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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 200ml 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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