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 200l 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.

Solvents
Glassware
Removal of contaminants
Practical 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 washed
efficiently 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-Therm System where reaction vials are heated in a
TM

thermostated aluminium block  and the Reacti-Vap Evaporator module

TM

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).