Lebensm.-Wiss. u.-Technol.

, 30, 202–209 (1997)

Simplified Method for the Simultaneous Gas

Chromatographic Determination of Fatty Acid
Composition and Cholesterol in Food
Eva Paterson and Renato Amadò*

Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zürich (Switzerland)
(Received April 22, 1996; accepted June 7, 1996)

A rapid, reliable and accurate gas chromatographic method for the determination of fatty acid composition in food was
developed and evaluated. The procedure consists of direct saponification of glycerides and hexane extraction of sterols from the
nonsaponifiable matter, followed by acidification of the residual soap and methylation of the free fatty acids. The substantial
advantage of this procedure is the possibility of simultaneous determination of the fatty acid composition and the cholesterol of
phytosterol content in food. In addition, it eliminates the use of chlorogenic chemicals and minimizes solvent use. This method
was compared to a slightly modified standard procedure, involving fat extraction prior to saponification and transmethylation of
the fatty acids. For both methods the coefficients of variation for analysed fatty acids (C ≥ 8) ranged from 0.6 to 7.4%. For the
majority of fatty acids no significant difference was observed between mean values at a 95% confidence level.

©1997 Academic Press Limited

Keywords: fatty acids; cholesterol; gas chromatography

Introduction benzene or other solvents (8–11). Takeuchi et al. (12)

The main interest from a nutritional point of view with
regard to fat fraction in food focuses on its fatty acid
composition and cholesterol content. Both these food
constituents have a very important influence on the
prevalence of cardiovascular disease (1). Although the
effects of the widely recommended low cholesterol,
high ratio of polyunsaturated to saturated fatty acids,
and low total fat content diet still remain controversial
(2), it is useful to have at hand fast, simple, reliable and
accurate methods for the estimation of these sub-
stances. According to recent publications (3,4), the
preferred methods for cholesterol assay use a direct
saponification of the food sample and extraction of the
unsaponifiable matter with an organic solvent like
hexane, rather than the initial lipid extraction usually
performed using a chloroform–methanol mixture or
diethylether as solvents, followed by saponification of
the polar fraction. The advantages of this approach
have been widely discussed (5). However, for quantita-
tive determination of the fatty acid content in food,
methods are still in use which are based on lipid
extraction as a first step (6). Nevertheless, in bio-
chemistry, attempts were undertaken to simplify the
quantitative determination of fatty acids in plasma by
omitting the lipid extraction step (7). Several one-step
extraction/methylation methods were described based
on the reaction with methanolic hydrochloric acid in
*To whom correspondence should be addressed.
0023-6438/97/020202 + 08$25.00/0/fs960166
recommended direct sample saponification in ethanolic
alkali for fish diets and fish faeces if all fatty acids
present are of interest.
The procedure introduced in this paper consists of the
direct saponification of food samples followed by the
separation of saponifiable and unsaponifiable matters
with hexane. These two fractions are used as starting
material for the quantitative determination of both
fatty acids and cholesterol in samples. This approach
allows the most important fat components to be
analysed in one procedure, thus saving time. Since no
chlorogenic solvents are required in the extraction step,
exposure to potentially hazardous vapours is reduced
and disposal costs associated with these solvents are
eliminated.
In order to verify obtained results, the developed
procedure was compared to the slightly modified
standard method with initial fat extraction as described
by Winter (13), followed by saponification and trans-
esterification according to Metcalfe et al. (14). The
modification consists of reducing the sample weight and
the quantity of extractants by a factor of ten. This
approach not only minimizes the use of hazardous
solvents but also enables the addition of an internal
standard at the beginning of the determination proce-
dure. The compensating nature of the internal stan-
dardization method minimized the error source in all
following steps. Due to the reduced amount of solvents,
it is possible to accelerate the separation of the
©1997 Academic Press Limited

202

chloroform–methanol–water mixture into two layers by
centrifugation thus saving analysis time.
The present paper describes the quantitative determi-
nation of the fatty acid composition of food samples.
This method is not suitable for the determination of
short chain fatty acids (C < 8). Quantification of choles-
terol content in food has been extensively treated in
another publication (15).
Experimental Details
Chemicals
All reagents were of analytical reagent grade unless
specified otherwise. Pyrogallol, methyl tridecanoate,
boron trifluoride ( ~ 1.3 mol/L) in methanol, n-hexane,
ethanol, methanol, chloroform, anhydrous sodium sul-
phate, seasand (acid purified) and Clara-Diastase
(No.10065) were obtained from Fluka Chemie AG
(Buchs, CH). 5α-Cholestane, cholesterol and trideca-
noic acid were supplied by Sigma Chemical Co.
(Munich, D). GLC Standard Mixtures of fatty acid
methyl esters (FAME) were purchased from Matreya
Inc. (Pleasant Gap, PA, U.S.A.). These products are
equal weight (10 or 12.5 mg) mixtures of FAME.
Thermoresistent α-amylase preparation Termamyl
120L was supplied by Novo Nordisk A/S. (Bagsvaerd,
DK).
Food sample
*enzymatic degradation of starch
saponification
separation
(hexane phase) (water phase)
nonsaponifiable fraction saponifiable fraction
cholesterol recovery of fatty
acids
phytosterols
tocopherols
separation of fatty
acids
esterification
fatty acid methyl
esters
Fig. 1 Direct method for quantitative determination of fatty
acids and sterols in foods. * = for starch-rich food samples
only
203
lwt/vol. 30 (1997) No. 2
Food sample
*enzymatic degradation of starch
lipid extraction
separation of lipids
saponification
esterification
fatty acid methyl esters
Fig. 2 Modified extraction method for determination of fatty
acids in foods. * = for starch-rich food samples only
Solutions
Calibration solution for FAME was prepared by
transferring the GLC Standard Mixtures No. GLC-101,
GLC-30, GLC-50, GLC-60 and GLC-100 into a 100 mL
volumetric flask and diluting to the mark with hexane.
The final concentration of the single FAME ranged
from 10 to 22.5 mg/100 mL. The solution was stored at
4 °C. Internal standard solution for FAME determina-
tion was prepared by dissolving approx. 100 mg ( ± 0.1
mg) tridecanoic acid (C13:0) in 50 mL n-hexane.
Internal standard solution for cholesterol determina-
tion was prepared by dissolving approx. 25 mg ( ± 0.1
mg) of 5α-cholestane in 50 mL n-hexane.
Potassium hydroxide solution was made by carefully
dissolving 50 g KOH pellets in 50 mL deionized
water.
Pyrogallol solution was prepared by dissolving 5 g
pyrogallol in 500 mL ethanol, methanol or water,
respectively, as required by the different procedures
(see below).
Sample preparation
Food samples for the analyses and all ingredients for
the meal were obtained in a local shop (Migros,
Rüschlikon, CH). The meal — pasta with meat sauce
— was prepared as follows: 152 g minced beef was
fried for 10 min in 10 g olive oil. The resulting weight
was 110 g. The tomato sauce contained 30 g cut onions
(fried for 3 min in 10 g butter) and 230 g of canned
tomatoes. The sauce was boiled for 5 min. The final
weight of the tomato sauce amounted to 245 g. Tomato
sauce and fried meat were mixed with 250 g of cooked
pasta (12 min in an excess of boiling water) and the
meal was sprinkled with 10 g of Parmesan cheese. The
finished test meal (615 g) was homogenized in a mixer,
divided into small (approx. 10 g) portions and kept in
the freezer at –18 °C until analysed.
lwt/vol. 30 (1997) No. 2

Instrumentation and operating conditions U.K.), heating block Reacti-Therm III™, heating/
A gas chromatograph (GC) Hewlett Packard Model stirring module (Pierce Chemical Co., Rockford, IL,
5890 Series II with a split/splitless capillary injection U.S.A.) and evaporator Rotavapor RE 111 with Water
system and a flame ionization detector (FID) equipped Bath 461, (Büchi Laboratoriums-Technik AG, Flawil,
with an autosampler HP 7673 (Hewlett Packard, CH).
Avondale, PA, U.S.A.) was used. Operating conditions
were: helium carrier gas at 1.7 mL/min, head pressure
100 kPa, 1:40 split ratio, injection port temperature 240 Procedure of the direct method (Fig. 1)
°C, detector temperature 280 °C, initial oven tem-
perature 160 °C held for 4 min, then increased up to 230 Homogenized food samples of 1.5 to 2.5 g, accurately
°C at a rate 4 °C/min and held for 3.5 min at this weighed, were placed in a 50 mL centrifuge tube. The
temperature. Injection volume was 2 µL. The GC sample weight of fat-rich food like butter or mayon-
separation was accomplished by using a fused silica naise must be reduced to approx. 0.2 g (the limiting
capillary column Supelcowax™ 10, 30 m 3 0.32 mm factor is the concentration of KOH). One millilitre
i.d. and 0.25 µm film thickness (Supelco, Inc., Belle- each of both internal standard solutions (5α-chol-
fonte, PA, U.S.A.). Peak areas were integrated and estane and C13:0), 9 mL pyrogallol in ethanol (1
calculated by Hewlett Packard 3365 ChemStation g/100 mL), 1 mL potassium hydroxide solution (50
software. g/100 g), a magnetic stirring bar, and approx. 1.5 g of
Saponification and separation of layers were carried seasand for a better fat dispersion were added and
out in 50 mL screw capped centrifuge tubes Cortex® the sample vortexed for 20 s. The capped tube was
Brand (Corning Inc., Corning, NY, U.S.A.). placed in a water-bath at 60 °C and saponified for 1 h
The following apparatus were also used: water-bath under continuous stirring. After cooling to room
Julabo U3 (Julabo Labortechnik GmbH, Seelbach, D), temperature, 5 mL of deionized water was added
magnetic stirrer/hot plate Heidolph MR 2002 (Hei- followed by 10 mL hexane and the sample vortexed
dolph GmbH, D), shaker Vortex Genie 2™ (Scientific at the highest setting for 2 min. The sample was then
Industries Inc., Bohemia, NY, U.S.A.), centrifuge MSE centrifuged for 3 min at 2200 3 g and the upper
Mistral 1000 (MSE Scientific Instruments, London, hexane layer transferred with a Pasteur pipette into a
C20:1 (11)
C10:0
C8:0

C12:0

C16:0

C20:0
C13:0 (I.S.)

C18:1 (9)
C18:0

C21:0
C19:0

C20:3 (11, 14, 17)

C20:2 (11,14)
C14:0

C18:3 (9, 12, 15)

C16:1 (9)

C18:2 (9, 12)

C15:0

C22:1 (13)

C24:1 (15)
C17:0

C22:0

0 5 10 15 20 25
Retention time (min)

Fig. 3 Gas chromatogram of the standard mixture of a fatty acid methyl esters. Column: Supelcowax™ 10, 30 m 3 0.32 mm
i.d., film thickness 0.25 µm, carrier gas helium at 1.7 mL/min; oven temperature programme: 160 °C (4 min), to 230 °C (4
°C/min), 230 °C (3.5 min), injection 2 µL, split ratio 1:40

204
 lwt/vol. 30 (1997) No. 2

clean 50 mL centrifuge tube. Another 10 mL hexane L). The tube was capped tightly and placed in a
was added to the water phase and the extraction and heating block for 4 min at 100 °C. Tubes were cooled
centrifugation step repeated. The combined hexane in cold tap water to room temperature and 4 mL
extracts can be directly used for cholesterol determi- hexane added, followed by 2 mL water. The sample
nation as described by Paterson et al. (15). was vortexed vigorously for 2 min and centrifuged for
The water phase was acidified with 3 mL hydrochloric 3 min at 2200 3 g. Approx. 1.5 mL of the hexane
acid (HCl) (25 g/100 g) to pH ≤ 1.5, and shaken extract was transferred into the GC-sample vials and
briefly. Released free fatty acids were extracted with 2 µL injected into the gas chromatograph.
10 mL hexane. The sample was vortexed at the In order to prevent incomplete extraction of fat and/
highest setting for 2 min and centrifuged for 3 min at or cholesterol in starch-rich food samples like cooked
2200 3 g. The upper hexane layer was transferred pasta it is necessary to perform an enzymatic degra-
with a Pasteur pipette into a clean 50 mL centrifuge dation of the gelatinized starch prior to saponifica-
tube and another 10 mL hexane added to the water tion: 0.25 mL of thermoresistent α-amylase prepara-
phase. The extraction and centrifugation steps were tion Termamyl 120L was mixed with 4.75 mL
then repeated. The first and second hexane extracts pyrogallol in deionized water (1 g/100 mL), and
were combined using the same pipette. In order to added to the sample prior to saponification. The tube
dry the extract, approx. 4 g of anhydrous sodium was capped and held for 15 min at 95 °C in a water-
sulphate was added to the centrifuge tube and shaken bath under continuous stirring. For extraction of the
vigorously for a short time. The extract was centri- unsaponifiable matter addition of water was omitted.
fuged for 3 min at 2200 3 g and 4 mL of the clear
extract (containing 10 to 50 mg of fatty acids) trans-
ferred into a 15 mL culture tube fitted with a screw Procedure of the modified extraction method (Fig. 2)
cap. The extract was concentrated in a rotatory evap-
orator under reduced pressure of 200 mbar for 3 min Well homogenized food samples of 1.5 to 2.5 g,
nearly to dryness. The residue was redissolved in 1 accurately weighed, were placed in a 50 mL cen-
mL of hexane and then a magnetic rod placed in the trifuge tube. Sample weight of fat-rich food like
tube. Fatty acids were converted into methyl esters by butter or mayonnaise must be reduced to approx. 0.5
adding 5 mL boron trifluoride in methanol (1.3 mol/ g. One millilitre of internal standard solution for
C13:0 (I.S.)

C18:1 (9)
C16:0

C19:0 and BHA

C18:0

C18:2 (9, 12)

C14:0
C10:0
C12:0

C18:3 (9, 12, 15)

C16:1 (9)
C8:0

C15:0

C17:0

0 5 10 15 20 25
Retention time (min)

Fig. 4 Representative gas chromatogram of fatty acid methyl esters in food sample (pasta with meat sauce). Chromatograph
conditions as in Fig. 3

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lwt/vol. 30 (1997) No. 2

Table 1 Fatty acid composition of minced beef, Parmesan cheese and mayonnaise in mg/100 g fresh sample
Minced Beef Parmesan Cheese Mayonnaise
Ext Dir Ext Dir Ext Dir
Fatty acid Meana CV Meana CV Meana CV Meana CV Meana CV Meana CV
C 8:0 – – 202.8 5.2 213.4 4.1 – –
C 10:0 – – 487.4 3.0 497.1 3.1 – –
C 12:0 – – 640.1 5.6 642.8 3.2 – –
C 14:0 163.1 2.2 161.0 2.6 2261.4 4.5 2235.2 3.2 59.4 2.4 62.0 4.9
C 15:0 27.1 1.9 26.4 2.3 268.5 4.0 263.1 3.3 – –
C 16:0 1507.9 2.7 1490.2 2.3 5950.9 3.9 5761.9 3.4 5275.3 0.6 5300.7 2.9
C 16:1(9) 214.7 3.3 211.4 1.9 267.5 4.5 261.7 3.7 125.4 1.2 125.1 5.8
C 17:0 73.3 4.3 69.7 4.0 170.6 4.2 166.1 3.7 – –
C 18:0 939.2 2.6 926.0 2.4 2308.2 4.2 2214.6 4.1 3736.9 0.7 3679.7 3.0
C 18:1(9) 2398.5 2.9 2350.8 2.4 3974.6 3.8 3821.2 3.8 16410.6 0.8 16214.2 2.8
C 18:2 (9,12) 214.2 1.0 220.9 2.9 400.4 3.6 386.6 3.8 45397.1 1.0 44818.4 2.2
C 18:3 (9,12,15) 54.8 2.1 55.6 1.8 131.6 3.2 125.7 3.6 169.6 1.1 171.7 4.9
C 19:0 – – 21.6 3.4 22.3 3.4 – –
C 20:0 – – 52.0 5.2 49.1 4.4 223.0 1.1 214.5 3.1
C 20:1(11) – – 48.4 5.8 48.8 5.6 100.0 0.9 99.2 3.6
C 20:2 (11,14) – – – – – –
C 20:3 (11,14,17) 37.4 3.7 39.0 3.5 32.2 5.9 34.2 3.3 – –
C 22:0 – – 24.6 6.8 23.1 3.1 581.7 1.6 551.4 2.4
C 22:1(13) – – – – – –
C 24:1(15) – – – – 156.3 1.5 142.7 2.8
Total fatty acids 5630.2 5551.0 17242.8 16766.9 72235.3 71379.6
a n= 4;CV = coefficient of variation in %.
– = content of fatty acid less than 10 mg/100 g fresh sample.
Ext = modified extraction method; DIR = direct method.

FAME (C13:0) was added and mixed with 8 mL filtered with slight suction through a sintered glass
chloroform and 8 mL pyrogallol in methanol (1 g/100 funnel coated with approx. 2 g of Celite and rinsed
mL). The sample was shaken vigorously for 2 min with 4 mL mixture of chloroform/methanol 2:1. For
and another 8 mL chloroform added. The sample was separation into two phases, z mL water was added,

Table 2 Fatty acid composition of cooked pasta in mg/100 g fresh sample determined without and with enzymatic starch
degradation
Ext Ext Ext Dir Ext Dir
Enzyme Clara-Diastase Termamyl 120 L Termamyl 120 L Termamyl 120 L
(concentration (0.15 g in 15 mL (0.25 mL in 5 mL (1.0 mL in 5 mL (0.25 mL in 5 mL
and reaction sodium acetate water, water, water,
conditions) none buffer, 40–45 °C, 1 h) 95 °C, 15 min) 95 °C, 30 min) none 95 °C, 15 min)
Fatty acid Meana CV Meana CV Meana CV Meana CV Meana CV Meana CV
C 8:0 – – – – – –
C 10:0 – – – – – –
C 12:0 – – – – – –
C 14:0 – – – – – –
C 15:0 – – – – – –
C 16:0 89.3 7.6 216.6 2.6 192.0 2.6 256.6 2.9 166.6 8.8 262.2 2.3
C 16:1(9) 6.9 8.3 13.7 3.2 11.6 3.2 15.7 3.3 13.4 10.8 15.1 2.4
C 17:0 – – – – – –
C 18:0 21.4 8.5 44.1 2.9 40.0 2.9 49.1 3.0 38.0 6.5 51.3 4.3
C 18:1(9) 121.5 9.0 248.7 2.8 216.0 2.8 288.7 2.6 238.6 10.7 286.0 2.3
C 18:2 (9,12) 162.8 5.4 356.1 2.7 302.5 2.7 426.1 2.8 341.3 9.1 428.3 3.2
C 18:3 (9,12,15) 8.4 4.1 19.0 5.3 14.0 5.3 19.3 4.8 16.1 8.6 19.8 1.6
C 20:0 – – – – – –
C 20:1(11) – – – – – –
C 20:2 (11,14) – – – – – –
C 20:3 (11,14,17) – 7.4 4.1 8.0 3.7 9.1 3.5 8.5 10.5 9.5 3.0
C 22:0 – – – – – –
C 22:1(13) – – – – – –
C 24:1(15) – – – – – –
Total fatty acids 410.3 905.6 784.1 1064.6 822.5 1072.2
a n=4;CV= coefficient of variation in %.
–= content of fatty acid less than 7 mg/100 g fresh sample.
Ext=modified extraction method; Dir=direct method.

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 lwt/vol. 30 (1997) No. 2

Table 3 Fatty acid composition of the prepared meal (pasta with meat sauce) in mg/100 g fresh sample determined without
and with enzymatic starch degradation with Termamyl 120L
With starch degradation

Ext Dir
Without starch degradation 1.0 mL in 5 mL 0.25 mL in 5 mL
water, water,
Ext Dir 95 °C, 30 min 95 °C, 15 min
Fatty acid Meanb CV Meanb CV Meana CV Meana CV
C 8:0 19.1 5.5 21.1 5.9 20.5 5.2 21.0 4.7
C 10:0 43.3 5.2 45.7 5.0 43.9 4.4 44.7 5.7
C 12:0 58.3 3.6 62.7 4.6 61.2 2.8 59.4 3.0
C 14:0 205.4 3.7 213.9 3.7 217.6 2.5 208.4 3.1
C 15:0 23.3 3.6 23.7 2.7 24.8 2.4 23.3 3.7
C 16:0 984.6 4.4 1020.8 2.5 1051.9 2.2 1005.8 3.7
C 16:1(9) 81.8 4.4 85.3 2.6 86.4 2.3 83.0 3.2
C 17:0 23.6 4.2 24.4 6.2 24.6 2.8 25.6 4.6
C 18:0 397.9 5.9 396.1 3.7 412.1 2.1 393.2 2.2
C 18:1(9) 1842.3 5.3 1888.5 2.1 1909.7 2.2 1896.5 3.4
C 18:2 (9,12) 324.1 6.6 339.3 6.2 310.3 3.2 337.0 3.3
C 18:3 (9,12,15) 36.9 5.2 40.3 5.4 38.6 2.3 39.0 4.6
C 20:0 – – – –
C 20:1(11) – – – –
C 20:2 (11,14) – – – –
C 20:3 (11,14,17) 10.2 4.3 10.7 5.4 10.6 7.4 12.2 2.1
C 22:0 – – – –
C 22:1(13) – – – –
C 24:1(15) – – – –
Total fatty acids 4050.8 4172.5 4212.2 4149.1
a n= 4; bn = 8;
CV=coefficient of variation in %.
– = content of fatty acid less than 10 mg/100 g fresh sample.
Ext = modified extraction method; Dir=direct method.

where z = 6 – (P 3 W/100), P = sample weight in g
and W = water content of sample in g/100 g. The
sample was vortexed for 2 min and centrifuged for 3
min at 2200 3 g. The upper layer was removed with a
Pasteur pipette and the remaining chloroform phase
dried by adding approx. 2 g of anhydrous sodium
sulphate. Four millilitre of the dried chloroform phase
(containing 10 to 50 mg of fatty acids) was trans-
ferred into a 15 mL culture tube fitted with a screw
cap and concentrated in a rotatory evaporator under
reduced pressure of 300 mbar for 5 min nearly to
dryness. The residue was redissolved in 1 mL hexane
and a magnetic rod placed in the tube. The content
was saponified by adding 4 mL of NaOH (0.5 mol/L)
in dry methanol and placing the capped tube for 5 to
10 min into the heating/stirring block at 100 °C under
continuous stirring. Conversion of the fatty acids into
methyl esters and separation of methyl esters was
made as described for the direct method, except that
(because of the larger end volume) the content of the
15 mL culture tube must be transferred into a larger
culture tube before separation.
For starch-rich food samples it is necessary to apply
an enzymatic degradation of gelatinized starch prior
to fat extraction: 1 mL of thermoresistent α-amylase
preparation Termamyl 120L was mixed with 4 mL
pyrogallol in deionized water (1 g/100 mL) and this
solution added to the sample. The tube was capped
and held for 30 min at 95 °C in a water-bath under
continuous stirring. For separation into two phases
z – 5 mL of water was added.
Optimization of the Termamyl treatment
In order to optimize the lipid extraction for starch-rich
food samples, incubation experiments with the α-amy-
lase preparation Termamyl 120L were carried out at
different enzyme concentrations and for different
reaction times. Various amounts of Termamyl 120L
(100, 200, 500 and 1000 µL) were added to the samples
and incubated at 95 °C for 15, 30 and 60 min. The
concentration of liberated D-glucose at the end of the
incubation period was chosen as an indicator of optimal
conditions for the Termamyl action. Determination was
performed using an enzymatic assay according to the
description given by the Food Analysis Boehringer
Mannheim Test No. 716251.
Statistical analysis
The measured values were evaluated by descriptive
statistics (means and coefficients of variation). Differ-
ences between the mean contents of each fatty acid in
all examined samples for both methods were analysed
with the statistical program of Microsoft® Excel
Version 5.0 (Microsoft Corporation, Redmond, WA,
U.S.A.). The paired two-sample t-test for means at the
95% confidence level was used.

207
lwt/vol. 30 (1997) No. 2
Results and Discussion
In order to compare the direct method (Fig. 1) with the
modified extraction method (Fig. 2) for the determina-
tion of fatty acid content and distribution in foodstuff, a
test meal (pasta with meat sauce), as well as some of its
ingredients, and mayonnaise were analysed. The lower
detection limit for the quantitative determination of
fatty acids in the meal was found to be approx. 10 mg
per 100 g fresh sample. The chromatogram of the
calibration solution of the standard mixture of FAME
(Fig. 3) and a representative chromatograph of the food
sample (Fig. 4) showed excellent separation of all fatty
acids of interest. The selection of fatty acids can be
extended for example by C20:4 and C20:5 without any
change of the operating conditions for GC (data not
shown). The data for minced beef, Parmesan cheese,
and mayonnaise are summarized in Table 1. The results
indicated a high degree of precision for both methods
as shown by low coefficients of variation (CV) for the
single fatty acids, ranging from 0.6 to 6.8. It was evident
that both methods led to equivalent results. Application
of the paired two-sample t-test at the 95% confidence
level revealed no significant differences between the
two methods for most of the fatty acids. A possible
explanation for the only fatty acid, Eicosatrienoic acid
(C 20:3), showing a different outcome of the statistical
analysis could be its low concentration in some food
samples making accurate integration of peaks more
difficult.
However, substantial differences resulted for cooked
pasta. The extraction method gave much lower yields of
fatty acids when compared to the direct method (Table
2). It was obvious that part of the lipids was ‘entrapped’
by the gelatinized starch and was thus not extracted.
Because of this problem, already described by the
method for determination of cholesterol (16), it was
supposed that even by the direct method not all lipids
were liberated and analysed. The data from Table 2
confirmed this assumption. The necessity to degrade the
gelatinized starch in order to release total lipids has
already been recognized. The official method described
in the Swiss Manual for Food Analysis (17) uses Clara-
Diastase (an α-amylase preparation from Aspergillus
oryzae) for starch degradation. In our experiments the
effectiveness of the Clara-Diastase was compared to
thermoresistent α-amylase Termamyl 120L. Clara-Dia-
stase at the recommended concentration level
improved lipid extraction from cooked pasta but the
amount of determined fatty acids was lower as com-
pared to the amount obtained by the use of Termamyl
120L (Table 2). Moreover, Clara-Diastase could not be
used in the direct method prior to the saponification
step because this enzyme needs buffered conditions for
its action. Another important advantage of using
Termamyl 120L in the extraction method is that only
half the time (30 min instead of 60) is needed for
sufficient starch degradation as compared to Clara-
Diastase. The need for a preliminary degradation of
starch for the test meal was also studied. The results are
summarized in Table 3 and indicate that this step could
208
be omitted. This finding was expected because the
percentage of fatty acids in pasta is low (Table 2) and
the share of pasta in the whole meal makes approx.
42%. The diminution of the total fatty acid content in
the meal caused by the insufficiently extracted pasta
was 6.5% for the extraction method and 2.5% for the
direct method only. It was therefore within the meth-
ods’ precision.
Preliminary experiments proved the necessity to pre-
vent the oxidation of unsaturated fatty acids in the
course of sample preparation. Addition of 3-tert-butyl-
4-hydroxyanisole (BHA) solution (1 g/100 g) in ethanol
(direct method) or methanol (modified extraction
method) led to two problems. Figure 4 reveals that
BHA coeluated with methyl ester of nonadecanoic acid
(C19:OMe) under the selected operating conditions.
This microbial fatty acid had been shown to be present
in Parmesan cheese used in the meal (Table 1). Second,
the insolubility of BHA in water prevented its use
during the starch degradation step. Therefore pyro-
gallol at a concentration of 1 g/100 mL was used as
antioxidant. This substance is soluble in water, ethanol
and methanol but insoluble in apolar solvents like
hexane or in chloroform. The protective effect of
pyrogallol is therefore limited to the enzymatic starch
degradation and to the saponification step. However,
these two procedures would most likely cause oxidation
because of the heating process involved.
The accuracy of both methods was also evaluated by
analysis of glycerine mono-oleate. Using the direct
method an oleic acid recovery of 99.6% was obtained,
whereas with the extraction method only 89.3% of the
theoretical value for oleic acid was recovered.
Conclusions
The proposed direct method represents an accurate,
precise, simple and time saving method which enables
quantitative determination of cholesterol and fatty acid
contents in food on the same sample. It eliminates the
use of hazardous chlorinated solvents. If the cholesterol
content of a sample is not to be determined, the even
simpler modified extraction method could be used for
the fatty acid analysis. The main advantage of this
modification is the possibility to introduce (like in the
direct method) the internal standard to the sample at
the very beginning of the analytical procedure which
improves its precision. Another advantage of the
procedure is a substantially reduced use of solvents.
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