (Battistutta F et al., J High Resol Chromatogr 1994, 17, 662). This method was also used to isolate fatty acid ethyl esters from alcoholic beverages. Shortly, the SPE cartridges are prepared in washing with methanol and water. 50 ml of liquid are passed through the column followed by a washing with acidified water. Analytes are eluted with 2 ml dichloromethane and 2.5 ml pentane.

The extraction of long-chain fatty acids from fermentation medium and industrial effluents with a 98 to 100% recovery was described (Lalman JA et al., JAOCS 2004, 81, 105). Maximal recovery was obtained by adding 2 ml of hexane/ter-butyl methyl ether (1/1), 80
ml of 50% H2SO4, and 0.05 g NaCl to 1 ml of the aqueous sample and mixing for 15 min at 200 rpm. A lower recovery was obtained only for caproic (C6:0) and caprylic (C8:0) acids : 27 and 76% recoveries, respectively.

The purification of free fatty acids has been done by solid-phase microextraction (SPME) (Tomaino RM et al. J Agric Food Chem 2001, 49, 3993). The fiber sheath of a 30
mm thick poly(dimethylsiloxane) fiber (Supelco) was incubated at 110°C for 80 min in the acidified medium and then placed into the injector of a gas chromatograph whose temperature was increased from 100°C to 245°C. Unfortunately, a progressive and rapid loss of sensitivity occurred with decreasing fatty acid chain length. Thus, it was necessary to determine the response factors for each fatty acid in relation to an internal standard (C17). Advantages of that extraction procedure are the little sample preparation, the absence of organic solvents, the detection of short chain fatty acids, and a good reproducibility.

A one-step extraction and derivatization method has been proposed, essentially based on a dispersive liquid-liquid microextraction (Pusvaskiene E et al., Chromatographia 2009, 69, 271). This simple and fast method using ethyl chloroformate as derivatization reagent was applied for the determination of free fatty acids in water (tap, lake, sea, river).
For many years, diazomethane was the reagent of choice to selectively derivatize and then detect free fatty acids due to its highly specific methylation of the carboxylic acid functional group. While its activity is very defined, it is dangerous and can be difficult to obtain. An important review has compiled a collection of methods which allow for the detection of hydroxy and non-hydroxy free fatty aicds without the use of diazomethane (Potter G et al., Eur J Lipid Sci Technol 2015, 117, 908).

A convenient, economic, and high throughput approach has been established to separating free from esterified fatty acids in using a chemical derivatization and immobilization on amino silica nano-paarticles (Chen J et al., J Chromatogr A 2016, 1431, 197).

Short-chain fatty acids (C1 to C5) in biological specimens need a special treatment taking into account their volatility. Thus a simple and efficient procedure using a vacuum transfer followed by HPLC enable the accurate determination of these acids in the nanomolar range in tissues and secretions (Stein J et al., J Chromatogr 1992, 576, 53). An eficient procedure using an extraction with a hollow fiber coupled with gas chromatography has been reported (Zhao G et al., J Chromatogr B 2007, 846, 202).
Application of gas chromatography coupled to mass spectrometry following headspace solid-phase microextraction was applied with great accuracy and sensitivity to the determination of free volatile fatty acids in aqueous samples (Abalos M et al., J Chromatogr A 2000, 891, 287). Valuable results were obtained for the determination of C2-C7 fatty acids in raw sewage.
Free medium-chain fatty acids in beer have been extracted using adsorption on a specific stir bar (Gerstel twister). The determination of caproic, caprylic, capric and lauric acids with solvent back extraction was described (Horak T et al., J Chromatogr A 2008, 1196-1197, 96). The procedure utilized 10ml of sample stirring with the stir bar with 1000rpm for 60min at room temperature. Solvent back extraction used 200
ml of solvent (dichloromethane/hexane, 50/50) at room temperature.

Bound fatty acids

When fatty acids are combined in more complex molecules such as acylglycerols, cholesterol esters, waxes and glycosphingolipids, they can be obtained free by saponification (inorganic or organic basic solution) or acidic hydrolysis and then derivatized. FAME may be also obtained directly by transesterification (alcoholysis or methanolysis) of the fatty acid-containing lipids. The extraction and methylation may also be combined in a one-step procedure, this is particularly recommended for very small samples in order to prevent any loss of fatty acids during the classical procedures. A usefull comparison of the various derivatization methods my be consulted (Ostermann A.I., et al., Prostagl, Leukotr Essential Fatty Acids 2014, 91, 235). A detailed protocol for the analysis of plasma and tissues is included in this article.


When fatty acids are required in free form for further analysis, lipids (present as glycerides, glycerophosphatides, glycosyldiglycerides, sterol esters or waxes) are first hydrolyzed in alkaline medium allowing to extract also the unsaponifiable material if present in the crude lipid mixture (sterols, alcohols, hydrocarbons, pigments, vitamins...). Glycosphingolipids are poorly hydrolyzed with the described procedure but, if any contribution of these complex lipids is to be avoided, a mild saponification process must be adopted.


Methanolic potassium hydroxide: mix 10 ml of 3M aqueous KOH to 90 ml methanol.
Hexane, diethyl ether, phenophthalein in ethanol, 6M HCl.


Pipet an aliquot of lipid extract (up to 30 mg) into a screw-capped tube (Teflon-lined). Evaporate the solvent and add 5 ml methanolic KOH. Warm for 1 h at 80°C in a water or a sand bath.
After cooling, extract the non-saponifiables with 2 washings of 5 ml diethyl ether. Add a few drops of phenolphthalein indicator to the lower phase and acidify with HCl (about 0.3 ml).
Extract the fatty acids with 2 washings of 5 ml hexane. When short-chain fatty acids are present in the lipid extract, it is necessary to extract more extensively with hexane (5 or 6 times). Do not evaporate too extensively the hexane phase (keep at a mild temperature) to prevent loss of these fatty acids.
Fatty acids may be weighed, titrated to determine their neutralization equivalent or converted to methyl esters before fractionation or GLC analysis..

An alternative method for saponification has been proposed using a microwave-assisted treatment (Pineiro-Avila G et al., Anal Chim Acta 1998, 371, 297). A closed reactor containing the lipid sample and an adapted volume of ethanolic KOH solution is irradiated for a short time (2-3 min) in a microwave oven at an exit power of about 350 W. The extraction of fatty acids is then processed as described above.

Saponification of dry powder may be done directly before the extraction of fatty acids or non-saponifiable compounds (Sanchez-Machado DI et al., J Chromatogr A 2002, 976, 277). 
250 mg of ground samle are mixed with 5 ml of 0.5M KOH in methanol. The tubes are incubated at 80°C for 15 min (vortexing every 5 min). After cooling in ice, 1 ml water and 5 ml hexane are added and the tubes are vortexed for 1 min. After a short centrifugation, 3 ml of the upper phase are transferred to another tube and dried under nitrogen before analysis.

Acidic hydrolysis

When the investigated lipid extract contains complex lipids as sphingolipids, an efficient procedure to free amide-bond fatty acids is needed. It is recommended to fractionate any crude lipid extract into glycerolipids and glycosphingolipids before applying an alkaline saponification to the former and an acidic hydrolysis to the later.
The procedure previously proposed for ceramides consists in a treatment with methanolic HCl in presence of water which is known to give rise to only minor amounts of by-products. It is noticeable that this procedure yields directly FAME ready to be fractionated or analyzed by GLC.

Organic basic hydrolysis

The organic basic solution, 1 M tetramethylammonium hydroxide (TMAH) was employed and recommended for the hydrolysis of extremely small amounts of lipids (lower than 1 mg) (Woo KL et al., J Chromatogr A 1999, 862, 199). That procedure was found excellent for small samples while saponification with ethanolic KOH was found unsuitable. Using TMAH, a 2 fold recovery of long-chain fatty acids was obtained as compared with the classical KOH hydrolysis and the reliability of data was very high. 

Deacylation of cerebrosides and sulfatides by a powerful microwave-mediated saponification was reported (Taketomi T et al. Biochem Biophys Res Comm 1996, 224, 462). The reaction was run in 0.1 M NaOH in methanol for 2 min in 500W microwave oven. After acidification the fatty acids are extracted in hexane and methylated.

Combined basic and acid hydrolysis

Another practical approach to the technical problem of the hydrolysis of sphingolipids has been described using a one-spot heating in a microwave oven with 0.1 M NaOH in methanol for 2 min followed by 1M HCl in methanol for 45 s (Itonori S et al., J Lipid Res 2004, 45, 574).


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Before GLC analysis it is necessary to prepare non-reactive derivatives of fatty acids (methyl esters or other derivatives) which are also more volatile than the free acid components. Acylated lipids are transformed by a transesterification reaction by which the glycerol moiety is displaced by another alcohol (methanol, butanol, propanol...) in acidic conditions (HCl or BF3).

The generation of methyl esters can be done in acidic or in alkaline conditions on isolated lipids or fatty acids but also directly by a one-step procedure combining lipid extraction and transesterification on small amounts of dried tissue.
On a large scale, fatty acid methyl esters, used as a substitute of diesel fuel (Biodiesel), are prepared by transesterification of vegetal oils with sodium methylate, NaOH or KOH in dry medium. 

Other fatty acid derivatives may be prepared as an answer to some specific problems

A - Acid-catalyzed esterification

The most common derivatives of fatty acids are the methyl esters obtained by heating free fatty acids with a large excess of anhydrous methanol in the presence of a catalyst, boron trifluoride (Morrison et al J Lipid Res 1964, 5,600). It must be noticed that O-acyl lipids are transesterified very rapidly with the same reagent. 


14% Boron trifluoride in methanol (Alltech or Sigma) (keep refrigerated under nitrogen and discard after 3 months or when solids appear at the bottom of the vial).
Pentane, chloroform.


As a general procedure, an aliquot of lipid extract (about 10 mg) is dried under nitrogen in a screw-capped glass tube and 1 ml of BF3/methanol is added.
If triacylglycerols or sterol esters are analyzed alone or are abundant in the extract, the dry lipids are dissolved in 0.75 ml of chloroform/methanol (1/1, v/v) and 0.25 ml BF3/methanol are added. If possible, the tube is closed after flushing with nitrogen.
Heat in boiling water (or at 100°C in a sand bath) the time indicated for the respective lipid:


Heating time (min)

Fatty acids




Sterol esters















After cooling, add 1 ml water and 2 ml pentane. Vortex for 1 min, centrifuge at low speed and collect the upper phase. Pentane is evaporated and the residue is immediately dissolved in 50-100 µl hexane. The solution is ready for injection in the gas chromatograph.

After TLC, spots containing fatty acid-based lipids may be scraped, collected and treated with the BF3/methanol solution directly in a glass tube. It was reported that selective loss of unsaturated fatty acids was observed oon certain brands of plates (Sowa JM et al., J Chromatogr B 2004, 813, 159). Thus, the authors determined that no loss occurred in both neutral and phospholipids with Alltech or Merck silica gel plates.

Alternative methods :

A sulfuric acid-methanol method was used with success to derivatize very long chain fatty acids (C24:0-C36:0) before gas chromatography analysis (Mendez Antolin E et al., J Pharm Biomed Anal 2008, 46, 194).
Acidic conditions generated by 3M HCl in dry methanol or methanolic sulfuric acid have been also described.

Preparation of fatty acid methyl esters from various sources using commercial aqueous HCl was also described (Ichihara K et al., J Lipid Res 2010, 51, 635). Yields of FAME were the same as those obtained with boron trifluoride method. Furthermore, the reagent is very convenient, safe and cheap.
Shortly, the reagent is made from 9.7 ml commercial concentrated HCI (35%, w/w) diluted with 41.5 ml of methanol and was stored in a refrigerator. A lipid sample was dissolved in 0.20 ml of toluene, then 1.50 ml of methanol and 0.30 ml of the reagent solution were added in this order. The tube was vortexed and then heated at 100°C for 1 h. After cooling, 1 ml of hexane and 1 ml of water were added for extraction of methyl esters in the hexane phase.

An improved method for determining medium- and long-chain in lipid samples using one-step transesterification with acetyl chloride has been reported (Xu Z et al., Lipids 2010, 45, 199). The data suggest that the method can be easily used to accurately determine fatty acids (C6–C24) in functional foods and lipid emulsions.

B - Base-catalyzed transesterification

Fatty esters form with a base (alcoholate) form an anionic intermediate which is transformed in the presence of a large excess of the alcohol into a new ester. Free fatty acids are not subject to nucleophilic attack by alcohols or bases and thus are not esterified in these conditions.

Derivatizations in the presence of basic catalysts have the advantages of speed and mild heating conditions. Thus this type of catalysis is recommended in samples with short-chain fatty acids or labile fatty acids (polyunsaturated, cyclopropane rings, conjugated unsaturations...). 

The most useful basic transesterifying agents are 1 to 2M Na or K methoxide in anhydrous methanol. These solutions are stable for several months at 4°C until a white precipitate of bicarbonate salt is formed. Glycerolipids are rapidly transesterified (2-5 min) at room temperature.

An improved rapid procedure to analyze fatty acid esters from triacylglycerols and phospholipids is described below (Ichihara K et al., Lipids 1996, 31, 535) :  


Hexane, 2 M methanolic KOH, capped plastic tubes.


Up to 10 mg of lipids are dissolved in 2 ml hexane followed by the addition of 0.2 ml of 2 M methanolic KOH. The tube is vortexed for 2 min at room temperature. After a light centrifugation, an aliquot of the hexane layer is collected for GC analysis.
It must be pointed out that sterol esters and waxes do not react under these conditions.

A modification and an adaptation of that procedure has been proposed allowing the direct preparation of fatty acid methyl esters from polar lipids (phospholipids) in lipid mixtures without prior isolation (Ichihara K et al., Lipids 2010, 45, 367). No obvious differences were found between the fatty acid compositions of phospholipids determined by that method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography.

An alternative base-catalyzed methodology in mild conditions was adapted for milk or seed lipids using K tert-butoxide and 2-methoxyethanol (Destaillats F et al., Lipids 2002, 37, 527): 


1 M K tert-butoxide in THF (Aldrich), 2-methoxyethanol, hexane, Na sulfate.


ml of a solution of K tert-butoxide in THF are added to 200 ml anhydrous 2-methoxyethanol in a closed vial. After homogenization, up to 10 mg of lipid in 1 ml hexane are added. Keep the mixture at 40°C for 15 min. After cooling, 1 ml water and 2 ml hexane are successively added. After 5 s vortexing and a short centrifugation, the organic phase is collected, dried over anhydrous Na sulfate and analyzed by GLC.

We have adopted another approach for some labile samples. A rapid and mild method which avoids the formation of oxidation products was described by Piretti et al. (Chem Phys Lipids 1988, 47, 149). We have most precisely adopted this procedure for the analysis of highly unsaturated lipids since higher amounts of polyunsaturated fatty acids were found when compared to the BF3/methanol procedure.
Furthermore, if hydroperoxy fatty acids are present, they are reduced into the corresponding hydroxy components.


2M NaOH, NaBH4, anhydrous Na2SO4.
Ethyl acetate, methanol, hexane.


2 mg of neutral lipids or up to 100 mg polar lipids are dried in a glass tube.
Add 1 ml of the reagent made in dissolving immediately before use 400 mg NaBH4 in 10 ml of the mixture methanol/2 M NaOH (19/1, v/v).
The mixture is stirred for 20 min at room temperature. After adding 2 ml water, the methanol is eliminated under nitrogen. The methyl esters are recovered from the aqueous phase by extracting 3 times with 1 ml ethyl acetate. The organic phase is then washed 3 times with 1 ml water and dried by adding Na2SO4. After vortexing and centrifugation, the ethyl acetate is evaporated and the residue dissolved in a small amount of hexane for GLC analysis.

Comments : A 30-min, micro-base-catalyzed method for vegetable oil fatty acid determination has been proposed using a novel fatty acid derivatization method (Lall RK et al., JAOCS 009, 86, 309). The sensitivity was improved for relatively small pure oil samples without loss of accuracy.

One step method combining saponification and methylation

A one-step method with saponification followed by 60 min of methylation time has been described as a simple, fast and accurate tool to quantitatively analyse fatty acids in human red blood cells (RBC) for for clinical and nutritional studies (Rodrigues RO et al., Chromatographia 2015, 78, 1271).
Shortly: In a 7 mL glass vial, 150 µL of RBC sample containing internal standard and BHT were added and mixed with 500 µL of methanolic KOH solution (0.2 M). Vials were capped and vigorously vortexed for 30 s followed by a nitrogen flushing. Saponification was performed at 90 °C during 10 min. After cooling the samples, 2 mL of BF3 methanolic solution was added and the vials were vortexed for 30 s followed by nitrogen flushing. Transmethylation was performed at 90 °C during 60 min. After cooling the samples, 1 mL of n-heptane was added and fatty acid methyl esters were extracted twice by vortex mixing
(30 s). The supernatant was transferred to a clean glass vial and evaporated under nitrogen gas. Finally, samples were resuspended in 100 µL of n-heptane and analysed by GLC.

Another one step and very rapid 10 seconds) method has been described using sodium methylate as the main reagent for the analysis of triacylglycerol composition (Ichihara K et al., Anal Biochem 2016, 495, 6). Shortly, methanolic CH3ONa (2 M) is prepared by diluting 25% (w/w, 4.37 M) CH3ONa with methanol. In a small glass test tube are placed 1 ml of hexane containing 20 mg or less of triacylglycerols and 0.5 ml of acetone. To the lipid solution is added 75 ml of 2M CH3ONa with vortexing. Methanolysis is completed within 10 s and the reaction is terminated with 1 ml of 0.5 M acetic acid. The upper organic layer containing methyl esters is washed with 1 ml of water. The hexane solution of FAMEs is then analyzed by gas chromatography. Under these conditions, trioleoylglycerol is converted to methyl oleate with an average yield of 99.3%.

C - Direct transmethylation without prior extraction

The concept of direct transesterification of techniques has been reported for small tissue samples (1-10 mg) or small volumes (about 50 ml) of biological fluids (blood, milk) and plant samples. 

Procedure for small tissue samples : 

A tissue sample containing as low as 10
mg of lipids is introduced at the bottom of a screw-capped tube (Teflon-lined). Then add 1 ml of methanolic HCl, 1 ml of methanol and 0.5 ml hexane. Close tightly the tube and heat at 100°C for 1 h (shake several times).
After cooling add 2 ml of hexane and 2 ml of water. Mix not too vigorously the tube and collect the hexane layer after a short centrifugation. Before GC analysis, the extract may be concentrated by evaporation under nitrogen if necessary.   

Comments : 

The total fatty acid composition of plasma was determined with a transesterification procedure similar to that described above (Glaser C et al., PlosOne 2010, 5, e12045). As claimed by the authors, the sample preparation time and analysis costs are reduced to a minimum. The method is an economically and ecologically superior alternative to conventional methods for assessing plasma fatty acid status in large studies.

In lipid-producing bacteria or microheterotrophs, the direct transesterification method was shown to be the most efficient to study the fatty acid profiles (Lewis T et al. J Microbiol Meth 2000, 43, 107). The proposed procedure consists in treating freeze-dried cells at 90°C for 60 min in the mixture methanol/conc HCl/chloroform (10/1/1, v/v)(3 ml). After addition of water (1 ml), fatty acid methyl esters are extracted by vortexing 3 times with 2 ml of hexane/chloroform (4/1, v/v).

A critical review on in situ transesterification avoiding the use of lipid extraction describes all aspects in order to achieve accurate and reliable results (Carrapiso AI et al. Lipids 2000, 35, 1167). An application of direct transmethylation to red blood cell membranes and cultured cell has been also described (Rise P et al., Anal Biochem 2005, 346, 182).
A comparative study of "direct" and "two steps" (extraction followed by derivatisation) methods has been done with plasma samples (Amusquivar E et al., Eur J Lipid Sci Technol 2011, 113, 711). The ‘‘two steps’’ method appears more appropriate and reliable, and C19:0 but not C15:0 should be used as the internal standard.

A quantitative and simple in situ method for the assessment of the fatty acid composition of solid samples (triturated seeds, lard, muscle) through their pentyl esters was described (Eras J et al., J Chromatogr A 2004, 1047, 157). The reaction was carried out using chlorotrimethylsilane and 1-pentenol as reagents for 40 min at 90°C. It permits major recoveries of the total saponifiable lipids present in solid samples, a 40 min reaction time ensuring the total conversion of lipids to the corresponding fatty acid pentyl esters.
A similar but more rapid (30 s) transesterification process using a one step carried out in a microwave reactor has been described for quantifying meat acylglycerides (Tomas A et al., J Chromatogr A 2009, 1216, 3290).

A comparative study between the direct methylation and the classic procedure has shown that, in eggs, the direct methylation procedure was less precise than the second procedure (Mazalli MR et al., Lipids 2007, 42, 483).

A rapid and efficient method for direct transesterification of lipids from plant sources has been described and compared with several other derivatization procedures (Alves SP et al., J Chromatogr A 2008, 1209, 212). The most efficient procedure was as follow :
1mL of internal standard (C17:0, 1mg/mL) and 1mL of toluene were added to 250mg of sample, followed by the addition of 3mL of 5% HCl solution in methanol (prepared by the addition of acetyl chloride to the methanol). After homogenization on vortex at slowspeed, sampleswere maintained for 2h at 70◦C in a water bath. After that, the solution was left to cool at room temperature and subsequently neutralized with 5mL of 6% K2CO3. FAMEs were extracted with 2mL of hexane, and 1 g of both Na2SO4 and activated carbon were added. Finally, samples were centrifuged for 5 min at 2500 rpm, the supernatantwas transferred to new tubes and the solvent removed under nitrogen at 37 ◦C. The final residue was dissolved in 1mL of hexane, and stored until GC analysis.
An additional step based on solid-phase extraction was necessary to produce clean samples.

An efficient direct transesterification has been described for assay of the fatty acid content of microalgae (Griffiths MJ et al., Lipids 2010, 45, 1053). Higher levels of fatty acid in the cells were obtained with that procedure in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume.

A micromethod for the fatty acid analysis of glycerophospholipids using a sodium methoxide solution has been described with cheek cell samplings (Klingler M et al., Lipids 2011, 46, 981-90).

Procedure for small amounts of bacteria :

The knowledge of the fatty acid composition of microorganisms is now recognized as essential for their taxonomic classification as well as for the evaluation of the nutritional quality of alternative microbial sources of fats. To guarantee a high recovery of fragile fatty acids, such as cyclopropane and conjugated linoleic acids, as well as a high degree of methylation for all types of fatty acids, a rapid and reliable method is needed. A direct methylation method representing a valuable alternative to other methylation procedures has been described (Dionisi F et al., Lipids 1999, 34, 1107).


One hundred milligrams of dried bacterial samples, with 500
mg of internal standard, is transesterified using 1 ml of methanolic HCl (1.5M) (from Supelco) and 1 ml methanol, at 80°C for 10 min. Water (2 ml) is added and after mixing and low speed centrifugation the upper phase is collected for gas chromatographic analysis.

Procedure for small amounts of fluid :

A convenient method was developed for preparation of fatty acid methyl esters in glycerolipids of blood or milk (Ichihara K et al., Lipids 2002, 37, 523).


About 50
ml of blood or milk are spotted onto a small piece of Whatman 3MM filter paper (1.5x1.5 cm) that has been previously washed with acetone containing 0.05 % BHT. Each piece, once dried for 30 min in vacuo is inserted into a small test tube, to which 2 ml hexane and 0.2 ml 2M KOH/methanol are added (alkali-catalyzed alcoholysis). After vigorous mixing or sonication for 2 min at room temperature, the solution is neutralized with acetic acid. To each tube is added 2 ml water with light mixing. An aliquot of the hexane layer was collected and evaporated to dryness; FAME are dissolved in 0.02 ml hexane or methyl acetate before GC analysis.
The presence of BHT on the filter paper allows the protection of unsaturated fatty acids for at least 7 days even exposed to the air.

A similar direct procedure using boron trifluoride-methanol as esterification reagent was described for the determination of fatty acids in human milk (Lopez-Lopez A et al., Chromatographia 2001, 54, 743).

A direct evaluation of the fatty acid status in a drop of blood was described (Marangoni F et al., Anal Biochem 2004, 326, 267). No more than 50
ml of blood were absorbed on a piece of chromatography paper and directly treated with 3 N methanol/HCl at 90°C for 1 h. The method was validated for reproducibility and satisfactorily compared with a conventional method.

Procedure for dried samples

A convenient method was developed for preparation of fatty acid methyl esters directly on freeze-dried milk samples. The extraction step is not required and the sample can be immediately subjected to the transesterification procedure (Gastaldi D et al., Chromatographia 2009, 70, 1485).
Briefly, a volume of 60
ml of a 500 mg per ml standard solution of linoleneaidic acid (internal standard) was evaporated to dryness under nitrogen in a 20 ml centrifuge tube provided with a Teflon-lined screw cap. Weighed amounts of sample were added to the residue. 3 ml of boron trifluoride–methanol reagent were added to the mixture under nitrogen. The tube was closed, heated at 80 °C for 45 min and cooled. For fatty acids methyl esters extraction, 1 ml of a NaCl saturated aqueous solution and 3 ml of n-hexane were added. The mixture was vigorously shaken and phase separation was achieved by centrifugation. 1.5 ml of clear supernatant was transferred into an autosampler vial for GLC analysis.

A very precise study was developed to analyze fatty acids in a capillary dried blood spot system in order to protect n-3 long-chain fatty acids from oxidation for up to 2 months at room temperature. The methodology has been validated for clinical applications through a direct comparison with established methods (Liu G. et al., Leukotr Essential Fatty Acids 2014, 91, 251).

D - Other fatty acid derivatives

Butylation - Propylation

Methylation is not efficient for analyzing carboxylic acids of medium or short chain (< C12) as their volability can lead to unquantifiable losses. Thus, derivatizations forming propyl or butyl esters have been used for a long time. Butyl esters are more frequently used for simultaneous analysis of low- and high-molecular weight fatty acids. The conversion efficiency of various carboxylic acids has been reported under different reaction conditions (Hallmann C et al., J Chromatogr A 2008, 1198-1199, 14). The most efficient recovery for fatty acids was obtained using n-butanol/BF3 (10%, w/w) from from Sigma–Aldrich at 100°C for 2 hours. Care must be taken when different types of carboxylic acids are to be analyzed.

The recovery of short-chain fatty acids in milk fat is improved when the analysis of the fatty acid composition by gas chromatography is conducted with the propyl derivatives, instead of the methyl esters (Sasaki R et al., J Oleo Sci 2015, 64, 1251). In this study, with the aim to identify minor fatty acids, the propyl esters were fractionated by Ag-ion solid phase extraction before gas chromatography.


If methyl esterification with BF3/methanol has been the most widely used derivatization method, other approaches were described to correct various defects such as reagent instability, destruction of epoxy, cyclic fatty acids, hydroxy groups, and non-derivatization of unsaponifiable materials. Trimethylsilyl derivatization is known to be an efficient method but it has some faults like thermal instability and partial hydrolysis of the derivatives. To overcome these defects, the ter-butyldimethylsilyl (tBDMSi) derivatization method for GC analysis was developed (Woo KL et al., J Chromatogr A 1999, 862, 199). These derivatives were shown to have a high thermal and hydrolytic stability and they improve the sensitivity and the selectivity of the analyses.

Procedure :

To fatty acids dissolved in 200 ml of hexane, a known amount of internal standard solution, 75 ml of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 ml of triethylamine are added. After tightly capping, the contents are maintained at 75°C for 30 min before injection.
The separation is done with a HP-1 capillary column (50 m x 0.2 mm ID) with a temperature program as follows : 40°C for 1 min and then after increasing to 70°C with 60°C/min, held for 2 mn. After increasing to 205°C with 5°C/min, held for 25 min and then increased to 285°C with 5°C/min and held for 1 min. Injector and detector are at 300°C.

Comments :

In all fatty acids, the peak responses for these derivatives are higher by 1.5-6.3-times than for methyl esters. In contrast, the stability was shown to be reduced practically to no more than 3 days.


 During cyanomethylation the carboxyl group of fatty acids is alkylated to cyanomethyl esters (R-COO-CH2-CN) and derivatives are detected with nitrogen-phosphorus detector. The method is rapid, inexpensive, and resistant to contaminants frequently found during the chromatographic separation of very-long-chain fatty acids (Paik MJ et al., J Chromatogr B 1999, 721, 3).