ANALYSIS OF FATTY ACIDS

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. 

History

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. 

STRATEGIES TO  STUDY FATTY ACIDS:

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

6- Global methods

 

PREPARATION OF FATTY ACIDS

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 l 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 m 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).

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 200l 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.

Saponification

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.

Reagents

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

Procedure

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).

 

DERIVATIZATION   BEFORE  GLC

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, ...) 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. Acidic conditions generated by 3M HCl in dry methanol or methanolic sulfuric acid have been also described. 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).

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.

Procedure

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.

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 (Ichibara K et al., Lipids 1996, 31, 535) :  

Reagents:

Hexane, 2 M methanolic KOH, capped plastic tubes.

Procedure:

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.

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 ): 

Reagents:

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

Procedure:

100 l of a solution of K tert-butoxide in THF are added to 200 l 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.

Reagents:

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

Procedure:

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.

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 l) of biological fluids (blood, milk) and plant samples. 

Procedure for small tissue samples : 

A tissue sample containing as low as 10 g 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.   

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 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).

Comments : 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.

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 ).

Procedure : 

One hundred milligrams of dried bacterial samples, with 500 g 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).

Procedure:

About 50 l 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 l 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.

D - Other fatty acid derivatives

Butylation

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.

Silylation

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 l of hexane, a known amount of internal standard solution, 75 l of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 l 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.

Cyanomethylation

 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).

LIPID EXTRACTION

The aim of all extraction procedures is to separate cellular or fluid lipids from the other constituents, proteins, polysaccharides, small molecules (amino acids, sugars...) but also to preserve these lipids for further analyses. The preservation of proteins involves special precautions found in delipidation procedures.

There is a great diversity of methodologies because biological tissues are not similar when considering their structure, texture, sensitivities and lipid contents. Removing the non-lipids without losing some lipids is a complex challenge, extracting some specific lipids is not always reliable for other kinds of lipids. The high sensitivity of the analytical methods needed for low amounts of extracted lipids requires the use of very pure solvents and clean glassware. The chemical nature of the extracted lipids must also be taken into consideration. Furthermore, all lipids must be protected against degradation through oxidation by solvent, oxygen, enzymes in combination with temperature and light.

If you want to learn more on lipid extraction : 

1. General methodologies  2. Practical aspects  3. Oilseed processing 4. Extraction techniques in chemistry

OILSEED PROCESSING

If we describe here the extraction on a laboratory scale of lipids from various biological materials to run analytical procedures, different processes may lead also to the extraction of oil from many seeds, nuts, and kernels for use as nutritional supplement, as well as raw material for industrial applications.

The type of instrument that is appropriate depends on the size of the operation. Oil processing operations range from "cottage industries" processing some kg per day, to factories processing several thousand tons of seed per day. In all cases, the sequence of operations includes cleaning, dehulling, grinding, and pressing. Furthermore, oils generally need refining to remove cloudiness, excess color and unpleasant flavors. As many crude oils contain variable amounts of mucilaginous compounds (gums) combined with phospholipids, a degumming process has to be run (Dijkstra AJ, OCL 1998, 5, 367). In several instances, and especially when seeds have low oil content (like soybeans) more complex methods including solvent extraction are processed. If hexane is the most commonly used solvent, isohexane appears to be the most likely candidate to replace n-hexane as the preferred oilseed extraction solvent, mainly in USA to avoid federal environmental regulations applying to the use of n-hexane. Several studies have shown that the extraction performances of the two isomers are quite similar (Inform 2002, 13, 282).  Those who are interested in oilseed processing will find a comprehensive description of basic processes with sources for additional information and a list of suitable raw material in the ATTRA (National Sustainable Agriculture Information Service) publication :

"Small-scale oilseed processing. Value-added and processing guide"

Aqueous enzymatic oil extraction is an emerging technology since it offers many advantages such as elimination of solvent consumption, degumming operations and removal of some toxins. A review of aqueous and enzyme-based processes may be consulted (Rosenthal A et al., Enzyme Microb Technol 1996, 19, 402). Applications of that methodology to the oil extraction of Irvangia seed kernels (Women HM et al., Eur J Lipid Sci technol 2008, 110, 232) and canola seeds (Latif S et al., Eur J Lipid Sci Technol 2008, 110, 887) have been reported.

Comparative experiments on tobacco seeds have demonstrated that the best oil recovery was obtained by cold pressing (Stanisavljevic IT et al., Eur J Lipid Sci Technol 2009, 111, 513). Shortly, seeds were pressed by a hydraulic press (Komet, Germany) through three nozzles (diameter of 15, 10 and 6 mm). The press does not involve mixing and tearing of the seeds. The seed cake was ground by an electrical mill and the residual oil was extracted by using n-hexane at a seeds-to-solvent ratio of 1 : 3 g/mL and 25°C for 60 min. This recovery technique is said to be more acceptable than the other methods, not only for economic but also for environmental, health and safety reasons.

 

GENERAL AND PRACTICAL ASPECTS

As carelessness at the preliminary stage of extraction may result in the loss of components of interest or in the production of artifacts, the lipidologist must be aware of various precautions that are summarized below.

SolventsGlassware

Removal of contaminantsPractical aspects

Storage

SOLVENTS

Neutral lipids or generally storage lipids are extracted with relatively non-polar solvents such as diethyl ether or chloroform but membrane-associated lipids are more polar and require polar solvents such as ethanol or methanol to disrupt hydrogen bondings or electrostatic forces.To avoid peroxidation of the extracted lipids, during the procedure or later, all solvents should be used peroxide-free. The most convenient is to use "pesticide grade" or HPLC grade" and to buy small quantities at once. Do not stock bottles for a year !! Manufacturers  often indicate an expiration date on bottles. To minimize possible deleterious effects due to peroxide formation, pay attention to the expiration dates and keep stocks for no more than 3 months.   Furthermore, to avoid contamination of extracted lipids by non-volatile material contained in the large volume of solvent used in the first step of extraction, it is important to use organic solvent containing less than 5 mg dry matter per liter, 1 mg/l is currently proposed.

Diethyl ether, dioxan, isopropyl ether and tetrahydrofuran form peroxides on storage and must be kept in dark bottles or in dark areas. These solvents should be  routinely tested to prove that peroxide levels are kept low before use. It must be emphasized that peroxides, even at low levels, may present an explosion hazard after concentration of solvents. Because of its low flammability and its natural origin, ethyl lactate was proposed as a substitute for ethyl acetate in exctracting carotenoids from foods (Ishida BK et al., J Agric Food Chem 2009, 57, 1051).

Alcohols are good solvents for most lipids, methanol and ethanol being the most popular. Ethanol can be contaminated on storage by aldehydes.

Chloroform is a popular solvent, particularly for lipids of intermediate polarity and when mixed with methanol it becomes a general extraction solvent. It is not very stable, forming phosgene and HCl in air, it should be stored in brown bottles. It is sold after addition of preservatives, ethanol, amylene or cyclohexene.  Dichloromethane (or methylene dichloride) is a similar extractant but less oxidizable. Chloroform and dichloromethane are currently stabilized by addition of ethanol (up to 1%) or methyl-2-butene-2 (about 20 mg/l).

Among hydrocarbons, hexane is the most popular but is a good solvent only for lipids of low polarity. Its main use is to extract neutral lipids from mixtures of water with alcohols. A mixture of isomers, called "hexanes", can be used for the same purpose. Hexane can be replaced by petroleum ether which is a mixtures of various hydrocarbons with 5 to 8 carbon atoms. Cyclohexane is sometimes used to store lipid extracts in the cold without danger of evaporation since it freezes at about 6°C. Benzene is no more used since it is now considered as a potent carcinogenic substance, it may be replaced by toluene which, nevertheless, is more difficult to evaporate.

A simple test for peroxides in water-immiscible solvents:

shake 2 ml of solvent with 1 ml of a freshly prepared KI solution (10% in water) and a drop of starch indicator solution, a blue color is rapidly developing if peroxides are present (a  faint yellow/brown color after addition of KI indicates low levels of peroxides).

GLASSWARE

The leitmotiv is to avoid any source of contamination by mineral oils, greases, plasticisers from plastics and detergents. Therefore, all operations should be carried out in glass equipment with, if required, ground-glass joints (do not grease !!). Separatory funnels or columns are best equipped with Teflon (PTFE) stopcocks.All vials or tubes should be closed if necessary (for incubation, agitation,

centrifugation) with screw caps including a Teflon-covered liner (Teflon inside the tube !!). Never use cork, rubber, polyethylene or Para film. Filtration of HPLC solvents should be made with an all-glass vacuum filtration apparatus equipped with filter membranes made of nylon or Teflon.Except Teflon, all plastics must be avoided because they leach contaminants into the solution which can be detected as unknown spots on thin-layer plates or extra-peaks in GLC chromatograms. Polypropylene tubes can be used if GLC is not necessary for the analysis.It should be emphasized that all the glassware must be cleaned using classical basic detergents in washing machine or sonicator bath but the washed vials or tubes should be first tested for the absence of contaminants with the current techniques.

REMOVAL OF CONTAMINANTS

In the presence of some water, small molecules are dissolved in organic solvents. Among these contaminants there are pigments, lipophilic amino acids, polypeptides and some hydrophobic proteins.The removal of contaminants can be done by various ways:

- A simple way to eliminate "proteolipids" is to evaporate the lipid extract under vacuum, add a small amount of methanol which is an excellent denaturation agent and evaporate again. Redissolve the dried lipid extract with a small volume of chloroform/methanol (2/1) and transfer into another clean tube.

- Use the washing procedure of Folch's method and repeat the process if necessary with the same volume of the reconstituted upper phase. Finally, a chloroform phase can be washedefficiently with water by vortexing and centrifugation.

- Use a liquid/liquid partitioning column. The Sephadex packing (G 25 superfine) is swollen (20 g/250 ml of solvent) in a mixture of chloroform/methanol/water (60/30/4.5) and washed three times (30 min each time). A small amount of suspension is poured in a small glass column (1 cm diameter, 2.5 cm height for filtration of up to 10 mg lipids).

The lipid sample is dissolved in the same solvent mixture and 2.5 ml are applied to the column followed by 5 ml of the same mixture, 2.5 ml of chloroform / methanol (2/1) and finally by 2.5 ml of chloroform / methanol / water (48/35/10). The total purified lipid extract (12.5 ml) is evaporated under a stream of nitrogen and redissolved in the required amount of solvent. This procedure is commonly used for gangliosides studies where colored contaminants frequently hide lipid spots on HPTLC (Dreyfus et al Anal Biochem 1997, 249, 67).

Alternatively, all lipids except gangliosides are separated from various impurities (bile salts, amino acids sugars) on a Sephadex G-25 column previously equilibrated with chloroform/methanol (19/1, v/v) saturated with water. A glass column with a Teflon stopcock and a fiberglass plug is used. The Sephadex beads are equilibrated in 1/1 methanol/water and added to a height of 10 to 20 cm. Five cm of washed sea sand is then added on the top. Lipids dissolved in chloroform/methanol (19/1, v/v) are applied on the column and eluted with 50 ml of the same solvent. The column is regenerated with 1/1 methanol/water and then with 19/1 chloroform/methanol saturated with water and thus can be used indefinitely.

Chlorophyll removal : High levels of chlorophyll-type compounds are frequently found in lipid extracts from plants. The presence of these pigments may interfere with further lipid fractionation. Thus, a method has been developed to chemically remove chlorophyll without modification of other extracted lipids (Bahmaei M et al., JAOCS 2005, 82, 679). Crude lipid extracts are mixed with a mechanical device at 200 rpm with a 0.4wt% mixture of phosphoric and sulfuric acids (2/0.75, v/v) for 5 min at 50°C. The precipitate is removed from the oily extract by centrifugation (4000 x g, 5 min) and the oil extract is washed according to the Folch procedure to remove acid traces. 

PRACTICAL CONSIDERATIONS

Glassware: Since all solvents may extract also some contaminants from containers or apparatus, only Teflon lined stoppers and clean glassware must be used. During extraction take care of any contact between solvent

and washers or greased bearings in your mechanical device.

Antioxidant: When fatty acid analyses are planned, it is advisable to add an antioxidant such as butylated hydroxytoluene (BHT) (about 100 mg per liter).

Lyophilized tissues: They are difficult to extract, thus it is recommended to rehydrate them with distilled water before extraction (about 5 ml per g dry material).

With some tissues: it is recommended to add first the required methanol amount during the homogenization to prevent clogging, this is currently the case with liver, muscle, blood. chloroform is then added before the last mixing and the true extraction step.

Evaporation of solvents: When a large amount of solvent must be evaporated, it should done in a rotary film evaporator, the flask containing the extract being maintained at no more than 50°C with a water bath. The reduced pressure must be generated by a pump running without oil (motorized pump with metallic or Teflon membranes) or conveniently with a water aspirator achieving a vacuum of 10-20 mm Hg. Last traces of water may be removed by adding 1 or 2 ml ethanol and evaporating again. 

The first description of that concentration device was made by Craig LC (Craig LC et al., Anal Chem 1950, 22, 1462), the famous inventor of countercurrent distribution. The diagram given in the Craig's paper (see below) shows clearly the principle of the device found on the market as yet.

   If small samples are extracted, the small solvent volumes (1-5 ml) are easily evaporated under nitrogen blowing and warming the tubes with the help of a water bath or a heating fan. With the later device, an excessive heating must be avoided as some free fatty acids or their esters (C14 and C16) escape from the tubes.

A very valuable and efficient evaporation equipment may be found from

Pierce, the Reacti-ThermTM System where reaction vials are heated in a thermostated aluminium block  and the Reacti-VapTM Evaporator module enabling a gentle nitrogen blow.

Lipids should not be left for any time in the dry state but should be taken up rapidly in an inert non-alcoholic solvent such as chloroform.If the weight of extracted lipids are needed, the desiccation time must be prolonged till a constant weight is reached. A convenient procedure is to evaporate a part of the total extract in a small flask and to keep it overnight under vacuum before weighing.

STORAGE OF LIPID EXTRACTS

Lipid extracts should not be stored in the dry state. Oxidative degradation is slower in solutions even in the absence of added antioxidants; This protection depends on the chemical nature of the solvent and the physical conditions of storage.Air and light should be avoided. The best storage conditions are a super freezer (-70°C) to store for a long time (up to one year) lipid extracts in

chloroform-methanol mixtures filling well stoppered glass vials (with Teflon liners), the cap being secured with a wide length of self-sticking tape. Pencil written labels should be protected by an outer layer of polyester tape. For long period of storage, it can be useful to flush vials or tubes with nitrogen before closing to prevent fatty acid oxidation. For the same purpose, a low amount of antioxidant such as BHT (butylated hydroxytoluene or 2,6-di-tert-butyl-4-methoxyphenol) or ethyl gallate (i.e. about 50 µg BHT/ml) may be added in the solvent if the natural antioxidant amount is estimated too low (purified extracts). The antioxidant is used as a concentrated solution in ethanol (10 mg/ml). After long periods of storage, the cleavage of ester lipids and plasmalogens must be expected with a concomitant rise in free fatty acids, diacylglycerols methyl or ethyl esters. These problems can be minimized by using neutral extracts and 2-propanol instead of methanol or ethanol in the solvent mixture. It must be remembered that best results are obtained with freshly prepared materials.

To prevent labile lipids from oxygen attack it is possible to use a commercially available oxygen absorber (Ageless, Mitsubishi Gas Chem Co) placed inside the sample package. After a short "pretreatment" at 2°C, the labile lipids appear preserved even at room temperature for a long time. This method allows transportation of biomaterial samples to a distant laboratory without utilizing any freezing system (Hirao S et al., J Oleo Sci 2003, 52, 583).

It has been demonstrated that after one year storage of plasma samples at -80°C various lipid parameters (cholesterol, triglycerides) were altered (Devanapalli B et al., Clin Chim Acta 2002, 322, 179). Thus, even at low temperature, intact biological samples must be extracted as soon as possible. In contrast, it was shown that after storage at -80°C up to 4 years, the fatty acid compositions of plasma triglycerides and erythrocyte phospholipids were practically unchanged (Hodson L et al., Clin Chim Acta 2002, 321, 63).

 

 

FREE OXYGEN AND THIYL RADICALS

 

Several reactive oxygen species (ROS) and one thiyl radical (RS.) are known. Among them, the most frequently studied are given below.

1 - Superoxide radical (O2.- )

This ROS is formed when oxygen takes up one electron and as leaks in the mitochondrial electron transport but its formation is easily increased when exogenous components (redox cycling compounds) are present. Its first production site is the internal mitochondrial membrane (NADH ubiquinone reductase and ubiquinone cytochrome C reductase). This species is reduced and forms hydrogen peroxide (H2O2). The production of superoxide radicals at the membrane level (NADPH oxidase) is initiated in specialized cells (oxidative burst) with phagocytic functions (macrophages) and contributes to their bactericid action. The flavin cytosolic enzyme xanthine oxidase found in quite all tissues and in milkfat globules generates superoxide radicals from hypoxanthine and oxygen and is supposed to be at the origin of vascular pathologies.While superoxide radical can be directly toxic (Fridovich I, Arch Biochem Biophys 1986, 247, 1), it has a limited reactivity with lipids, raising questions about its true toxicity. Thus, its action is frequently considered to

result from a secondary production of the far-more reactive .OH species by the iron-catalyzed Haber-Weiss reaction. As that production may be limited in vivo, it was proposed that nitric oxide reacting with O2

.- generates secondary cytotoxic species (peroxinitrite anion).

2 - Hydrogen peroxide (H2O2)

Hydrogen peroxide is mainly produced by enzymatic reactions. These enzymes are located in microsomes, peroxysomes and mitochondria. Even in normoxia conditions, the hydrogen peroxide production in relatively important and leads to a constant cellular concentration between 10-9 and 10-7 M.

In plant and animal cells, superoxide dismutase is able to produce H2O2 by dismutation of O2

.- , thus contributing to the lowering of oxidative reactions. The natural combination of dismutase and catalase contributes to remove H2O2 and thus has a true cellular antioxidant activity. H2O2 is also able to diffuse easily through cellular membranes.

3 - Hydroxyl radical (.OH)

In the presence of Fe2+, H2O2 produces the very active species .OH by the Fenton reaction (described in 1894):

Fe2+ + H2O2 ----> Fe3+ + .OH + OH-

This iron-catalyzed decomposition of oxygen peroxide is considered the most prevalent reaction in biological systems and the source of various deleterious lipid peroxidation products.It must be noticed that an important part of hydroxyl radicals is also produced (with .NO2)  by the decay of peroxinitrite or peroxynitrous acid

Another reaction involving myeloperoxidase and Cl- ions represent an

important .OH production process in neutrophils during phagocytosis.

4 - Nitric oxide (.NO)

Nitric oxide is produced in various types of cells and is well studied in vascular endothelium. While this species is not too reactive (poorly oxidizing function), even antioxidant under physiological concentrations (up to 100 nM), it reacts rapidly with oxygen to yield nitrogen dioxide (.NO2) which in turn may react with .NO to yield nitrogen trioxide (N2O3). The rapid

reaction of O2.-, produced in different pathological states, with .NO gives the

extremely reactive peroxinitrite (ONOO-) which mediates oxidation,

nitrosation, and nitration reactions.  In alkaline solutions, ONOO- is stable but decays rapidly once protonated into peroxynitrous acid. Its

decomposition forms .OH and nitrogen dioxide (NO2.), radicals important in

the formation of acid rain. The high rate and broad distribution of .NO production, combined with its facile reactions with oxygen radical species, assure that it plays a central role in regulating oxidant reactions. Multiple mechanisms account for the nitration of lipids by .NO-derived species (O’Donnell V B et al., Circ Res 2001, 88, 12). In acidic conditions, protonation of NO2

- to HNO2 can mediate the nitration of polyunsaturated fatty acids and lipid hydroperoxides giving nitrated lipid products whose structure and function are incompletely characterized. Nitric oxide is naturally formed in activated macrophages and endothelial cells and is considered as an active agent in several pathologies based on inflammation, organ reperfusion and also may play an important role in atherosclerosis.

5 - Singlet oxygen (1O2)

This chemical form of oxygen is not a true radical but is reported to be an important ROS in reactions related to ultraviolet exposition (UVA, 320-400 nm). Its toxicity is reinforced when appropriate photoexcitable compounds (sensitizers) are present with molecular oxygen. Several natural sensitizers are known to catalyzed oxidative reactions such as tetrapyrroles (bilirubin), flavins, chlorophyll, hemoproteins and reduced pyridine nucleotides (NADH). Some of these sensitizers are also found in foods and cosmetics. Some others are used for therapeutic purposes (anticancer treatments) and are sensitive to visible light. The presence of metals contributes to increase the production of singlet oxygen, as well as anion superoxide, and thus accelerates the oxidation of unsaturated lipids generating hydroperoxides.

It has been suggested that singlet O2 may be formed during the degradation of lipid peroxides and thus may cause the production of other peroxide molecules. This singlet O2 formation may account for the chemiluminescence observed during lipid peroxidation.

6 - Ozone (O3)

This natural compound present in the higher atmosphere and in the lower atmosphere of our polluted cities is a major pollutant formed by photochemical reactions between hydrocarbons and nitrogen oxides. Ozone is not a free radical but, as singlet oxygen, may produce them, stimulates lipid peroxidation and thus induces damages at the lipid and protein levels in vivo mainly in airways.

The exact chemistry of ozone-mediated stimulation of peroxidation is not entirely known. Ozone may add on across a double bond and decomposes to form a free radical. The proposed mechanism is given below.

7 - Thiyl radicals (RS.)

Aliphatic thiols (RSH) are contained in living organisms in high concentrations. Typical levels of intracellular of glutathione are about of 5 to 10 mM. Furthermore, the level of protein SH may exceed that of GSH. The thiolate specie RS– is one of the most reactive functional groups found in proteins. It can react as a nucleophile and attack a disulfide bond. In the absence of oxygen, a thiyl radical was shown to induced cis/trans-isomerization of linoleic acid and led to several isomers. (Schwinn J, Int J Radiat Biol 1998, 74, 359). A review of the effects of thiyl radicals on lipid structures and metabolisms may be consulted (Ferreri C et al., Cell Mol Life Sci 2005, 62, 834).

Thiol compounds (RSH) are frequently oxidized in the presence of iron or copper ions:

RSH + Cu2+ ----> RS. + Cu+ + H+

These thiyl radicals have strong reactivity in combining with O2 :

RS. + O2 ---> RSO2.

Furthermore, they are able to oxidize NADH into NAD. , ascorbic acid and

to generate various free radicals (.OH and O2.-). These thiyl radicals may

also be formed by homolytic fission of disulfide bonds in proteins.

8 - Carbon-centered radicals

The formation of these reactive free radical is observed in cells treated with CCl4 . The action of the cytochrome P450 system generates the

trichloromethyl radical (.CCl3) which is able to react with oxygen to give

several peroxyl radicals (i.e. .O2CCl3).