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Mass spectrometry of fatty acid derivatives: preparation of methyl esters

MASS SPECTROMETRY OF FATTY ACID DERIVATIVES

PREPARATION OF METHYL ESTERS

1. Introduction

Although methyl esters are not the best derivatives for the mass spectrometric analysis of lipids,
they are so simple in structure and in such wide-spread use for fatty acid analysis in general that
they are inevitably much used for mass spectrometry. Without further derivatization, they are of
little worth for structural analysis of mono- or dienoic fatty acids, but they can often be of value for
polyunsaturated fatty acids, and those with functional groups other than double bonds, especially
when spectra of authentic compounds are available for comparison purposes. Following a brief
description of the methodology, experimental protocols for the two most important methods are
given here. More detailed discussion of the mechanisms of esterification and alternative methods
are given in a review article that is now available online here-

W.W. Christie, Preparation of ester derivatives of fatty acids for chromatographic analysis. In:
Advances in Lipid Methodology - Two, pp. 69-111 (1993) (Ed. W.W. Christie, Oily Press,
Dundee).

- or in my recent book (Christie, W.W. Lipid Analysis (3rd edition) (Oily Press, Bridgwater) (2003)) –
not to forget “Gas Chromatography and Lipids” available online here. Other useful derivatization
techniques, e.g. hydrogenation, deuteration, etc., are described in the section of these pages
dealing with “Mass spectrometry of methyl esters – further derivatization”.

Full practical details are given elsewhere on this site for preparation of the nitrogen-containing
derivatives, i.e. picolinyl esters, 4,4-dimethyloxazoline (DMOX) derivatives and pyrrolidides, which
are most useful for mass spectrometric analysis of fatty acids.

There is no need to hydrolyse lipids to obtain the free fatty acids before preparing esters, as most
lipids can be transesterified directly. No single reagent will suffice, however, and one must be
chosen that best fits the circumstances. Esters prepared by any of the following methods can be
purified if necessary by adsorption chromatography (see below). Care should be taken in the
evaporation of solvents as appreciable amounts of esters up to C14 (or even C16) can be lost if this
step is performed carelessly, for example by an over vigorous use of a nitrogen stream. The main
procedures are acid- or base-catalysed esterification. Diazomethane was once used frequently as
a mild method for esterification of free acids, but concerns over the toxicity of reagents now limit its
use. The methods described below are suitable for the common esterified lipids and their fatty acid
constituents. Special methods are required for amide-bound fatty acids, and those with of short
chain-length or with sensitive functional groups, for which the review and books cited above should
be consulted.

2. Acid-Catalysed Esterification and Transesterification

Free fatty acids are esterified and O-acyl lipids transesterified by heating them with a large excess
of anhydrous methanol in the presence of an acidic catalyst.

If water is present, it may inhibit the reaction. The reagent is most easily prepared by adding acetyl
chloride (5 mL) slowly to cooled dry methanol (50 mL). Methyl acetate is formed as a by-product,
but it does not interfere with methylations at this concentration. A solution of 1-2% (v/v)

© W.W. Christie www.lipidlibrary.co.uk 1


Mass spectrometry of fatty acid derivatives: preparation of methyl esters

concentrated sulfuric acid in methanol


transesterifies lipids in the same a
manner and at much the same rate; it is O R'OH O -H+ O
R C R C R C
very easy to prepare whenever it is
required (fresh reagent is best). It is
OH H+ + O R' OR'
usual to heat the lipid sample in the H a
reagent under reflux for about two
hours, but equally effective esterification is obtained if the reaction mixture is heated in a stoppered
tube at 50°C overnight (also incidentally reducing the glassware requirements). While boron
trifluoride in methanol (12-14% w/v) has also been much used as a transesterification catalyst and
for esterifying free fatty acids, I have considerable reservations about its use (see the specific web
page).

Laboratory protocol: The lipid sample (up to 5 mg) is dissolved in toluene (1 mL) in a
test tube fitted with a condenser, and 1% sulfuric acid in methanol (2 mL) is added, before
the mixture is refluxed for 2 hours (or alternatively the mixture can be left overnight in a
stoppered tube at 50°C). Water (5 mL) containing sodium chloride (5%) is added and the
required esters are extracted with hexane (2 x 5 mL), using Pasteur pipettes to separate
the layers. The hexane layer is washed with water (4 mL) containing potassium
bicarbonate (2%) and dried over anhydrous sodium sulfate. The solution is filtered and
the solvent removed under reduced pressure in a rotary film evaporator or in a stream of
nitrogen.

No solvent other than methanol is necessary if free fatty acids alone are to be methylated, or if
polar lipids such as phospholipids are to be transesterified. The same method is used to prepare
dimethylacetals from aliphatic aldehydes or plasmalogens.

3. Base-Catalysed Transesterification

O-Acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic
catalyst. Free fatty acids are not normally esterified, however, and care must be taken to exclude
water from the reaction medium to prevent their formation by hydrolysis of lipids.

a _
O O O
_ _
R C + OR'' R C OR'' R C + OR'
OR' OR' OR'' a

0.5M Sodium methoxide in anhydrous methanol, prepared simply by dissolving fresh clean sodium
in dry methanol, is the most popular reagent, but potassium methoxide or hydroxide have also
been used as catalysts. The reagent is stable for some months at room temperature, if oxygen-free
methanol is used in its preparation. The reaction is very rapid; phosphoglycerides, for example, are
completely transesterified in a few minutes at room temperature, but cholesterol and wax esters
take longer. It is commonly performed as follows:

Laboratory protocol: The lipid sample (up to 10 mg) is dissolved in dry toluene (1 mL) in
a test-tube, 0.5M sodium methoxide in anhydrous methanol (2 mL) is added, and the
solution is maintained at 50°C for 10 min. Glacial acetic acid (0.1 mL) is then added,
followed by water (5 mL). The required esters are extracted into hexane (2 x 5 mL), using
a Pasteur pipette to separate the layers. The hexane layer is dried over anhydrous
sodium sulfate and filtered, before the solvent is removed under reduced pressure on a
rotary film evaporator.

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Mass spectrometry of fatty acid derivatives: preparation of methyl esters

As with acid-catalysed transesterification procedures, an additional solvent, such as toluene or


tetrahydrofuran, is necessary to solubilize non-polar lipids like cholesterol esters or triacylglycerols.
Aldehydes are not liberated from plasmalogens with basic reagents.

4. Clean-up of Methyl Esters

It is sometimes necessary to purify methyl esters after transesterification has been carried out in
order to eliminate troublesome impurities prior to analysis by gas chromatography. For example,
cholesterol should be removed in this way from animal tissue preparations. This can be
accomplished by adsorption chromatography with a short column (approx. 2 cm) of silica gel or
TM
Florisil in a Pasteur pipette plugged with glass wool, and pre-conditioned with hexane. Methyl
esters are eluted with hexane-diethyl ether (95:5, v/v; 10 mL), while cholesterol and other polar
impurities remain on the column. Commercial solid-phase extraction columns can be used in the
same way. Methyl esters can also be purified by preparative thin-layer chromatography with
hexane-diethyl ether (9:1, v/v) as the mobile phase.

5. Choice of a Column for Gas Chromatography-Mass Spectrometry of Fatty Acid


Derivatives

The choice of columns for GC analysis in general is discussed elsewhere on this website. It is not
always possible to use the more popular analytical columns for GC-MS analysis of lipids, as
bleeding of the stationary phase can lead to troublesome backgrounds. However, a number of
manufacturers now make columns with cross-linked highly stable phases specifically for use with
TM
mass spectrometry. We have made extensive use of a column of Supelcowax 10 (25 m in
length; from Supelco-Sigma Inc.) in our laboratory. It is typical of columns of the Carbowax type in
its elution properties, and can cover with ease a wide range of fatty acid methyl esters or DMOX
derivatives (up to C30 in chain-length).

Counts
solvent

16:0

16000
18:1(n-9)

20:4(n-6)

14000
18:0

12000
18:2(n-6)

10000
C18 acetals
C16 acetal

8000
BHT

22:5(n-3)
22:6(n-3)
{

20:3(n-6)

22:4(n-6)
18:1(n-7)

20:5(n-3)

6000
18:3(n-3)

20:4(n-3)
18:3(n-6)

20:0

22:5(n-6)

24:0
} 16:1

22:0
14:0

18:4

24:1
20:1

4000

5 10 15 20 Time (min)

© W.W. Christie www.lipidlibrary.co.uk 3


Mass spectrometry of fatty acid derivatives: preparation of methyl esters

The figure above illustrates a typical chromatogram of the fatty acids of human erythrocytes as
methyl esters on a column of the Carbowax type (25 m) with a flame-ionization detector. Excellent
separations are achieved of fatty acid esters of a given chain-length that differ both by degree of
unsaturation and in the positions of the double bonds. For example, two isomers of 18:1 and of
18:3 are separated, as are three isomers of 20:3, two of 20:4 and two of 22:5. With the methyl
esters of the more common families of polyunsaturated fatty acids, the shorter the distance
between the last double bond and the end of the molecule, the longer the retention time of the
isomer. Even better separations of positional isomers might be obtained with a more polar phase,
though the order of elution relative to components of a different chain length might vary and there
can be other problems of components overlapping. By a judicious use of two or more columns
differing in polarity in this way, most of the fatty acids of metabolic importance can be separated
and estimated.

Picolinyl esters and pyrrolidides are more polar or have higher molecular weights, and the
Supelcowax column can cope with the common range up to about C22. When a more polar phase
TM
is required, we have used a column of BPX-70 (from SGE Ltd). Of course, other manufacturers
will also have columns available that fully meet the requirements for GC-MS, but we no experience
of any other than those listed.

It is not possible to expect the same quality of resolution in mass spectrometry applications as with
a flame-ionization detector (FID), mainly because of the greater dead volume in a mass
spectrometer in comparison with an FID. This depends largely on the specific instrument in use, so
further comment is impossible here. Also, it is not possible to expect the same resolution with
picolinyl esters and pyrrolidides as with methyl esters and DMOX derivatives, because of the high
molecular weight and polarity of the former. However, pyrrolidides do have some interesting GC
properties in their own right (see the appropriate web page).

Finally, we have a requirement for a column coated with a low polarity silicone phase, because of
its high thermal stability. Such columns lack the resolution of phases of higher polarity, but are
adequate for many purposes. We use a DB5TM column for fatty acids of longer than usual chain-
length (especially as picolinyl esters), for hydroxy fatty acids, and for other lipids of high molecular
weight, including sterols and waxes. It is essential for any fatty acid derivative or other lipid that has
been silylated.

© W.W. Christie
Scottish Crop Research Institute (and Mylnefield Lipid Analysis), Invergowrie,
Dundee (DD2 5DA), Scotland

Last updated: 14/4/2008

© W.W. Christie www.lipidlibrary.co.uk 4