ANALYSIS OF FATTY ACIDS
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
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
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
ml of 50%
H2SO4, and 0.05 g NaCl to 1 ml of the aqueous
sample and mixing for 15 min at 200 rpm. A lower recovery was obtained only for
caproic (C6:0) and caprylic (C8:0) acids : 27 and 76% recoveries, respectively.
The purification of free fatty acids has been done by
solid-phase microextraction (SPME) (Tomaino RM et al. J Agric Food Chem 2001,
49, 3993). The fiber sheath of a 30
mm
thick poly(dimethylsiloxane) fiber (Supelco) was incubated at 110°C for 80 min
in the acidified medium and then placed into the injector of a gas chromatograph
whose temperature was increased from 100°C to 245°C. Unfortunately, a
progressive and rapid loss of sensitivity occurred with decreasing fatty acid
chain length. Thus, it was necessary to determine the response factors for each
fatty acid in relation to an internal standard (C17). Advantages of that
extraction procedure are the little sample preparation, the absence of organic
solvents, the detection of short chain fatty acids, and a good reproducibility.
Short-chain fatty acids (C1 to C5) in biological specimens need a special
treatment taking into account their volatility. Thus a simple and efficient
procedure using a vacuum transfer followed by HPLC enable the accurate
determination of these acids in the nanomolar range in tissues and secretions (Stein
J et al., J Chromatogr 1992, 576, 53). An eficient procedure using an
extraction with a hollow fiber coupled with gas chromatography has been reported
(Zhao
G et al., J Chromatogr B 2007, 846, 202).
Application of gas chromatography coupled to mass spectrometry following
headspace solid-phase microextraction was applied with great accuracy and
sensitivity to the determination of free volatile fatty acids in aqueous samples
(Abalos
M et al., J Chromatogr A 2000, 891, 287). Valuable results were obtained
for the determination of C2-C7 fatty acids in raw sewage.
Free medium-chain fatty acids in beer have been extracted using adsorption on a
specific stir bar (Gerstel
twister). The determination of caproic, caprylic, capric and lauric acids
with solvent back extraction was described (Horak
T et al., J Chromatogr A 2008, 1196-1197, 96). The procedure utilized
10ml of sample stirring with the stir bar with 1000rpm for 60min at room
temperature. Solvent back extraction used 200ml
of solvent (dichloromethane/hexane, 50/50) at room temperature.
Bound fatty acids
When fatty acids are combined in more complex
molecules such as acylglycerols, cholesterol esters, waxes and glycosphingolipids, they
can be obtained free by saponification (inorganic or organic basic
solution) or acidic hydrolysis and then derivatized. FAME may
be also obtained directly by transesterification (alcoholysis or methanolysis) of the fatty acid-containing lipids.
The extraction and methylation may also be combined in a one-step
procedure, this is particularly recommended for very small samples in order to prevent
any loss of fatty acids during the classical procedures.
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 ml
of a solution of K tert-butoxide in THF are added to 200 ml
anhydrous 2-methoxyethanol in a closed vial. After homogenization, up to 10 mg
of lipid in 1 ml hexane are added. Keep the mixture at 40°C for 15 min. After
cooling, 1 ml water and 2 ml hexane are successively added. After 5 s vortexing
and a short centrifugation, the organic phase is collected, dried over anhydrous
Na sulfate and analyzed by GLC.
We have adopted
another approach for some labile samples. A rapid and mild method which avoids the formation of oxidation products
was described by Piretti et al. (Chem Phys
Lipids 1988, 47, 149). We have most precisely adopted this procedure for the analysis of highly
unsaturated lipids since higher amounts of polyunsaturated fatty acids were found when
compared to the BF3/methanol procedure.
Furthermore, if hydroperoxy fatty acids are present, they are reduced into the
corresponding hydroxy components.
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.
C - Direct transmethylation without prior extraction
The concept of direct transesterification of
techniques has been reported for small tissue
samples (1-10 mg) or small volumes (about 50 ml)
of biological fluids (blood, milk).
Procedure for small tissue samples :
A tissue sample containing as low as 10 mg
of lipids is introduced at the bottom of a screw-capped tube (Teflon-lined). Then add 1 ml of methanolic HCl, 1 ml of methanol and 0.5 ml hexane. Close
tightly the tube and heat at 100°C for 1 h (shake several times).
After cooling add 2 ml of hexane and 2 ml of water. Mix not too vigorously the
tube and collect the hexane layer after a short centrifugation. Before GC
analysis, the extract may be concentrated by evaporation under nitrogen if
necessary.
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.
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 mg
of internal standard, is transesterified using 1 ml of methanolic HCl (1.5M)
(from Supelco) and 1 ml methanol, at 80°C for 10 min. Water (2 ml) is added and
after mixing and low speed centrifugation the upper phase is collected for gas
chromatographic analysis.
Procedure for small amounts of fluid :
A convenient method was developed for preparation of fatty acid methyl
esters in glycerolipids of blood or milk (Ichihara K et al., Lipids 2002, 37,
523).
Procedure:
About 50 ml
of blood or milk are spotted onto a small piece of Whatman 3MM filter paper
(1.5x1.5 cm) that has been previously washed with acetone containing 0.05 % BHT.
Each piece, once dried for 30 min in vacuo is inserted into a small test tube,
to which 2 ml hexane and 0.2 ml 2M KOH/methanol are added (alkali-catalyzed
alcoholysis). After vigorous mixing or sonication for 2 min at room temperature,
the solution is neutralized with acetic acid. To each tube is added 2 ml water
with light mixing. An aliquot of the hexane layer was collected and evaporated
to dryness; FAME are dissolved in 0.02 ml hexane or methyl acetate before GC
analysis.
The presence of BHT on the filter paper allows the protection of unsaturated
fatty acids for at least 7 days even exposed to the air.
A similar direct procedure using boron trifluoride-methanol as esterification
reagent was described for the determination of fatty acids in human milk (Lopez-Lopez
A et al., Chromatographia 2001, 54, 743).
A direct evaluation of the fatty acid status in a drop of blood was described (Marangoni
F et al., Anal Biochem 2004, 326, 267). No more than 50
ml of
blood were absorbed on a piece of chromatography paper and directly treated with
3 N methanol/HCl at 90°C for 1 h. The method was validated for reproducibility
and satisfactorily compared with a conventional method.
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 ml
of hexane, a known amount of internal standard solution, 75 ml
of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 ml
of triethylamine are added. After tightly capping, the contents are maintained
at 75°C for 30 min before injection.
The separation is done with a HP-1 capillary column (50 m x 0.2 mm ID) with a
temperature program as follows : 40°C for 1 min and then after increasing to
70°C with 60°C/min, held for 2 mn. After increasing to 205°C with 5°C/min,
held for 25 min and then increased to 285°C with 5°C/min and held for 1 min.
Injector and detector are at 300°C.
Comments :
In all fatty acids, the peak responses for these derivatives are higher by
1.5-6.3-times than for methyl esters. In contrast, the stability was shown to be
reduced practically to no more than 3 days.
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).
