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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


estimate nutritional values in samples. Different ways of declaring
In recent years, polyunsaturated fatty acids (PUFAs) have fatty acid contents in food is discussed by Hyvönen (1996).
gained increasing attention, especially the n–3 (omega-3) and n–6 The FA content in a lipid extract is usually determined by gas
(omega-6) fatty acids, because of their various biological functions chromatography (GC). In order to improve volatility and to gain
related to health and disease (Mazza et al., 2007). These fatty acids better GC peak shapes, transformation of FA into FA methyl ester
(FAs) play important roles in the treatment and prevention of (FAME), i.e. methylation, is required. FAME can be prepared either
cardiovascular, autoimmune, and nervous disease and in the by multistep methods (Sirot et al., 2008; Kozlova and Khotim-
improvement of learning ability (Nielsen et al., 2005). The group of chenko, 2000) involving lipid extraction followed by transmethy-
marine lipids is an excellent source of essential polyunsaturated lation, or by direct methylation (DM). DM is also called one-step
FAs, in particular the n–3 family of FAs. extraction/methylation, and has been applied for FA analysis in a
The content of total lipid and FA composition are two key wide range of samples (Lepage and Roy, 1986; Ulberth and
indices utilized for assessment of nutritional value of food. The Henninger, 1992; Carrapiso et al., 2000; Dickey et al., 2002; Meier
lipid content is traditionally gravimetrically determined by solvent et al., 2006; Araujo et al., 2008). DM has several advantages over
extractions. There are a number of methods for lipid extraction. the extraction-based methods, such as small sample sizes and low
Soxhlet (AOCS Ba 3-38), acid hydrolysis (ISO 6492, 1999) and Bligh solvent consumption, leading to a cost-efficient and environmen-
and Dyer (B&D) (Bligh and Dyer, 1959) are the dominating extraction tally friendly methodology. DM methodology has also been shown
methods for the assessment of lipid content in food and feed to have high recovery for polar lipids that are tightly bound to the
ingredients. The total lipid content obtained by solvent extraction matrix and difficult to recover by extraction (Meier et al., 2006).
represents the content of crude fat, possibly including non-fat 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
* Corresponding author at: Department of Chemistry, University of Bergen, P.O.
Box 7803, NO-5020 Bergen, Norway. FAs in samples before extraction and in extracts and residues after
E-mail address: svein.mjos@kj.uib.no (S.A. Mjøs). 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) 198–207 199

analyzed by DM were used as a reference for calculation of the Section 2.2.1. In order to calculate the quantity of total extracted
mass balances of the extractions. 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,
2. Experimental and weighed. The residue was weighed after the solvent had
evaporated under vacuum. Two aliquots of the residues (approx.
2.1. Samples 200 mg each) were collected, weighed, and methylated using the
same procedure as described in Section 2.2.1.
The study of extraction efficiencies was conducted on dry
powders of marine origin that are typically used as food and feed 2.2.3. Acid hydrolysis extraction and methylation
ingredients; 8 samples, covering a wide range of lipid composi- An initial Soxhlet extraction was performed according to the
tions, were analyzed; sample type and origin are described in Table procedure described in Section 2.2.2, with the exception that
1. Prior to extraction all samples were ground into fine powders on extraction time was 2 h instead of over night. The residue in the
a Retch Grindomix GM 200 laboratory mill for about 20 s at extraction tube was dried and transferred into a 250 mL round-
8000 rpm, except dried salmon filet (Sample C). The latter was bottomed flask. 100 mL of 3 M aqueous HCl was added and the
subject to agglomeration due to high lipid content. In order to solution was refluxed for an hour. The solution was cooled down,
avoid degradation of the samples during the experiment period, and filter aid (kiselgur or hyflo-supercel) was added. The sample
each sample was divided into three aliquots, vacuum packed and was filtered through a pre-wetted, fat-free 150 mm white band
stored at 5 8C; each aliquot being used for evaluation of one of the filter and washed with cold water until the filtrate became neutral.
extraction methods. The filter cake was thereafter isolated and dried for 2 h at 103 8C,
Samples B, C and D contain all the lipids originally present in the and transferred to the same extraction tube that was used in the
raw material, while the majority of triacylglycerols (TAGs) in the initial extraction. The extraction tube was placed in the Soxhlet
other samples had been removed during the production process. extractor together with the flask containing the initial extract.
Products commercially available were supplied by Nofima Feed Petroleum ether was added to ca. 100 mL and the sample was
Production Facility, Titlestad, Norway. Samples B, C and D were extracted over night at a condensation rate of approximately 2–3
purchased locally as frozen products, freeze dried, and treated as drops per second. After extraction, the extract and residue were
described above. Sample H was prepared at Nofima Ingredients, treated, as described in Section 2.2.2.
Bergen, Norway, under conditions similar to a typical fishmeal
process. 2.2.4. Modified B&D extraction and methylation
Approximately 2.5 g of sample with known water content was
2.2. Extraction and methylation of samples weighed and transferred into a 250 mL flask. The appropriate
amount of water, making the total water content equal to 16 mL,
Each sample was treated by four different methods: (a) direct was added, followed by the addition of methanol (40 mL) and
methylation; (b) Soxhlet extraction followed by methylation; (c) chloroform (20 mL). The mixture was homogenized for 60 s on a
acid hydrolysis extraction followed by methylation; and (d) B&D T25 Ultra-Turrax homogenizer (IKA, Stanfen, Germany). Chloro-
extraction followed by methylation. Experimental methods are form (20 mL) and water (20 mL) were added in sequence followed
described below. by homogenization for 30 s after each addition. The mixture was
cooled down on ice bath and filtered. The filtrate was collected in a
2.2.1. Direct methylation 200 mL graduated cylinder. The volume of the water/methanol
Samples that were not treated by extraction were directly phase (upper phase) was recorded and two 3 mL aliquots were
transmethylated into FAME. Approximately 200 mg were accu- collected, evaporated to dryness under a stream of nitrogen at
rately weighed and mixed with 1.0 mL of methylation reagent, 100 8C, and methylated as described in Section 2.2.1. The
which consisted of 75% of 2.5 M methanolic HCl and 25% of toluene remaining water/methanol phase was removed from the cylinder
and prepared as described in the literature (Meier et al., 2006). The using a water aspirator. The filter cake was transferred into the
sample was thereafter heated at 100 8C for 2 h, cooled down to original flask and 20 mL of chloroform was added, followed by
room temperature, and concentrated to 50% by evaporation under homogenization for 15 s. The mixture was filtered and the filtrate
a stream of nitrogen at 70 8C. 1.0 mL of water was added and the was collected in the same graduated cylinder. The total volume of
sample was extracted twice with 1.0 mL of isooctane. The extracts the chloroform phase was recorded and two aliquots were taken,
were combined and diluted with isooctane to an appropriate evaporated to dryness under a stream of nitrogen at 100 8C, and
concentration suitable for GC analysis. methylated as described in Section 2.2.1. 20 mL of the chloroform
Prior to methylation of the sample the saturated FAME 23:0 phase was transferred into a tray, evaporated to dryness under an
(methyl tricosanoate, Larodan AB, Malmø, Sweden, 99.5%) in infrared lamp, and weighed. The total extracted lipid was
isooctane was added to the reaction tube and used as internal calculated through multiplication of this weight by ratio of the
standard for quantification of FAME by GC. The amount of added total volume of the chloroform phase to 20 mL (gravimetry). An
23:0 was varied according to the possible lipid content of sample. aliquot of the extract was analyzed by liquid chromatography (LC)
in order to analyze the lipid classes (see Section 2.4). The solid
2.2.2. Soxhlet extraction and methylation residue was collected, dried in vacuum for 2 h, and weighed. Two
Samples weighing 4–7 g were accurately weighed, packed with portions of the residue (approximately 200 mg each) were
cotton wool, and transferred into the extraction tube, which was weighed and methylated as described in Section 2.2.1.
equipped with a pre-dried Soxhlet flask. Approximately 100 mL of
petroleum ether (boiling point 40–60 8C) was added and the 2.3. Fatty acid analysis by GC
sample was extracted overnight (16–18 h) at a condensation rate
of 2–3 drops per second. The extract was diluted to approximately The GC analyses was conducted on a Trace GC gas chromato-
100 mL with petroleum ether and the solution was weighed. Two graph (Thermo Fisher Scientific) with flame ionization detector
aliquots (approx. 4 mL each) were collected, weighed, and (GC–FID), equipped with a 60 m  0.25 mm BPX-70 cyanopropyl
evaporated to dryness under a stream of nitrogen at 70 8C, column with 0.25 mm film thickness (SGE, Ringwood, Victoria,
followed by methylation using the same procedure as described in 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 or residue (Fig. 1, 2–8), methylation and FA analysis was performed


Sample details.
in two replicates. Mass balances were calculated by comparing the
Sample Type Water amounts found in extracts and residues (Fig. 1, 2–8) with amounts
content found after DM (1 in Fig. 1).
(%)

A Fishmeal (unspecified species), commercial 7.84 3. Results and discussion


B Cod (Gadus morhua) filet, freeze dried and milled 2.31
C Salmon (Salmo salar) filet, freeze dried and milled 6.49
3.1. Precision of the direct methylation method
D Herring (Clupea harengus) roe, freeze dried and milled 1.50
E Krill (Euphausia superba) meal, commercial 6.09
F Fishmeal, blue whiting (Micromesistius poutassou), 10.48 The repeatability (within-day variation) of the FA analysis by
commercial DM was evaluated by analyzing 7 replicates of Sample A within the
G Fishmeal (unspecified species), commercial 7.47
same day. In order to evaluate the reliability of FA analysis for the
H Krill (Euphausia superba) meal, produced by Nofima 6.39
extract (solution phase), 6 replicates of Soxhlet extract of Sample A
were also treated using the same method. The coefficient of
pressure of 2.20 bar. The injector temperature was 280 8C and the variation was around 5.1% for the powder and 1.2% for the extract.
detector temperature was 260 8C. The oven was programmed as The higher deviation in the former presumably results from the
follows: 60 8C for 4 min, 30 8C/min to 166 8C, and then 1.1 8C/min lower homogeneity. In addition, the solid sample contains a large
to 213 8C, and 120 8C/min to 250 8C/min where the temperature number of other components (e.g. minerals and proteins), and a
was held for 10 min. The sample solution (1.0 ml) was injected wider range of lipid classes compared to the extract, which may
splitless and the split was opened after 2 min. The FAMEs were also affect the methylation reaction. For both the solid sample and
identified by comparing the elution pattern and relative retention the extract, the experimental deviation for each specific FA was
time with the reference FAME mixture (GLC-793, Nu-Chek Prep close to that of total FAs. The methylation reaction showed no
Inc., Elysian, MN, USA). Additional identifications were achieved by selectivity for specific FAs. Further details are given as supplemen-
comparison with a control oil, where the FA content has been tary material (Table S2).
identified by gas chromatography–mass spectrometry (GC–MS) The intermediate precision (between-day variation) was also
methodology as described in the literature (Mjøs, 2003, 2004). evaluated by analyzing the control oil and the samples on the
Chromatographic peak areas were corrected by empirical response different days during the experimental period. The coefficient of
factors calculated from the areas of the GLC-793 mixture. variation for total FAs of the control oil was around 1.9%, indicating
a high intermediate precision, while coefficient of variation for
2.4. Lipid class analysis by liquid chromatography 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
Lipid class composition of B&D extracts was analyzed by liquid close to those of the other FAs, suggesting that degradation by
chromatography (LC) using a modified version of a previously oxidation was minimal. No other trends related to time were seen,
described method (Homan and Anderson, 1998). Analyses were indicating that the samples were stable throughout the experi-
carried out on a Series 200 HPLC system (Perkin Elmer, Shelton, CT, mental period. There was no pronounced variation observed for
USA) equipped with a charged aerosol detector (ESA, Chelmsford, the FA classes including saturated FA (SFA), monounsaturated FA
MA, USA) and a 125  4 mm LiChrospher diol column with 5 mm (MUFA) and polyunsaturated FA (PUFA). Further details are given
particle diameter (Merck, Darmstadt, Germany). The column as supplementary material (Table S3).
temperature was kept at 45 8C. The sample was separated in a
single chromatographic run by using a ternary solvent gradient 3.2. FA compositions and lipid classes of samples
program. Further details are given in supporting information
(Table S1). The compositions of the FAs of 8 marine samples were
determined by GC and the results are reported in Table 2. Among
2.5. Repeatability and precision 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
The repeatability of DM was checked by analyzing 6 replicates sample). The dried cod filet (Sample B) had the lowest content of
of Sample A and 7 replicates of a Soxhlet extract of Sample A on the total FAs (2.2 g/100 g sample). Between these two extremes the
same day. The intermediate precision and sample stability were other samples ranged from 5.2 to 14.2 g FA per 100 g of the powder.
evaluated by measuring the control oil (NorsalmOil fish oil, Vedde Lipid classes analyzed as described in Section 2.4 are reported in
AS, Langevåg, Norway) and Samples A–H on different days Table 3. There are large variations in lipid class composition
throughout the study. between different samples. TAG varied from 2.3 to 87 weight % of
extracted fat. The main phospholipids, phosphatidyl choline and
2.6. Analysis of data phosphatidyl ethanolamine, varied from less than 1% to 49% and
from less than 1% to 12%, respectively. Free FAs varied from 0.8% to
Analysis of variance (ANOVA), F-test and t-test were used to 36%. Cholesterol varied from less than 0.5% to 8.2%. Other lipid
analyze the data at P = 0.05 level. Principal component analysis classes were below 2% in all samples. With the exception of
(PCA) was performed using the Sirius 8.0 software (Pattern Samples B and D, total neutral (apolar) lipid contents were higher
Recognition Systems, Bergen, Norway). 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
2.7. Replicates and outline of the experiment found by gravimetry.
The dried cod filet (Sample B) and one of the fish meals (Sample
Fig. 1 provides an overview of the experimental set up. DM F) had high content of free FAs (25.0% and 36.0%), implying the
(Fig. 1, 1) was conducted in three replicates and the three occurrence of hydrolysis of lipids in these two samples. Free FAs in
extraction methods were carried out in two replicates. The three Samples B and F were also determined by titration according to
replicates of the DM analyses were conducted at the start, middle (AOCS Ca 5A-40), which gave values of 27.5% and 28.6%
and end of the experimental period, respectively. For each extract 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 extracts (29–99% relative to DM), only account for a part of the
freeze-drying (Sample B), or during long term storage in the case of total FAs, and the other FAs still remain in the residues (2.3–66%).
the fishmeal (Sample F). The sum of FAs in the extracts and residues match well with those
obtained by DM; total recoveries ranged from 96% to 112%.
3.3. Mass balances Dried salmon filet (Sample C), containing a high content of TAG
and a very small amount of polar lipids, had the highest extraction
The FA results obtained by DM are reported in Table 2. When efficiency (98.0–99%), whereas dried cod filet (B) and herring roe
normalized to the total sample weight, the sums of the extracted and (D), bearing small quantities of TAG and high contents of
residual FAs after the three extraction methods should ideally be phosphatidylcholine and phosphatidylethanolamine, showed the
equal to the DM results, which are used as benchmark in the study. lowest extraction efficiencies (37.3% and 38.2% for B, and 29.1% and
31.4% for D, respectively).
3.3.1. Mass balance for the Soxhlet method The FA contents in the extracts and residues varied clearly as a
The mass balances for the Soxhlet method are presented in function of sample composition. Percent extracted lipids were
Table 4. It is obvious that the FAs recovered from the Soxhlet 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 solvents. In addition, the acid treatment may facilitate lipid
extracted. It is also well known that the Soxhlet extraction with extraction by breaking down proteins and other compounds in
apolar solvents is sensitive to the polarity of lipids and has low the matrix. In the case of the acid hydrolysis method used in this
recovery for phospholipids (Sahasrabudhe and Smallbone, 1983; work, the residues after the initial Soxhlet extraction were
Hole et al., 1996; Johnson and Barnett, 2003). It should be pointed hydrolyzed and extracted again with petroleum ether. The
out that the neutral lipids are not always recovered 100% by combined extracts and final residues were analyzed and their
Soxhlet extraction, and that some polar lipids can also be extracted mass balances relative to DM were established. The results are
(Ewald et al., 1998). listed in Table 5.
Total lipids determined by gravimetry are also reported in Table Compared to the Soxhlet method, it can be seen that acid
4. Total fat determined by gravimetry varied from 52% to 123% of hydrolysis substantially improves the extraction efficiency, which
FAME determined by DM. The weight of FAs is determined as FAME is in accordance with results previously reported (de Koning et al.,
and gravimetric lipid content can be expected to be similar only in 1985). Extracted lipids varied from 82% to 112% relative to DM,
cases where the lipids are close to 100% TAG. Free cholesterol, while residual lipids varied from 0.7% to 17%. Sum of the extracts
which has no FAs, contributes only to the gravimetric weight. and residues varied from 99% to 115%. Even though extraction
Cholesterol esters and phospholipids, where the FAs constitute a efficiency was substantially increased for the phospholipid-rich
smaller part of the molecule than in TAG, will also contribute more samples, it should be emphasized that residual lipids were still
to the total lipid weight than to the weight of FAME after above 5% in three samples, and as high as 17% in the dried cod filet.
methylation. This can explain the values above 100%. Values below This could indicate either that the hydrolysis is not complete after
100% are explained by low extraction efficiencies and other the acidic treatment, or that the free FAs are extracted with low
analytical errors. efficiency.
The total lipid content determined by gravimetry varied from
3.3.2. Mass balance for the acid hydrolysis method 108% to 145% relative to FAs determined as FAME by DM. All krill
In order to improve the low efficiency of Soxhlet extraction, and fish meals had values above 130%. The acid hydrolysis method
samples are often hydrolyzed prior to extraction. This will is frequently applied for the determination of lipid content in food
release the FAs from the matrix in the form of free FAs. As long and feed ingredients. In this respect it is worth noting that there is
as the matrix is not alkaline, free FAs can be extracted by apolar 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; residual FA varied from 1.4% to 10%, and FA in the water phase
Hyvönen, 1996). varied from 0% to 2.8%. Sum of extracts and residues varied from
91% to 121%.
3.3.3. Mass balance for the B&D method There are significant amounts of FA left in the residue after the
The mass balances for the B&D method are reported in Table 6. B&D extraction as well. Since B&D is a batch extraction, the FA in
There are two fundamental differences between this method and the residue can be ascribed either to non-extracted FA or to
the Soxhlet and acid hydrolysis method: (1) the B&D employs extracted FAs present in solvent that are trapped in the residue.
batch extraction, while the other methods are continuous However, if there were substantial amounts of dissolved lipids left
extractions; and (2) after extraction, solvent in the B&D method in the trapped solvent, one should find larger amounts of FA
are separated into an apolar (basically chloroform) and a polar measured in absolute values (g/100 g sample) for Sample C
fraction (methanol/water). The lipids are assumed to be in the (salmon powder), which is much richer in extractable FAs than any
apolar phase only, while the polar phase is expected to contain other sample. One should also expect a general correlation
matrix interferents. The presence of polar lipids in the water phase between absolute amounts of FAs in the extracts and residues.
has been reported (Kolarovic and Fournier, 1986; Shaikh, 1994). This, however, was not the case. It can also be seen that the largest
FAs were therefore analyzed also in this phase in addition to the amounts in the residues are found in the same samples that had
apolar extract and the residue. low extraction efficiencies in the Soxhlet and acid hydrolysis
The mass balances for the B&D extraction are reported in Table methods, and the largest values are found for Sample B (dried cod
6. Extracted FA varied from 86% to 115% relative to DM, while 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- profiles, i.e. the relative distribution of FAs. FA profiles were
mined as FAME after DM in Fig. 2b. These values should constructed by normalizing the FAs in the different samples and
theoretically be close to 100% only in the case of TAG, because extracts against total FAs, so that the sum of FAs is 100%. In
the weight of three FAME molecules created from TAG by addition to the FA profiles of the extracts and DM, reconstructed FA
methylation will be roughly equal to the weight of the original profiles were created by summing the FAs in extracts and residues
TAG. Values close to 100% are also observed for the dried salmon (including water phase in B&D) before normalization. While the FA
filet (Sample B), where the lipids are basically TAG. Large profile of an extract may differ from the profile of corresponding
deviations are observed for the other samples. It can be seen that DM as a result of extraction efficiencies, the reconstructed profile
the lipid contents obtained by B&D are generally higher than those should in theory differ only if there is a net loss of some of the FAs,
by acid hydrolysis, which is consistent with that observed by de i.e. by oxidation.
Koning et al., 1985. This can partly be explained by slightly higher The similarity of FA profiles was evaluated by Euclidean
extraction efficiency for B&D. However, a possible explanation may distances and by principal component analysis (PCA). The
also be that polar lipids extracted after acid hydrolysis of the Euclidean distance is defined as:
samples are recovered as free FAs, while these lipids are acquired qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
intact in the B&D method. A phospholipid will therefore contribute dð p; qÞ ¼ ð p1  q1 Þ2 þ ð p2  q2 Þ2 þ   ð pn  qn Þ2
to more mass when extracted by the B&D method than extracted vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u n
after acid hydrolysis. Rader et al. (1995) compared gravimetric uX
¼ t ð pi  qi Þ2 ; (1)
lipid content by hydrolytic extraction with extracted FAs for 24 i¼1
types of food and found that total lipids were higher than the total
FAs determined chromatographically. This agrees with our where pi and qi are two points in an n-dimensional coordinate
observations as well. system; p and q are in this case different profiles of the same
The FAs are energy stores which are oxidized to release the sample, while i signifies the different FAs and n is the number of
energy. The energy value of a food product is generally described FAs in the profiles. Two identical profiles will have a Euclidean
by the contents of total FAs. Research studies also indicate that the distance of zero, and the distance increases with dissimilarity of
dietary energy values of a food product also depend on the profiles.
saturation degree of the FAs, free FA contents and chain length of The Euclidean distances from the DM profile to the extracts and
the FAs (Wiseman et al., 1998). Undoubtedly, an accurate reconstructed profiles are shown in Table 7. It can be seen that the
estimation of the energy value of a food product should be carried distance between Soxhlet extracts and DM is substantially larger
out by determining the FA contents instead of crude lipid weight. than that between DM and the other profiles. The distance is also
However, due to the lack of sufficient data about the FAs of larger for samples that typically had low extraction efficiencies,
different matrices, the gravimetric lipid contents determined by showing that the low extraction efficiency has a profound
extraction methods are often used to evaluate the dietary energy influence on the relative distribution of the FAs in the extracts.
value of a food product (Aksnes and Opstvedt, 1998; Smit et al., The average distance between the reconstructed Soxhlet profile
2004). and the DM is only 15% of the distance between Soxhlet extract and
In this study, total extracted lipids varied from 52% to 160% of DM, and of the same magnitude as the distance between the other
the FA content determined as FAME. The poor correspondence profiles and DM. This shows that the differences between DM and
between total extracted lipids determined by gravimetry and the the Soxhlet extract are accounted for by the FAs left in the residue,
actual FA content in the samples shows that gravimetric lipid and that there is no net loss of FAs that have significant impact on the
content can be a poor estimate for the actual energy content in the profiles. The profiles of acid hydrolysis extracts and B&D extracts
lipids. In this respect it should also be emphasized that different are much closer to the DM than the Soxhlet extract. But also for these
extraction methods may give highly different results for total extractions there is a small reduction of distances when the FAs in
extracted lipids when applied on the same sample. the residues are included in the reconstructed profiles.
Factors to convert between different lipid extraction methods, A PCA score plot of all FA profiles is shown in Fig. 3a. This plot
and between fatty acid content and gravimetrically determined basically illustrates similarities and dissimilarities between the
lipids, have been calculated for a variety of foods (Weihrauch et al., samples. The different profiles for the same samples can be found
1975; Hyvönen, 1996). However, for fish meals it has been shown in rather dense groups, compared to the differences between the
that the differences between methods are dependent on oxidation samples, illustrating that differences caused by the chosen sample
status of the sample, indicating that such correction factors cannot preparation method is substantially smaller than the differences
be applied with accuracy on fish meal and similar products (de between the samples. However, the Soxhlet extracts are outliers in
Koning and Mol, 1989). all groups except in the case of the TAG rich salmon powder
(Sample C).
3.5. Influence of the extraction methods on the fatty acid profiles 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
Because different lipid classes have different FA distributions, each type of profile. This illustrates the difference between the
the differences in extraction efficiencies may also influence the FA 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 extracts were below 50% in some samples. Acid hydrolysis has a
is grouped together with the other. Among the profiles of the clear effect on the release of FAs bound in polar lipids, but there
extracts, the B&D is the one that is nearest DM along the first were still substantial amounts of FAs left in the residue (up to 17%)
principal component, which account for 95% of the variance. This of some of the samples. The Bligh and Dyer methodology seems to
also corresponds to the conclusions that can be drawn from the be slightly more efficient in releasing the polar lipids, but up to 10%
Euclidean distances. Full FA profiles are presented as supporting of the FAs were left in the residue of some of the samples after two
information in Tables S4–S10. B&D extractions.
In marine lipids, special attention are usually paid to the highly The FA profiles of the Soxhlet extracts were substantially
unsaturated long-chain omega-3 fatty acids EPA (20:5 n–3) and different from the other extracts and the profile obtained by DM.
DHA (22:6 n–3), both because of their nutritional significance, and The highly unsaturated FAs EPA and DHA were particularly low in
because of they are easily oxidized or degraded by other reactions. the Soxhlet extracts. The use of a reconstructed FA profile, where
Low recovery of these FAs in sample preparation is often seen as an the FAs left in the residue were taken into account, showed that the
indicator of oxidation. However, because polar lipids are usually cause was low extraction efficiency and not a net loss (e.g.
rich in these FAs, low extraction efficiencies may have a similar oxidation) of these FAs. Extracts after acid hydrolysis and B&D had
effect. In the FA profiles of the Soxhlet extracts, the amounts of EPA similar FA profiles as obtained with DM. B&D was marginally closer
were only 77–96% (average 89%) of the amounts in the DM profile, to the DM method than acid hydrolysis. Both methods seem
and corresponding values for DHA was as low as 56–89% (average suitable for FAs analysis of dry marine powders.
77%). When the FAs left in the residue are included in the
reconstructed Soxhlet profile, the amounts of EPA varied from 96% Acknowledgement
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 The European Commission Erasmus Mundus Project EMQAL is
net loss of unsaturated FAs in the extraction. gratefully acknowledged for financial support of L.X.
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 Appendix A. Supplementary data
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 Supplementary data associated with this article can be found, in
slightly higher values, from 101% to 106% for EPA and from 97% to the online version, at doi:10.1016/j.jfca.2011.08.003.
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. References

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