Journal of Food Composition and Analysis 25 (2012) 198–207

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Journal of Food Composition and Analysis

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Original Article

Efficiencies of three common lipid extraction methods evaluated by calculating

mass balances of the fatty acids
Liping Xiao a, Svein Are Mjøs a,b,*, Bjørn Ole Haugsgjerd b
a
Department of Chemistry, University of Bergen, P.O. Box 7803, NO-5020 Bergen, Norway
b
Nofima BioLab, Kjerreidviken 16, NO-5141 Fyllingsdalen, Norway

A R T I C L E I N F O A B S T R A C T

Article history: Efficiencies of three common lipid extraction methods have been evaluated by analyzing fatty acids in
Received 7 June 2011 residues and extracts and calculating the mass balance for the fatty acids. Fatty acids were analyzed by
Received in revised form 30 August 2011 an acid catalyzed direct methylation procedure followed by gas chromatography of fatty acid methyl
Accepted 31 August 2011
esters. This procedure was also used as the benchmark for the calculation of mass balances. The three
Available online 16 September 2011
extraction principles investigated were Soxhlet extraction with petroleum ether, Soxhlet extraction after
acid hydrolysis, and the Bligh and Dyer method. All samples were dry powders of marine origin; most
Keywords:
samples had high ratios of polar to nonpolar lipids. Significant amounts of fatty acids were detected in
Food analysis
Food composition
the residues after extraction. The lowest extraction efficiencies were 30% for the Soxhlet method, 83% for
Fatty acids the acid hydrolysis method and 90% for the Bligh and Dyer extraction. The lowest extraction efficiencies
Lipid extraction were typically found in samples with high ratio of polar to nonpolar lipids.
Lipid content ß 2011 Elsevier Inc. All rights reserved.
Soxhlet extraction
Bligh and Dyer extraction
Marine lipids
Phospholipids

1. Introduction components. Therefore, lipid content often fails to accurately

In recent years, polyunsaturated fatty acids (PUFAs) have
gained increasing attention, especially the n–3 (omega-3) and n–6
(omega-6) fatty acids, because of their various biological functions
related to health and disease (Mazza et al., 2007). These fatty acids
(FAs) play important roles in the treatment and prevention of
cardiovascular, autoimmune, and nervous disease and in the
improvement of learning ability (Nielsen et al., 2005). The group of
marine lipids is an excellent source of essential polyunsaturated
FAs, in particular the n–3 family of FAs.
The content of total lipid and FA composition are two key
indices utilized for assessment of nutritional value of food. The
lipid content is traditionally gravimetrically determined by solvent
extractions. There are a number of methods for lipid extraction.
Soxhlet (AOCS Ba 3-38), acid hydrolysis (ISO 6492, 1999) and Bligh
and Dyer (B&D) (Bligh and Dyer, 1959) are the dominating extraction
methods for the assessment of lipid content in food and feed
ingredients. The total lipid content obtained by solvent extraction
represents the content of crude fat, possibly including non-fat
* Corresponding author at: Department of Chemistry, University of Bergen, P.O.
Box 7803, NO-5020 Bergen, Norway.
E-mail address: svein.mjos@kj.uib.no (S.A. Mjøs).
estimate nutritional values in samples. Different ways of declaring
fatty acid contents in food is discussed by Hyvönen (1996).
The FA content in a lipid extract is usually determined by gas
chromatography (GC). In order to improve volatility and to gain
better GC peak shapes, transformation of FA into FA methyl ester
(FAME), i.e. methylation, is required. FAME can be prepared either
by multistep methods (Sirot et al., 2008; Kozlova and Khotim-
chenko, 2000) involving lipid extraction followed by transmethy-
lation, or by direct methylation (DM). DM is also called one-step
extraction/methylation, and has been applied for FA analysis in a
wide range of samples (Lepage and Roy, 1986; Ulberth and
Henninger, 1992; Carrapiso et al., 2000; Dickey et al., 2002; Meier
et al., 2006; Araujo et al., 2008). DM has several advantages over
the extraction-based methods, such as small sample sizes and low
solvent consumption, leading to a cost-efficient and environmen-
tally friendly methodology. DM methodology has also been shown
to have high recovery for polar lipids that are tightly bound to the
matrix and difficult to recover by extraction (Meier et al., 2006).
The purpose of this work was to evaluate extraction efficiencies
and calculate mass balances of FAs in lipid extraction methods in
samples of marine origin, specifically in fishmeal, cod and salmon
filets, herring roe and krill meal. This has been done by analyzing
FAs in samples before extraction and in extracts and residues after
extraction. The amounts of FAs in the unextracted samples

0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2011.08.003
 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207
analyzed by DM were used as a reference for calculation of the
mass balances of the extractions.
2. Experimental
2.1. Samples
The study of extraction efficiencies was conducted on dry
powders of marine origin that are typically used as food and feed
ingredients; 8 samples, covering a wide range of lipid composi-
tions, were analyzed; sample type and origin are described in Table
1. Prior to extraction all samples were ground into fine powders on
a Retch Grindomix GM 200 laboratory mill for about 20 s at
8000 rpm, except dried salmon filet (Sample C). The latter was
subject to agglomeration due to high lipid content. In order to
avoid degradation of the samples during the experiment period,
each sample was divided into three aliquots, vacuum packed and
stored at 5 8C; each aliquot being used for evaluation of one of the
extraction methods.
Samples B, C and D contain all the lipids originally present in the
raw material, while the majority of triacylglycerols (TAGs) in the
other samples had been removed during the production process.
Products commercially available were supplied by Nofima Feed
Production Facility, Titlestad, Norway. Samples B, C and D were
purchased locally as frozen products, freeze dried, and treated as
described above. Sample H was prepared at Nofima Ingredients,
Bergen, Norway, under conditions similar to a typical fishmeal
process.
2.2. Extraction and methylation of samples
Each sample was treated by four different methods: (a) direct
methylation; (b) Soxhlet extraction followed by methylation; (c)
acid hydrolysis extraction followed by methylation; and (d) B&D
extraction followed by methylation. Experimental methods are
described below.
2.2.1. Direct methylation
Samples that were not treated by extraction were directly
transmethylated into FAME. Approximately 200 mg were accu-
rately weighed and mixed with 1.0 mL of methylation reagent,
which consisted of 75% of 2.5 M methanolic HCl and 25% of toluene
and prepared as described in the literature (Meier et al., 2006). The
sample was thereafter heated at 100 8C for 2 h, cooled down to
room temperature, and concentrated to 50% by evaporation under
a stream of nitrogen at 70 8C. 1.0 mL of water was added and the
sample was extracted twice with 1.0 mL of isooctane. The extracts
were combined and diluted with isooctane to an appropriate
concentration suitable for GC analysis.
Prior to methylation of the sample the saturated FAME 23:0
(methyl tricosanoate, Larodan AB, Malmø, Sweden, 99.5%) in
isooctane was added to the reaction tube and used as internal
standard for quantification of FAME by GC. The amount of added
23:0 was varied according to the possible lipid content of sample.
2.2.2. Soxhlet extraction and methylation
Samples weighing 4–7 g were accurately weighed, packed with
cotton wool, and transferred into the extraction tube, which was
equipped with a pre-dried Soxhlet flask. Approximately 100 mL of
petroleum ether (boiling point 40–60 8C) was added and the
sample was extracted overnight (16–18 h) at a condensation rate
of 2–3 drops per second. The extract was diluted to approximately
100 mL with petroleum ether and the solution was weighed. Two
aliquots (approx. 4 mL each) were collected, weighed, and
evaporated to dryness under a stream of nitrogen at 70 8C,
followed by methylation using the same procedure as described in
199
Section 2.2.1. In order to calculate the quantity of total extracted
lipids by gravimetry, the solvent of the remaining extract was
distilled off, and the residue was dried in the oven at 103 8C for 2 h,
and weighed. The residue was weighed after the solvent had
evaporated under vacuum. Two aliquots of the residues (approx.
200 mg each) were collected, weighed, and methylated using the
same procedure as described in Section 2.2.1.
2.2.3. Acid hydrolysis extraction and methylation
An initial Soxhlet extraction was performed according to the
procedure described in Section 2.2.2, with the exception that
extraction time was 2 h instead of over night. The residue in the
extraction tube was dried and transferred into a 250 mL round-
bottomed flask. 100 mL of 3 M aqueous HCl was added and the
solution was refluxed for an hour. The solution was cooled down,
and filter aid (kiselgur or hyflo-supercel) was added. The sample
was filtered through a pre-wetted, fat-free 150 mm white band
filter and washed with cold water until the filtrate became neutral.
The filter cake was thereafter isolated and dried for 2 h at 103 8C,
and transferred to the same extraction tube that was used in the
initial extraction. The extraction tube was placed in the Soxhlet
extractor together with the flask containing the initial extract.
Petroleum ether was added to ca. 100 mL and the sample was
extracted over night at a condensation rate of approximately 2–3
drops per second. After extraction, the extract and residue were
treated, as described in Section 2.2.2.
2.2.4. Modified B&D extraction and methylation
Approximately 2.5 g of sample with known water content was
weighed and transferred into a 250 mL flask. The appropriate
amount of water, making the total water content equal to 16 mL,
was added, followed by the addition of methanol (40 mL) and
chloroform (20 mL). The mixture was homogenized for 60 s on a
T25 Ultra-Turrax homogenizer (IKA, Stanfen, Germany). Chloro-
form (20 mL) and water (20 mL) were added in sequence followed
by homogenization for 30 s after each addition. The mixture was
cooled down on ice bath and filtered. The filtrate was collected in a
200 mL graduated cylinder. The volume of the water/methanol
phase (upper phase) was recorded and two 3 mL aliquots were
collected, evaporated to dryness under a stream of nitrogen at
100 8C, and methylated as described in Section 2.2.1. The
remaining water/methanol phase was removed from the cylinder
using a water aspirator. The filter cake was transferred into the
original flask and 20 mL of chloroform was added, followed by
homogenization for 15 s. The mixture was filtered and the filtrate
was collected in the same graduated cylinder. The total volume of
the chloroform phase was recorded and two aliquots were taken,
evaporated to dryness under a stream of nitrogen at 100 8C, and
methylated as described in Section 2.2.1. 20 mL of the chloroform
phase was transferred into a tray, evaporated to dryness under an
infrared lamp, and weighed. The total extracted lipid was
calculated through multiplication of this weight by ratio of the
total volume of the chloroform phase to 20 mL (gravimetry). An
aliquot of the extract was analyzed by liquid chromatography (LC)
in order to analyze the lipid classes (see Section 2.4). The solid
residue was collected, dried in vacuum for 2 h, and weighed. Two
portions of the residue (approximately 200 mg each) were
weighed and methylated as described in Section 2.2.1.
2.3. Fatty acid analysis by GC
The GC analyses was conducted on a Trace GC gas chromato-
graph (Thermo Fisher Scientific) with flame ionization detector
(GC–FID), equipped with a 60 m  0.25 mm BPX-70 cyanopropyl
column with 0.25 mm film thickness (SGE, Ringwood, Victoria,
Australia). Helium 4.6 was used as mobile phase under the
200 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207
Table 1
Sample details.
Sample Type
A Fishmeal (unspecified species), commercial
B Cod (Gadus morhua) filet, freeze dried and milled
C Salmon (Salmo salar) filet, freeze dried and milled
D Herring (Clupea harengus) roe, freeze dried and milled
E Krill (Euphausia superba) meal, commercial
F Fishmeal, blue whiting (Micromesistius poutassou),
commercial
G Fishmeal (unspecified species), commercial
H Krill (Euphausia superba) meal, produced by Nofima
pressure of 2.20 bar. The injector temperature was 280 8C and the
detector temperature was 260 8C. The oven was programmed as
follows: 60 8C for 4 min, 30 8C/min to 166 8C, and then 1.1 8C/min
to 213 8C, and 120 8C/min to 250 8C/min where the temperature
was held for 10 min. The sample solution (1.0 ml) was injected
splitless and the split was opened after 2 min. The FAMEs were
identified by comparing the elution pattern and relative retention
time with the reference FAME mixture (GLC-793, Nu-Chek Prep
Inc., Elysian, MN, USA). Additional identifications were achieved by
comparison with a control oil, where the FA content has been
identified by gas chromatography–mass spectrometry (GC–MS)
methodology as described in the literature (Mjøs, 2003, 2004).
Chromatographic peak areas were corrected by empirical response
factors calculated from the areas of the GLC-793 mixture.
2.4. Lipid class analysis by liquid chromatography
Lipid class composition of B&D extracts was analyzed by liquid
chromatography (LC) using a modified version of a previously
described method (Homan and Anderson, 1998). Analyses were
carried out on a Series 200 HPLC system (Perkin Elmer, Shelton, CT,
USA) equipped with a charged aerosol detector (ESA, Chelmsford,
MA, USA) and a 125  4 mm LiChrospher diol column with 5 mm
particle diameter (Merck, Darmstadt, Germany). The column
temperature was kept at 45 8C. The sample was separated in a
single chromatographic run by using a ternary solvent gradient
program. Further details are given in supporting information
(Table S1).
2.5. Repeatability and precision
The repeatability of DM was checked by analyzing 6 replicates
of Sample A and 7 replicates of a Soxhlet extract of Sample A on the
same day. The intermediate precision and sample stability were
evaluated by measuring the control oil (NorsalmOil fish oil, Vedde
AS, Langevåg, Norway) and Samples A–H on different days
throughout the study.
2.6. Analysis of data
Analysis of variance (ANOVA), F-test and t-test were used to
analyze the data at P = 0.05 level. Principal component analysis
(PCA) was performed using the Sirius 8.0 software (Pattern
Recognition Systems, Bergen, Norway).
2.7. Replicates and outline of the experiment
Fig. 1 provides an overview of the experimental set up. DM
(Fig. 1, 1) was conducted in three replicates and the three
extraction methods were carried out in two replicates. The three
replicates of the DM analyses were conducted at the start, middle
and end of the experimental period, respectively. For each extract
or residue (Fig. 1, 2–8), methylation and FA analysis was performed
in two replicates. Mass balances were calculated by comparing the
Water amounts found in extracts and residues (Fig. 1, 2–8) with amounts
content found after DM (1 in Fig. 1).
(%)
7.84 3. Results and discussion
2.31
6.49
3.1. Precision of the direct methylation method
1.50
6.09
10.48 The repeatability (within-day variation) of the FA analysis by
DM was evaluated by analyzing 7 replicates of Sample A within the
7.47
same day. In order to evaluate the reliability of FA analysis for the
6.39
extract (solution phase), 6 replicates of Soxhlet extract of Sample A
were also treated using the same method. The coefficient of
variation was around 5.1% for the powder and 1.2% for the extract.
The higher deviation in the former presumably results from the
lower homogeneity. In addition, the solid sample contains a large
number of other components (e.g. minerals and proteins), and a
wider range of lipid classes compared to the extract, which may
also affect the methylation reaction. For both the solid sample and
the extract, the experimental deviation for each specific FA was
close to that of total FAs. The methylation reaction showed no
selectivity for specific FAs. Further details are given as supplemen-
tary material (Table S2).
The intermediate precision (between-day variation) was also
evaluated by analyzing the control oil and the samples on the
different days during the experimental period. The coefficient of
variation for total FAs of the control oil was around 1.9%, indicating
a high intermediate precision, while coefficient of variation for
total FAs of the solid samples varied from 1.9% to 6.8%. The
experimental deviation for 22:6 n–3 (the most unsaturated FA) is
close to those of the other FAs, suggesting that degradation by
oxidation was minimal. No other trends related to time were seen,
indicating that the samples were stable throughout the experi-
mental period. There was no pronounced variation observed for
the FA classes including saturated FA (SFA), monounsaturated FA
(MUFA) and polyunsaturated FA (PUFA). Further details are given
as supplementary material (Table S3).
3.2. FA compositions and lipid classes of samples
The compositions of the FAs of 8 marine samples were
determined by GC and the results are reported in Table 2. Among
the 8 samples, the salmon powder (Sample C), being from a typical
fatty fish, contained the highest amount of total FAs (36.2 g/100 g
sample). The dried cod filet (Sample B) had the lowest content of
total FAs (2.2 g/100 g sample). Between these two extremes the
other samples ranged from 5.2 to 14.2 g FA per 100 g of the powder.
Lipid classes analyzed as described in Section 2.4 are reported in
Table 3. There are large variations in lipid class composition
between different samples. TAG varied from 2.3 to 87 weight % of
extracted fat. The main phospholipids, phosphatidyl choline and
phosphatidyl ethanolamine, varied from less than 1% to 49% and
from less than 1% to 12%, respectively. Free FAs varied from 0.8% to
36%. Cholesterol varied from less than 0.5% to 8.2%. Other lipid
classes were below 2% in all samples. With the exception of
Samples B and D, total neutral (apolar) lipid contents were higher
than those of polar lipids. The sum of lipid classes determined by LC
account for approximately 78.0–92.3% of the amounts that were
found by gravimetry.
The dried cod filet (Sample B) and one of the fish meals (Sample
F) had high content of free FAs (25.0% and 36.0%), implying the
occurrence of hydrolysis of lipids in these two samples. Free FAs in
Samples B and F were also determined by titration according to
(AOCS Ca 5A-40), which gave values of 27.5% and 28.6%
respectively. Because enzymatic activity in dry powders is
 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207 201

Fig. 1. Outline of the experiment.

minimal, hydrolysis in these samples has probably occurred before
freeze-drying (Sample B), or during long term storage in the case of
the fishmeal (Sample F).
3.3. Mass balances
The FA results obtained by DM are reported in Table 2. When
normalized to the total sample weight, the sums of the extracted and
residual FAs after the three extraction methods should ideally be
equal to the DM results, which are used as benchmark in the study.
3.3.1. Mass balance for the Soxhlet method
The mass balances for the Soxhlet method are presented in
Table 4. It is obvious that the FAs recovered from the Soxhlet
extracts (29–99% relative to DM), only account for a part of the
total FAs, and the other FAs still remain in the residues (2.3–66%).
The sum of FAs in the extracts and residues match well with those
obtained by DM; total recoveries ranged from 96% to 112%.
Dried salmon filet (Sample C), containing a high content of TAG
and a very small amount of polar lipids, had the highest extraction
efficiency (98.0–99%), whereas dried cod filet (B) and herring roe
(D), bearing small quantities of TAG and high contents of
phosphatidylcholine and phosphatidylethanolamine, showed the
lowest extraction efficiencies (37.3% and 38.2% for B, and 29.1% and
31.4% for D, respectively).
The FA contents in the extracts and residues varied clearly as a
function of sample composition. Percent extracted lipids were
highly correlated with sum of percent TAG and free FAs (FFA) with

Table 2
Fatty acid compositions of samples determined by direct methylation (average of 3 replicates). Fatty acids that are below 1% of total fatty acids are not listed individually, but
contribute to the total fatty acids. Values are given in g fatty acids per 100 g sample.

FAs Aa Ba Ca Da Ea Fa Ga Ha

14:0 0.48 0.03 1.04 0.42 1.69 0.18 0.61 1.23

16:0 1.24 0.42 3.96 2.16 3.47 0.88 1.55 3.03
18:0 0.16 0.11 1.03 0.23 0.23 0.17 0.19 0.17
16:1 0.28 0.03 1.04 0.36 0.99 0.23 0.35 0.51
18:1 0.93 0.27 13.81 1.59 2.96 0.93 1.36 2.43
20:1 0.82 0.05 1.82 0.25 0.20 0.67 1.03 0.16
22:1 1.14 0.01 1.19 0.11 0.14 0.71 1.51 0.13
24:1 0.11 0.03 0.20 0.14 0.03 0.13 0.14 0.01
18:2 n–6 0.10 0.02 4.02 0.12 0.24 0.06 0.11 0.24
18:3 n–3 0.07 0.01 1.69 0.09 0.15 0.03 0.07 0.38
18:4 n–3 0.17 0.01 0.26 0.10 0.29 0.06 0.12 0.72
20:4 n–3 0.05 0.01 0.40 0.07 0.08 0.02 0.04 0.10
20:5 n–3 0.67 0.29 1.34 1.19 2.06 0.36 0.56 2.67
22:5 n–3 0.07 0.03 0.79 0.11 0.06 0.04 0.07 0.06
22:6 n–3 1.36 0.80 2.31 3.04 1.20 0.60 1.10 1.49

SFAb 1.91 0.56 6.22 2.82 5.44 1.25 2.39 4.47

MUFAb 3.28 0.37 18.08 2.45 4.32 2.68 4.34 3.23
PUFAb 2.62 1.28 11.92 4.85 4.42 1.25 2.20 6.00
Total FAsb 7.81 2.20 36.22 10.13 14.18 5.17 8.92 13.71
a
Sample codes are given in Table 1.
b
Minor fatty acids that contribute to the sums are 10:0, 12:0, 20:0, 22:0, 16:2 n–4, 16:3 n–4, 20:2 n–6, 20:3 n–6, 22:4 n–6, 20:3 n–3 and 21:5 n–3.
202 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207

Table 3
Lipid classes of samples obtained by LC. Values are given as g/(100 g extracted fat), obtained by analyses of the Bligh and Dyer extracts.

Lipids Aa Ba Ca Da Ea Fa Ga Ha

Triacylglycerol (TAG) 54.0 2.3 87.0 17.0 45.0 25.0 57.0 39.0
Diacylglycerol <0.5 <0.5 <0.5 <0.5 1.1 1.8 <0.5 <0.5
Monoacylglycerol <1.0 <1.0 <1.0 <1.0 <1.0 1.4 <1.0 <1.0
free FAs 3.4 25.0 0.8 2.6 3.6 36.0 5.3 13.6
Cholesterol 2.1 6.7 <0.5 8.2 2.0 6.2 4.1 1.2
Cholesterol ester <0.5 <0.5 0.5 <0.5 0.8 0.7 <0.5 <0.5
Phosphatidylethanolamine 6.2 12.0 <1.0 7.0 <1.0 <1.0 <1.0 1.2
Phosphatidylcholine 16.0 29.0 <1.0 49.0 34.0 8.0 13.0 35.0
Lyso-phosphatidylcholine 1.2 1.8 <1.0 <1.0 2.8 1.8 1.0 1.3
Total polar lipids 23.6 42.8 1.1 56.6 37.9 10.1 14.6 37.8
Total neutral lipids 59.7 35.3 88.6 27.6 52.1 71.2 67.2 54.4
Total lipid 83.3 78.0 89.7 84.2 90.0 80.9 81.8 92.3
a
Sample codes are given in Table 1.

R2 of 0.86, indicating that it is basically these FAs that were
extracted. It is also well known that the Soxhlet extraction with
apolar solvents is sensitive to the polarity of lipids and has low
recovery for phospholipids (Sahasrabudhe and Smallbone, 1983;
Hole et al., 1996; Johnson and Barnett, 2003). It should be pointed
out that the neutral lipids are not always recovered 100% by
Soxhlet extraction, and that some polar lipids can also be extracted
(Ewald et al., 1998).
Total lipids determined by gravimetry are also reported in Table
4. Total fat determined by gravimetry varied from 52% to 123% of
FAME determined by DM. The weight of FAs is determined as FAME
and gravimetric lipid content can be expected to be similar only in
cases where the lipids are close to 100% TAG. Free cholesterol,
which has no FAs, contributes only to the gravimetric weight.
Cholesterol esters and phospholipids, where the FAs constitute a
smaller part of the molecule than in TAG, will also contribute more
to the total lipid weight than to the weight of FAME after
methylation. This can explain the values above 100%. Values below
100% are explained by low extraction efficiencies and other
analytical errors.
3.3.2. Mass balance for the acid hydrolysis method
In order to improve the low efficiency of Soxhlet extraction,
samples are often hydrolyzed prior to extraction. This will
release the FAs from the matrix in the form of free FAs. As long
as the matrix is not alkaline, free FAs can be extracted by apolar
solvents. In addition, the acid treatment may facilitate lipid
extraction by breaking down proteins and other compounds in
the matrix. In the case of the acid hydrolysis method used in this
work, the residues after the initial Soxhlet extraction were
hydrolyzed and extracted again with petroleum ether. The
combined extracts and final residues were analyzed and their
mass balances relative to DM were established. The results are
listed in Table 5.
Compared to the Soxhlet method, it can be seen that acid
hydrolysis substantially improves the extraction efficiency, which
is in accordance with results previously reported (de Koning et al.,
1985). Extracted lipids varied from 82% to 112% relative to DM,
while residual lipids varied from 0.7% to 17%. Sum of the extracts
and residues varied from 99% to 115%. Even though extraction
efficiency was substantially increased for the phospholipid-rich
samples, it should be emphasized that residual lipids were still
above 5% in three samples, and as high as 17% in the dried cod filet.
This could indicate either that the hydrolysis is not complete after
the acidic treatment, or that the free FAs are extracted with low
efficiency.
The total lipid content determined by gravimetry varied from
108% to 145% relative to FAs determined as FAME by DM. All krill
and fish meals had values above 130%. The acid hydrolysis method
is frequently applied for the determination of lipid content in food
and feed ingredients. In this respect it is worth noting that there is
often little correspondence between the gravimetric lipid content

Table 4
Mass balance for the Soxhlet method.

Samplea,e Total Total fatty acidsc Mass balanced

lipidb
Extract Residue Sum Extract Residue Sum

A1 (fish meal) 9.37 6.88 1.32 8.20 88.1 16.9 105.0

A2 (fish meal) 9.16 7.08 1.41 8.49 90.7 18.1 108.7
B1 (cod powder) 1.43 0.82 1.46 2.28 37.3 66.4 103.6
B2 (cod powder) 1.30 0.84 1.44 2.28 38.2 65.5 103.6
C1 (salmon powder) 38.62 35.50 0.83 36.33 98.0 2.3 100.3
C2 (salmon powder) 38.96 35.98 0.83 36.81 99.3 2.3 101.6
D1 (herring roe) 5.35 3.18 7.59 10.77 31.4 74.9 106.3
D2 (herring roe) 5.18 2.95 6.75 9.70 29.1 66.6 95.8
E1 (krill meal) 15.89 11.37 3.02 14.40 80.2 21.3 101.6
E2 (krill meal) 16.36 10.81 2.93 13.74 76.2 20.7 96.9
F1 (fish meal) 6.41 4.52 1.06 5.58 87.4 20.5 107.9
F2 (fish meal) 6.34 4.41 1.17 5.57 85.3 22.6 107.7
G1 (fish meal) 10.86 8.24 1.57 9.81 92.4 17.6 110.0
G2 (fish meal) 10.81 8.29 1.68 9.98 92.9 18.8 111.9
H1 (krill meal) 16.59 11.57 2.75 14.32 84.4 20.1 104.4
H2 (krill meal) 16.04 11.18 2.83 14.01 81.5 20.6 102.2
a
Each sample was determined twice.
b
g/(100 g sample), determined by gravimetry.
c
g/(100 g sample).
d
%, percentage of FAs obtained by Soxhlet extraction in the total FAs by DM.
e
Additional sample details are given in Table 1.
 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207 203

Table 5
Mass balance for the acid hydrolysis method.

Samplea,e Total Total fatty acidsc Mass balanced

lipidb
Extract Residue Sum Extract Residue Sum

A1 (fish meal) 10.55 8.04 0.18 8.22 102.9 2.3 105.2

A2 (fish meal) 10.77 8.79 0.23 9.02 112.5 2.9 115.5
B1 (cod powder) 2.58 1.81 0.38 2.20 82.3 17.3 100.0
B2 (cod powder) 2.58 1.97 0.38 2.35 89.5 17.3 106.8
C1 (salmon powder) 39.21 37.16 0.34 37.5 102.6 0.9 103.5
C2 (salmon powder) 39.25 37.02 0.24 37.26 102.2 0.7 102.9
D1 (herring roe) 12.24 9.41 0.59 10.00 92.9 5.8 98.7
D2 (herring roe) 12.43 10.06 0.68 10.75 99.3 6.7 106.1
E1 (krill meal) 18.95 13.83 0.26 14.09 97.5 1.8 99.4
E2 (krill meal) 18.77 13.61 0.59 14.20 96.0 4.2 100.1
F1 (fish meal) 7.58 5.39 0.30 5.69 104.3 5.8 110.1
F2 (fish meal) 7.45 5.40 0.36 5.76 104.4 7.0 111.4
G1 (fish meal) 12.73 9.64 0.18 9.82 108.1 2.0 110.1
G2 (fish meal) 12.58 9.66 0.26 9.92 108.3 2.9 111.2
H1 (krill meal) 19.08 13.56 0.35 13.91 98.9 2.6 101.5
H2 (krill meal) 19.24 14.2 0.29 14.49 103.6 2.1 105.7
a
Each sample was determined twice.
b
g/(100 g sample), determined by gravimetry.
c
g/(100 g sample).
d
%, percentage of FAs obtained by acid hydrolysis in the total FAs by DM.
e
Additional sample details are given in Table 1.

and FA content, i.e. digestible energy (Weihrauch et al., 1975;
Hyvönen, 1996).
3.3.3. Mass balance for the B&D method
The mass balances for the B&D method are reported in Table 6.
There are two fundamental differences between this method and
the Soxhlet and acid hydrolysis method: (1) the B&D employs
batch extraction, while the other methods are continuous
extractions; and (2) after extraction, solvent in the B&D method
are separated into an apolar (basically chloroform) and a polar
fraction (methanol/water). The lipids are assumed to be in the
apolar phase only, while the polar phase is expected to contain
matrix interferents. The presence of polar lipids in the water phase
has been reported (Kolarovic and Fournier, 1986; Shaikh, 1994).
FAs were therefore analyzed also in this phase in addition to the
apolar extract and the residue.
The mass balances for the B&D extraction are reported in Table
6. Extracted FA varied from 86% to 115% relative to DM, while
residual FA varied from 1.4% to 10%, and FA in the water phase
varied from 0% to 2.8%. Sum of extracts and residues varied from
91% to 121%.
There are significant amounts of FA left in the residue after the
B&D extraction as well. Since B&D is a batch extraction, the FA in
the residue can be ascribed either to non-extracted FA or to
extracted FAs present in solvent that are trapped in the residue.
However, if there were substantial amounts of dissolved lipids left
in the trapped solvent, one should find larger amounts of FA
measured in absolute values (g/100 g sample) for Sample C
(salmon powder), which is much richer in extractable FAs than any
other sample. One should also expect a general correlation
between absolute amounts of FAs in the extracts and residues.
This, however, was not the case. It can also be seen that the largest
amounts in the residues are found in the same samples that had
low extraction efficiencies in the Soxhlet and acid hydrolysis
methods, and the largest values are found for Sample B (dried cod
filet), which is particularly difficult to extract. This indicates that

Table 6
Mass balance for the Bligh and Dyer method.

Samplea,e Total Total fatty acidsc Mass balanced

lipidb
CHCl3 H2O Residue Sum CHCl3 H2O Residue Sum

A1 (fish meal) 10.75 8.95 0.03 0.46 9.43 114.6 0.4 5.9 120.7
A2 (fish meal) 10.67 7.97 0.03 0.52 8.52 102.0 0.4 6.7 109.1
B1 (cod powder) 3.39 2.20 0.01 0.21 2.42 100.0 0.5 9.5 110.0
B2 (cod powder) 3.29 1.98 0.01 0.23 2.23 90.0 0.5 10.5 101.4
C1 (salmon powder) 37.29 34.57 0.01 0.52 35.11 95.4 0.0 1.4 96.9
C2 (salmon powder) 37.46 33.17 0.02 0.66 33.85 91.6 0.1 1.8 93.5
D1 (herring roe) 16.17 10.87 0.05 0.68 11.60 107.3 0.5 6.7 114.5
D2 (herring Roe) 16.3 9.87 0.02 0.61 10.50 97.4 0.2 6.0 103.7
E1 (krill meal) 19.46 12.95 0.20 0.61 13.77 91.3 1.4 4.3 97.1
E2 (krill meal) 19.08 12.17 0.39 0.52 13.08 85.8 2.8 3.7 92.2
F1 (fish meal) 7.68 4.91 0.06 0.56 5.54 95.0 1.2 10.8 107.2
F2 (fish meal) 7.68 4.78 0.05 0.40 5.23 92.5 1.0 7.7 101.2
G1 (fish meal) 12.81 8.62 0.05 0.58 9.25 96.6 0.6 6.5 103.7
G2 (fish Meal) 12.77 8.28 0.05 0.50 8.83 92.8 0.6 5.6 99.0
H1 (krill meal) 17.62 13.07 0.17 0.63 13.87 95.3 1.2 4.6 101.2
H2 (krill meal) 17.67 11.88 0.15 0.41 12.45 86.7 1.1 3.0 90.8
a
Each sample was determined twice.
b
g/100 g sample, determined by gravimetry.
c
g/100 g sample.
d
%, percentage of FAs obtained by B&D in the total FAs by DM.
e
Additional sample details are given in Table 1.
204 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207

the majority of the residual FAs after B&D are FAs that were not assumes that the solvent that is trapped in residues and filters has
extracted. Since the lipid class results (Table 3) are acquired from a the same lipid concentration as the sampled solvent, and that
B&D extract, it is possible that a large portion of the non-extracted total solvent volume in the apolar phase is equal to added
FA is bound in molecules that are not detected by the lipid class chloroform. This is more accurate than measured subsampling
method. when a single extraction is applied. But the assumption that the
Except in Samples E, F and H, the contents of the FAs in the concentration in the trapped liquid is identical to the concentra-
aqueous phase were less than 1.0%. FAs in the water phase may be tion in the collected lipid cannot be valid if repeated extractions
bound in very polar lipids, such as lyso-phosphatidylcholine, lyso- are applied. Blind subsampling will also be affected by evapora-
phosphatidylethanolamine, and sphingomyelins. Loss of these tion of solvents. The reason for using measured subsampling in
lipids to the polar phase has been reported previously (Kolarovic this study is that it was necessary with a second wash of the
and Fournier, 1986; Shaikh, 1994). The largest amounts of FA were residue for the evaluation of the residual FAs. Then blind
found in the water phase from Sample E (krill meal). This is also the subsampling is not an option.
sample that had largest value of lyso-phosphatidylcholine.
Another explanation for the FAs in the water phase is that they 3.4. Comparison of the extraction methods
can be trapped in micelles or droplets of apolar solvent that is
dispersed in the polar phase. Extracted FA and total extracted lipids for the three methods are
Total lipids determined by gravimetry varied from 103% to shown in Fig. 2. Fig. 2a compares extracted FAs with FAs
160% relative to the weight of FAME determined by DM, which is a determined by DM. This value should be close to 100% for an
slightly wider range than what was observed for the acid efficient extraction. Except for the dried salmon filet (Sample C),
hydrolysis method. The sum of the mass balance, which should which contains basically TAG, the Soxhlet method is lower than the
ideally be close to 100%, also covers a wider range than observed others, and the differences are particularly large in the samples
for the Soxhlet and acid hydrolysis methods. These deviations can that had the highest relative proportions of phospholipids
probably be ascribed to a larger analytical error for the B&D (Samples B and D). The B&D method had slightly higher extraction
method. While mass balances of the two other methods are efficiency than the acid hydrolysis method for the samples that
basically based on weights, the B&D method applied in this study were most difficult to extract, but ANOVA analysis showed no
involves a volumetric determination of the two phases, and phase general differences between these two methods when all samples
separation is not always clear. were considered.
The wash with polar phase in B&D is basically used for removal
of non-lipid material when total lipids are determined by
gravimetry. As long as loss of polar lipids are observed in Folch
or B&D methods, one can question the use of the two-phase
systems prior to the determination of FAs or polar lipid classes by
chromatographic methods that are not influenced by the presence
of small amounts of non-lipids. Alternative methods based on
single-phase solvent systems have proven to be more efficient than
the biphasic extraction procedures on the isolation of PL from
biological sources (Kolarovic and Fournier, 1986).
The Bligh and Dyer method was originally developed for the
analysis of fresh tissues, and is basically used for this purpose.
There exist several variants of this method, as well as other
methods applying chloroform and methanol as extraction medium
(Folch et al., 1957; Smedes and Thomasen, 1996; Phillips et al.,
2008). Factors that have been shown to affect extraction
efficiencies in these methods are: proper adjustment of water
content (Bligh and Dyer, 1959), pre-treatment of samples (Phillips
et al., 2008), the ratio between fat and extraction medium (Iverson
et al., 2001), adjustment of pH and ion strength (Phillips et al.,
2008) and the sampling method of the apolar phase (Smedes and
Thomasen, 1996). It should therefore be emphasized that
the relatively high levels of residual fatty acids seen in some of
the samples may not have been found by other variants of the
methodology, or with fresh samples.
The results given in Table 6 are determined by ‘‘measured
subsampling’’, where the recovered organic phase volume is
measured and an aliquot, in which the lipid content is determined,
is sampled. The lipid content for the sample is then calculated for
the total volume of the recovered organic phase. The alternative to
measured subsampling is ‘‘blind subsampling’’ where the volume
of the organic phase is expected to be equal to added chloroform.
Advantages and disadvantages of the two principles are discussed
in Smedes and Thomasen (1996).
A single extraction and measured subsampling will underes-
Fig. 2. Comparison of the extraction methods. (a) Percentage of FAs obtained by
timate the lipid content because of the amount of solvent that is
Soxhlet (white), acid hydrolysis (gray) and B&D (black) relative to total fatty acids
trapped in the residue, filters and other places. Blind subsampling by direct methylation. (b) Percentage of gravimetric lipids obtained by Soxhlet
is commonly applied with the B&D method (Roose and Smedes, (white), acid hydrolysis (gray) and Bligh and Dyer (black) relative to total fatty acids
1996; Smedes and Thomasen, 1996). In blind subsampling one by direct methylation.
 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207 205

Values for total extracted lipids are compared to FAs deter-
mined as FAME after DM in Fig. 2b. These values should
theoretically be close to 100% only in the case of TAG, because
the weight of three FAME molecules created from TAG by
methylation will be roughly equal to the weight of the original
TAG. Values close to 100% are also observed for the dried salmon
filet (Sample B), where the lipids are basically TAG. Large
deviations are observed for the other samples. It can be seen that
the lipid contents obtained by B&D are generally higher than those
by acid hydrolysis, which is consistent with that observed by de
Koning et al., 1985. This can partly be explained by slightly higher
extraction efficiency for B&D. However, a possible explanation may
also be that polar lipids extracted after acid hydrolysis of the
samples are recovered as free FAs, while these lipids are acquired
intact in the B&D method. A phospholipid will therefore contribute
to more mass when extracted by the B&D method than extracted
after acid hydrolysis. Rader et al. (1995) compared gravimetric
lipid content by hydrolytic extraction with extracted FAs for 24
types of food and found that total lipids were higher than the total
FAs determined chromatographically. This agrees with our
observations as well.
The FAs are energy stores which are oxidized to release the
energy. The energy value of a food product is generally described
by the contents of total FAs. Research studies also indicate that the
dietary energy values of a food product also depend on the
saturation degree of the FAs, free FA contents and chain length of
the FAs (Wiseman et al., 1998). Undoubtedly, an accurate
estimation of the energy value of a food product should be carried
out by determining the FA contents instead of crude lipid weight.
However, due to the lack of sufficient data about the FAs of
different matrices, the gravimetric lipid contents determined by
extraction methods are often used to evaluate the dietary energy
value of a food product (Aksnes and Opstvedt, 1998; Smit et al.,
2004).
In this study, total extracted lipids varied from 52% to 160% of
the FA content determined as FAME. The poor correspondence
between total extracted lipids determined by gravimetry and the
actual FA content in the samples shows that gravimetric lipid
content can be a poor estimate for the actual energy content in the
lipids. In this respect it should also be emphasized that different
extraction methods may give highly different results for total
extracted lipids when applied on the same sample.
Factors to convert between different lipid extraction methods,
and between fatty acid content and gravimetrically determined
lipids, have been calculated for a variety of foods (Weihrauch et al.,
1975; Hyvönen, 1996). However, for fish meals it has been shown
that the differences between methods are dependent on oxidation
status of the sample, indicating that such correction factors cannot
be applied with accuracy on fish meal and similar products (de
Koning and Mol, 1989).
3.5. Influence of the extraction methods on the fatty acid profiles
Because different lipid classes have different FA distributions,
the differences in extraction efficiencies may also influence the FA
profiles, i.e. the relative distribution of FAs. FA profiles were
constructed by normalizing the FAs in the different samples and
extracts against total FAs, so that the sum of FAs is 100%. In
addition to the FA profiles of the extracts and DM, reconstructed FA
profiles were created by summing the FAs in extracts and residues
(including water phase in B&D) before normalization. While the FA
profile of an extract may differ from the profile of corresponding
DM as a result of extraction efficiencies, the reconstructed profile
should in theory differ only if there is a net loss of some of the FAs,
i.e. by oxidation.
The similarity of FA profiles was evaluated by Euclidean
distances and by principal component analysis (PCA). The
Euclidean distance is defined as:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
dð p; qÞ ¼ ð p1  q1 Þ2 þ ð p2  q2 Þ2 þ   ð pn  qn Þ2
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u n
uX
¼ t ð pi  qi Þ2 ; (1)
i¼1
where pi and qi are two points in an n-dimensional coordinate
system; p and q are in this case different profiles of the same
sample, while i signifies the different FAs and n is the number of
FAs in the profiles. Two identical profiles will have a Euclidean
distance of zero, and the distance increases with dissimilarity of
profiles.
The Euclidean distances from the DM profile to the extracts and
reconstructed profiles are shown in Table 7. It can be seen that the
distance between Soxhlet extracts and DM is substantially larger
than that between DM and the other profiles. The distance is also
larger for samples that typically had low extraction efficiencies,
showing that the low extraction efficiency has a profound
influence on the relative distribution of the FAs in the extracts.
The average distance between the reconstructed Soxhlet profile
and the DM is only 15% of the distance between Soxhlet extract and
DM, and of the same magnitude as the distance between the other
profiles and DM. This shows that the differences between DM and
the Soxhlet extract are accounted for by the FAs left in the residue,
and that there is no net loss of FAs that have significant impact on the
profiles. The profiles of acid hydrolysis extracts and B&D extracts
are much closer to the DM than the Soxhlet extract. But also for these
extractions there is a small reduction of distances when the FAs in
the residues are included in the reconstructed profiles.
A PCA score plot of all FA profiles is shown in Fig. 3a. This plot
basically illustrates similarities and dissimilarities between the
samples. The different profiles for the same samples can be found
in rather dense groups, compared to the differences between the
samples, illustrating that differences caused by the chosen sample
preparation method is substantially smaller than the differences
between the samples. However, the Soxhlet extracts are outliers in
all groups except in the case of the TAG rich salmon powder
(Sample C).
Fig. 3b shows a PCA score plot where the data matrix has been
augmented so that all 8 samples are merged into one object for
each type of profile. This illustrates the difference between the
various types of profiles. While the Soxhlet profile is clearly

Table 7
Euclidean distances from the fatty acid profiles obtained by direct methylation to the fatty acid profiles obtained by extraction or the reconstructed profiles (sum of extracts
and residues).

A B C D E F G H Average

Soxhlet extract 5.08 13.46 1.18 17.08 5.50 2.79 4.97 4.40 6.81
Soxhlet reconstr. 0.86 1.80 0.25 1.47 0.88 0.84 1.11 1.21 1.05
Acid hydr. extract 1.37 3.61 0.60 3.02 1.44 0.60 1.09 1.01 1.59
Acid hydr. reconstr. 0.94 2.00 0.35 2.88 1.00 1.06 0.71 0.77 1.22
Bligh and dyer extract 1.01 1.74 0.38 1.37 0.92 1.42 1.55 0.70 1.13
Bligh and dyer reconstr. 1.01 1.13 0.37 1.05 0.87 1.43 1.06 0.61 0.94
206 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207
Fig. 3. Principal component analysis scoreplot of the fatty acid profiles. SE: Soxhlet extracted; SR: Soxhlet reconstructed; AE: acid hydrolysis extracted; AR: acid hydrolysis
reconstructed; BE: Bligh and Dyer extracted; BR: Bligh and Dyer reconstructed.
different from the other profiles, the reconstructed Soxhlet profile
is grouped together with the other. Among the profiles of the
extracts, the B&D is the one that is nearest DM along the first
principal component, which account for 95% of the variance. This
also corresponds to the conclusions that can be drawn from the
Euclidean distances. Full FA profiles are presented as supporting
information in Tables S4–S10.
In marine lipids, special attention are usually paid to the highly
unsaturated long-chain omega-3 fatty acids EPA (20:5 n–3) and
DHA (22:6 n–3), both because of their nutritional significance, and
because of they are easily oxidized or degraded by other reactions.
Low recovery of these FAs in sample preparation is often seen as an
indicator of oxidation. However, because polar lipids are usually
rich in these FAs, low extraction efficiencies may have a similar
effect. In the FA profiles of the Soxhlet extracts, the amounts of EPA
were only 77–96% (average 89%) of the amounts in the DM profile,
and corresponding values for DHA was as low as 56–89% (average
77%). When the FAs left in the residue are included in the
reconstructed Soxhlet profile, the amounts of EPA varied from 96%
to 101% (average 99%) and the amounts of DHA varied from 93% to
98% (average 96%), which shows that there have been little or no
net loss of unsaturated FAs in the extraction.
Compared to the Soxhlet extract, acid hydrolysis had a profound
impact on the release of EPA and DHA. The amounts in the extracts
relative to DM were from 94% to 102% for EPA and 92% to 98% for
DHA. In the reconstructed extracts these figures increased to 96%
to 105% for EPA and 92% to 102% for DHA. The B&D method had
slightly higher values, from 101% to 106% for EPA and from 97% to
105% for DHA. The residues after B&D had little residual fat and the
reconstructed profiles were therefore in the same range as the
extracts.
4. Conclusions
By applying a direct methylation procedure on samples,
extracts and residues it was possible to achieve mass balances
for the lipid extraction methods of marine samples close to 100%.
This shows that the concept can be suitable for evaluation of
extraction efficiency of lipid extraction methods.
Soxhlet extraction with apolar solvents has low extraction
efficiencies for polar lipids, and the recovery of fatty acids in the
extracts were below 50% in some samples. Acid hydrolysis has a
clear effect on the release of FAs bound in polar lipids, but there
were still substantial amounts of FAs left in the residue (up to 17%)
of some of the samples. The Bligh and Dyer methodology seems to
be slightly more efficient in releasing the polar lipids, but up to 10%
of the FAs were left in the residue of some of the samples after two
B&D extractions.
The FA profiles of the Soxhlet extracts were substantially
different from the other extracts and the profile obtained by DM.
The highly unsaturated FAs EPA and DHA were particularly low in
the Soxhlet extracts. The use of a reconstructed FA profile, where
the FAs left in the residue were taken into account, showed that the
cause was low extraction efficiency and not a net loss (e.g.
oxidation) of these FAs. Extracts after acid hydrolysis and B&D had
similar FA profiles as obtained with DM. B&D was marginally closer
to the DM method than acid hydrolysis. Both methods seem
suitable for FAs analysis of dry marine powders.
Acknowledgement
The European Commission Erasmus Mundus Project EMQAL is
gratefully acknowledged for financial support of L.X.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jfca.2011.08.003.
References
Aksnes, A., Opstvedt, J., 1998. Content of digestible energy in fish feed ingredients
determined by the ingredient-substitution method. Aquaculture 161, 45–53.
AOCS Ba 3-38, 1998a. Official Methods and Recommended Practices of the Ameri-
can Oil Chemists’ Society. American Oil Chemists’ Society, Urbana, IL, USA.
AOCS Ca 5A-40, 1998b. Official Methods and Recommended Practices of the
American Oil Chemists’ Society. American Oil Chemists’ Society, Urbana, IL,
USA.
Araujo, P., Nguyen, T.T., Frøyland, L., Wang, J., Kang, J.X., 2008. Evaluation of a rapid
method for the quantitative analysis of fatty acids in various matrices. Journal of
Chromatography A 1212, 106–113.
Bligh, Dyer, W.J., 1959. A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology 37, 911–917.
 L. Xiao et al. / Journal of Food Composition and Analysis 25 (2012) 198–207
Carrapiso, A.I., Timón, M.L., Petrón, M.J., Tejeda, J.F., Garcı́a, C., 2000. In situ
transesterification of fatty acids from Iberian pig subcutaneous adipose tissue.
Meat Science 56, 159–164.
De Koning, A.J., Evans, A.A., Heydenrych, C., Purcell, C.J.D., Wessels, J.P.H., 1985. A
critical investigation of a number of different methods of lipid determination
in fish meal, with particular emphasis on corrections required in these
determinations. Journal of the Science of Food and Agriculture 36, 177–
185.
De Koning, A., Mol, T.H., 1989. Lipid determination in fish meal: an investigation of
three standard methods applied to stabilised and non-stabilised anchovy meals
at increasing stages of maturity. Journal of the Science of Food and Agriculture
46, 259–266.
Dickey, L.A., Teter, B.B., Sampugna, J., Woods, L.C., 2002. Comparison of a direct
transesterification method and the bligh and dyer method to determine fatty
acid content in striped bass tissues and diet. North American Journal of
Aquaculture 64, 158–163.
Ewald, G., Bremle, G., Karlsson, A., 1998. Differences between Bligh and Dyer and
Soxhlet extractions of PCBs and lipids from fat and lean fish muscle: implica-
tions for data evaluation. Marine Pollution Bulletin 36, 222–230.
Folch, J., Lees, M., Stanley, S., 1957. A simple method for the isolation and purifica-
tion of total lipids from animal tissues. Journal of Biological Chemistry 226,
497–509.
Hole, S., Hole, M., Taylor, K.D.A., 1996. Methods of extraction composition and
stability of vitamin A and other components in dogfish (Squalus acanthias) liver
oil. Food Chemistry 55, 215–220.
Homan, R., Anderson, M.K., 1998. Rapid separation and quantitation of combined
neutral and polar lipid classes by high-performance liquid chromatography and
evaporative light-scattering mass detection. Journal of Chromatography B:
Biomedical Sciences and Applications 708, 21–26.
Hyvönen, L., 1996. Approach to fat analysis of foods. Food Chemistry 23, 23–26.
ISO 6492:1999 (E), 1999. Animal Feeding Stuffs-determination of Fat Content.
International Organization for Standardization, Geneva, Switzerland.
Iverson, S.J., Lang, S.L.C., Cooper, M.H., 2001. Comparison of the Bligh and Dyer and
Folch methods for total lipid determination in a broad range of marine tissue.
Lipids 36, 1283–1287.
Johnson, R.B., Barnett, H.J., 2003. Determination of fat content in fish feed by
supercritical fluid extraction and subsequent lipid classification of extract by
thin layer chromatography-flame ionization detection. Aquaculture 216, 263–
282.
Kolarovic, L., Fournier, N.C., 1986. A comparison of extraction methods for the
isolation of phospholipids from biological sources. Analytical Biochemistry 156,
244–250.
Kozlova, T.A., Khotimchenko, S.V., 2000. Lipids and fatty acids of two pelagic cottoid
fishes (Comephorus spp.) endemic to Lake Baikal. Comparative Biochemistry
and Physiology B 126, 477–485.
Lepage, G., Roy, C.C., 1986. Direct transesterification of all classes of lipids in a one-
step reaction. Journal of Lipid Research 27, 114–120.
207
Mazza, M., Pomponi, M., Janiri, L., Bria, P., Mazza, S., 2007. Omega-3 fatty acids and
antioxidants in neurological and psychiatric diseases: an overview. Progress in
Neuro-Psychopharmacology and Biological Psychiatry 31, 12–26.
Meier, S., Mjøs, S.A., Joensen, H., Grahl-Nielsen, O., 2006. Validation of a one-step
extraction/methylation method for determination of fatty acids and cholesterol
in marine tissues. Journal of Chromatography A 1104, 291–298.
Mjøs, S.A., 2003. Identification of fatty acids in gas chromatography by application
of different temperature and pressure programs on a single capillary column.
Journal of Chromatography A 1015, 151–161.
Mjøs, S.A., 2004. The prediction of fatty acid structure from selected ions in electron
impact mass spectra of fatty acid methyl esters. European Journal of Lipid
Science and Technology 106, 550–560.
Nielsen, N.S., Göttsche, J.R., Holm, J., Xu, X., Mu, H., Jacobsen, C., 2005. Effect of
structured lipids based on fish oil on the growth and fatty acid composition in
rainbow trout (Oncorhynchus mykiss). Aquaculture 250, 411–423.
Phillips, K.M., Ruggio, D.M., Amanna, K.R., 2008. Extended validation of a simplified
extraction and gravimetric determination of total fat to selected foods. Journal
of Food Lipids 15, 309–325.
Rader, J.I., Angyal, G., O‘Dell, R.G., Weaver, C.M., Sheppard, A.J., Bueno, M.P., 1995.
Determination of total fat and saturated fat in foods by packed column gas–
liquid chromatography after acid hydrolysis. Food Chemistry 54, 419–427.
Roose, P., Smedes, F., 1996. Evaluation of the results of the QUASIMEME lipid
intercomparison: the Bligh & Dyer total lipid extraction method. Marine
Pollution Bulletin 32, 674–680.
Sahasrabudhe, M.R., Smallbone, B.W., 1983. Comparative evaluation of solvent
extraction methods for the determination of neutral and polar lipids in beef.
Journal of the American Oil Chemists’ Society 60, 801–805.
Shaikh, N.A., 1994. Assessment of various techniques for the quantitative extraction
of lysophospholipids from myocardial tissues. Analytical Biochemistry 216,
313–321.
Sirot, V., Oseredczuk, M., Bemrah-Aouachria, N., Volatier, J.L., Leblanc, J.C., 2008.
Lipid and fatty acid composition of fish and seafood consumed in France:
CALIPSO study. Journal of Food Composition and Analysis 21, 8–16.
Smedes, F., Thomasen, T.K., 1996. Evaluation of the Bligh & Dyer lipid determination
method. Marine Pollution Bulletin 32, 681–688.
Smit, L.E., Schönfeldt, H.C., de Beer, W.H.J., 2004. Comparison of the energy values of
different dairy products obtained by various methods. Journal of Food Compo-
sition and Analysis 17, 361–370.
Ulberth, F., Henninger, M., 1992. One-step extraction/methylation method for
determining the fatty acid composition of processed foods. Journal of the
American Oil Chemists’ Society 69, 174–177.
Weihrauch, J.L., Posati, L.P., Anderson, B.A., Exler, J., 1975. Lipid conversion factors
for calculating fatty acid contents of foods. Journal of the American Oil Che-
mists’ Society 54, 36–40.
Wiseman, J., Powles, J., Salvador, F., 1998. Comparison between pigs and poultry in
the prediction of the dietary energy value of fats. Animal Feed Science and
Technology 71, 1–9.