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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:


Lipids Heating time (min)
Fatty acids 5
Triacylglycerols 45
Sterol esters 45
Monoacylglycerols 15
Diacylglycerols 15
Glycerophospholipids 15
Glyceroglycolipids 15
Sphingomyelin 90
Glycosphingolipids 90



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.


100 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


Animal tissues

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). 

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 method for the direct preparation of fatty-acid methyl esters was proposed for fatty-acid analysis of a single fish larva using gas chromatography (Matsumoto Y et al., Lipids 2018, 53, 919). Briefly, a little piece of a single larva was dehydrated in 99.5% ethanol (50 μL), followed by blowing through a stream of nitrogen and dried in vacuo in the same vial. Methyl tricosanoate in toluene and 1 M KOH in 95% ethanol were added into the vial. The mixture was directly saponified overnight in the dark at room temperature. The reaction was stopped by adding 2 M HCl to acidify the saponification products. Solvents and excess HCl were evaporated by passing through a stream of nitrogen until KCl was crystalized. Fatty acids released in the vial were methylated in a mixture of toluene, methanol, and a 10% solution of trimethylsilyldiazomethane in hexane at 23 C for 15 min. The reaction was stopped by adding acetic acid, and all solvents were again removed by passing through a stream of nitrogen. Fatty-acid methyl esters were purified by column chromatography using the silica gel 60. At present, this method has the highest sensitivity for fatty-acid analysis of a single larva.

Plant sources

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).

A rapid and routine direct transesterification method for the evaluation of the fatty acid composition of microalgae biomass has been presented. The transesterification is being done in two steps as sonication assisting base‐catalyzed methanolysis followed by BF3 methylation reactions. Total preparation time is very fast (14 min), while the fatty acid compositions are virtually identical to those prepared by classical methods for various species of microalgae as well as fish and krill biomasses (Safafar H et al., Eur J Lipid Sci technology 2019, 121,1700409).

Procedure for 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 (1.5×1.5 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). Similar fatty acid patterns to blood extracted and derivatives in the traditional way have been described. The authors claimed that the fatty acids in the dried blood spots were stable for long periods and the test was sensitive enough to detect changes in dietary fat consumption (Marangoni F et al., Prostaglandins Leukot Essent Fatty Acids 2007, 76, 87). No more than 50 μl of blood were absorbed on a piece of chromatography paper and directly treated with 3 N methanol/HCl at 90°C for 1 h. A range of parameters that might affect the accuracy of the results have been studied (Liu G et al., Prostaglandins Leukot Essent Fatty Acids 2014, 91, 251). The best-known example of the use of this technique is the fatty acid analysis expressed as Omega-3 Index defined by Harris and co-workers (Harris WS et al., Atherosclerosis 2017, 262, 51).

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).


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