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LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 2, 1996 487
FOOD COMPOSITION AND ADDITIVES
A Simple and Rapid Solvent Extraction Method for Determining
Total Lipids in Fish Tissue
CHONG M. LEE, BELZAHET TREVINO, and MAYUREE CHAIYAWAT
University of Rhode Island, Department of Food Science and Nutrition, 530 Liberty Ln, West Kingston, RI 02892
(3) used 2:1 chloroform-methanol at a 20:1 ratio of solvent to Downloaded from https://academic.oup.com/jaoac/article/79/2/487/5684567 by guest on 14 September 2022
Solvent systems that have been developed for lipid tissue (by weight) for simplicity and rapidity.
extraction include chloroform-methanol, n-hexane- Hara and Radin (4) used 3:2 hexane-isopropyl alcohol. They
isopropyl alcohol, and methylene chloride-metha- blended 1 g tissue with 18 mL solvent and removed the nonlipid
nol. The extraction methods are labor intensive, fraction with 12 mL 6.25% sodium sulfate. However, extraction
lack precision, or require a large volume of solvent. of proteolipid protein and gangliosides was incomplete.
Correct computation of lipid content calls for full re-
covery of solvent after extraction, but recovery al- Swaczyna and Montag (5) used methylene chloride-metha-
ways is incomplete because of unaccounted sol- nol. Methylene chloride poses a problem because of its low
vent residue that remains in jar, filter paper, and ho- boiling point (39.7°C). The low boiling point may result in sol-
mogenized tissue. A rapid and simple extraction vent losses due to rapid evaporation during blending and filter-
method coupled with correct computation was de- ing, leading to erroneously higher yield.
veloped for determining total lipids in fish tissue. Recently, Erickson (6) used catfish tissue to compare 9 sol-
The method uses chloroform-methanol and an vent systems for lipid extraction with a screw-cap test tube (16
Eberbach blending jar. Variables examined were x 125 mm) and a Vortex mixer. The size of the test tube limits
chloroform-methanol ratio, solvent-to-sample ra- sample sizes to 0.5-1 g, based on solvent-to-tissue ratios of 7-
tio, and phase separation time. Precision was 30. Chloroform-methanol was preferred over other solvent
within 0.5%. Conventional computation of lipid con- systems (hexane-isopropyl alcohol and chloroform-isopropyl
tent depends on the volume of chloroform meas- alcohol), which required longer evaporation times. Exposure of
ured after filtration. This volume does not include tissue to methanol prior to chloroform is necessary for this sol-
unaccounted solvent residue. Thus, a time-consum- vent extraction system. Although the method uses a low vol-
ing second extraction is required for complete re- ume of solvent and is simple and rapid, it has some drawbacks.
covery. The mass balance of each extraction and fil- One disadvantage is limited sample size (not more than 1 g),
tration step confirmed that the correct volume of thus requiring an extremely uniform sample to minimize vari-
chloroform (measured plus unaccounted) was ability. Potential safety hazards from pressure buildup inside
close to the theoretical volume. The procedure the tube from a 2 min agitation on a Vortex mixer are another
eliminates problems associated with laborious fil- disadvantage. Also, use of a Vortex mixer may result in inade-
tration and variation in chloroform volume read- quate disintegration of tissue, compared with mixing in a blade-
ings and does not require an exact reading of chlo- equipped blender. Inadequate breakup of tissue may prevent
roform volume. Instead it allows use of a theoreti- complete extraction of lipid from tissue.
cal volume, which depends on solvent volume and Earlier methods are labor intensive and have other draw-
ratio used. backs, including lack of precision and use of a large volume of
solvent. A rapid and simple extraction method for lipid analysis
offish tissue based on the chloroform-methanol solvent system
olvent extraction systems that have been available for was developed.
Sfish lipid extraction include chloroform-methanol.
Folch et al. (1) used 2:1 chloroform-methanol to extract Experimental
40 g tissue in 2 steps: first with 760 mL and then with 400 mL Apparatus
solvent. Bligh and Dyer (2) used 1:1 chloroform-methanol in
a 2-step extraction. Tissue (100 g) was blended first with The blending unit consisted of an Eberbach blending jar
200 mL methanol and 100 mL chloroform and then with (250 mL, Ref. No. 8580; Eberbach Corp., Ann Arbor, MI),
100 mL chloroform. The residue was blended with 100 mL Waring blender base (Model 33BL79; Waring Products, New
chloroform and rinsed with 50 mL chloroform. Hubbard et al. Hartford, CT), and variable autotransformer (Model 3PN1010;
Staco Energy Products, Dayton, OH). Unlike other typical
Received April 11, 1995. Accepted by JL August 2, 1995. blending jars, the Eberbach jar is tapered such that added sol-
488 LEE ET AL. : JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 2199, 6
vent remains in the narrow lower part (60 mL holding capac- To separate the filtrate into 2 phases (methanol-water and
ity), and no spillage and evaporation losses occur during vigor- chloroform), 20 mL 0.5% NaCl was added. The mixture was
ous blending with sample. gently shaken by tilting the graduated cylinder 4 times and then
allowed to stand for 30 min or until a clear separation was vis-
Reagents ible. The NaCl solution was added to prevent formation of a
The following reagents were used: chloroform (Fisher, Ref. stable emulsion and to remove proteinaceous matter from the
No. C298-4), methanol (Fisher, Ref. No. A452-4), and sodium chloroform fraction, as reported by Palmer (7). At this point, the
chloride (crystal; Fisher, Ref. No. S271-500). volume of the chloroform layer should be equal to the theoreti-
cal value minus losses that occurred during blending and filtra-
Sample Preparation tion. The theoretical volume of chloroform in 2:1 chloroform-
methanol is 50 x 2/3 = 33 mL.
Cod (Gadus morhua) and mackerel {Scomber scombrus) To determine the amount of lipid extracted, a 5 mL chloro- Downloaded from https://academic.oup.com/jaoac/article/79/2/487/5684567 by guest on 14 September 2022
were chosen as representatives of lean and fatty species, respec- form layer was removed with a 10 mL pipet, transferred to a
tively. Fish was filleted, cut into small pieces, and homogenized preweighed (to 1 mg) 10 mL beaker, and evaporated for ca
in a kitchen Waring blender. The resulting paste free of bones, 30 min on a Corning hot plate (PC-35) set between low and 2
skins, and scales was used as sample for solvent extraction. to avoid excessive heating and drying.
Method Development Calculation
A single extraction with chloroform-methanol was used The lipid content was calculated with the following formula:
throughout the study. The following variables were examined Lipid content, % lipid extracted(g)
to determine optimum extraction conditions: solvent-to-sample sample weight (g)
ratio, chloroform-methanol ratio, and phase separation time.
The ratio of solvent volume to sample weight was varied from [chloroform volume (read + unaccounted)] (mL)
2 to 14. The chloroform-methanol ratio was varied from 0.9 to 5 mL xlOO
18. Phase separation time was varied from 30 min to 6 h.
Statistical Analysis
Lipid Extraction Procedure A 3 x 5 factorial analysis of variance (n = 3) was used to
Paste weighing 5 ± 0.1 g was placed into a 250 mL Eber- determine the significance of variations due to solvent ratio and
bach homogenizer with a narrow stem and a wide upper body sample size. Method precision was determined from the vari-
and opening. Such geometry allows efficient blending with a ability (coefficient of variation) from the mean.
small amount of solvent and a large headspace that prevents
solvent spillage. Various amounts of solvent were added to alter Results and Discussion
solvent-to-sample ratios. The mixture was blended for 1.5 min
at moderate speed; a variable transformer was used to maintain Tables 1 and 2 presents the effects of solvent ratio and sam-
a constant speed. High speed resulted in solvent vaporization ple size on extraction of lipids from lean (cod) and fatty (mack-
and a rise in temperature. The homogenate was filtered through erel) fish, respectively. For lean fish, the more polar solvent
a coarse, fast-speed filter paper (12.5 mm id, Fisher P8) and system (1:2 chloroform-methanol) was more effective than the
funneled into a 100 mL glass-stoppered graduated cylinder. less polar solvent system (2:1 chloroform-methanol), espe-
The wet cake was pressed with the round tip of a spatula to cially as the sample size increased. Compared with that of lean
squeeze the remaining solvent. The final volume of filtrate var- fish, extractability of lipids from fatty fish responded sharply to
ied with the batch of sample and species. Solvent losses from changes in solvent ratio and sample size. As solvent polarity
residue left in the homogenizer after blending and pouring and increased with increased sample size, efficiency of lipid extrac-
absorbed on the filter paper after filtration were taken into ac- tion dropped drastically. With 2:1 chloroform-methanol, lipid
count in yield determination. extracted from mackerel ranged from 11.1 to 9.96%. With 1:2
Table 1. Effects of solvent ratio and sample size on lipid extracted from cod tissue (lean fish)
Lipid extracted (%) at indicated sample size (g) and solvent-to-sample ratio3
Chloroform-methanol ratio 1(50) 3(16.7) 5(10) 7(7.1) 9 (5.5) 4,30
£ DC c c c
1:2 1.20 1.12 ' 1.05 1.04 1.03 4.23 0.01
c c c c
1:1 1.12* 0.94 0.93 0.93 0.90 32.79 0.001
c d e e
2:1 1.34* 1.04 0.86 0.74 0.72 36.43 0.001
a Solvent-to-sample ratios are indiated in parentheses. For all sample sizes, 50 mL solvent was used. Effect of sample size (F ) = 51.28 (P«
0.001). Effect of solvent ratio (F ) = 30.12 (P < 0.001). Interaction (F ) = 10.94 (P< 0.001). 430
230 830
b,c,d,e |\/| ( _ 3) j vvith different superscripts are significantly different (P< 0.05).
eans n n tne same row
LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 2,1996 489
Table 2. Effects of solvent ratio and sample size on lipid extracted from mackerel tissue (fatty fish)
3
Lipid extracted (%) at indicated sample size (g) and solvent-to-sample ratio
Chloroform-methanol ratio 1(50) 3(16.7) 5(10) 7(7.1) 9 (5.5)
C c e
1:2 10.6 10.1° 4.42 2.53 1.90 901 0.001
C c c e
1:1 10.8* 10.2 10.1 6.59 5.65 432 0.001
C c C c
2:1 11.1* 10.3 10.1 10.0 9.96 12.8 0.001
a
Solvent-to-sample ratios are indicated in parentheses. For all sample, sizes 50 mL solvent was used. Effect of sample size (F ) = 827 (P<
0.001). Effect of solvent ratio (F ) = 1021 (P< 0.001). Interaction (F ) = 284 (P< 0.001). 430
230 830
b.c.d.e M ( _ 3) jt different superscripts in the same row are significantly different (P< 0.05).
eans n w n
chloroform-methanol, it dropped from 10.6 to 1.90%. This re- Chloroform-methanol (2:1) was selected as the optimum Downloaded from https://academic.oup.com/jaoac/article/79/2/487/5684567 by guest on 14 September 2022
sult reflected greater F values for effects of solvent ratio, sam- solvent for fatty fish, such as mackerel, with lipid contents
ple size, and their interaction for fatty fish than for lean fish. higher than 6% and composed largely of triacylglycerols. This
Better extraction of lean fish with a polar solvent (1:2 chlo- system used the least amount of chloroform among solvent ra-
roform-methanol) is due to the preponderance of membrane- tios that gave maximum extraction. Chloroform-methanol
bound phospholipids (88.1%) in lean fish (8). By contrast, fatty (1:2) was selected for lean fish, such as cod, with lipid contents
fish is composed mostly of triacylglycerols (86.8%) (9). less than 2% and composed mostly of phospholipids. A solvent-
Maximum lipid extraction of mackerel (about 45% lipid on to-sample ratio of 10:1 (50 mL solvent to 5 g sample) was op-
a solid weight basis, equivalent to 15% lipid on a wet weight timum for both lean and fatty fish. Incomplete extraction is pos-
basis and 34% solid) was achieved with chloroform-methanol sible at this ratio when the lipid content is very high (>20%),
at ratios ranging from 2:1 to 5:1 (Figure 1) and a solvent-to- unless the solvent-to-sample ratio is increased to >10, regard-
sample ratio >10 (Figure 2). Extraction efficiency decreased as less of the composition of the chloroform-methanol system.
the solvent mixture approached a biphasic state at solvent ratios Method precision is supported by low variability with re-
higher than 5:1. Bligh and Dyer (2) reported that the monopha- spect to volume of chloroform read and lipid extracted (Ta-
sic state is necessary for efficient lipid extraction, because it ble 3). The mass balance of solvent during the whole procedure
provides more contact areas for extraction and thus helps the (Table 4) proves there was no apparent loss other than the re-
solvent dissolve the lipids. No significant increase in lipid ex- maining solvent residue. By using the mass balance given in
traction was noted at solvent-to-sample ratios ranging from Table 4, the actual measured and unaccounted volumes of chlo-
roform were computed (Table 5). The combined volume was
10:1 to 14:1, but at 50:1 (50 mL solvent to 1 g sample), a sig- close to the theoretical volume: 33, 25, and 16.7 mL for 2:1,
nificant increase in lipid extraction (Tables 1 and 2) was ob- 1:1, and 1:2 chloroform-methanol, respectively. Therefore, if
served at most solvent ratios. However, use of such a small all steps are carefully controlled, the theoretical volume can be
sample may reduce precision, unless sample homogeneity is used on a routine basis without measuring the precise volume
ensured. of chloroform by hand squeezing or vacuum filtering every
drop out of the wet homogenate. Theoretical and unaccounted
volumes were 33.0 and 3.2; 33.3 and 3.2; and 16.8 and 1.8 mL
LU O
|_ CO
If x 5
UJ V)
Q Ol
E2
-' 0.1 -
Figure 1. Effect of solvent composition on lipid SOLVENT to SAMPLE RATIO
extraction of mackerel tissue. Extraction was done at a
10:1 ratio of solvent to wet sample and 5% water. The 5% Figure 2. Effect of solvent-to-sample ratio on lipid
water (2.5 mL out of 50 mL solvent mixture) was extraction of mackerel tissue. Extraction was done with
estimated from a minimum contribution of 2.5 mL from 5 g wet sample and various volumes of at 75:20:5
5 g sample. chloroform-methanol-water.
490 LEE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, No. 2,1996
Table 3. Method precision determined with a single mackerel sample'
read, mL Lipid extracted, mg Lipid content, %
Sample weight, g Chloroform layer
5.01 29 141.68 18.66
5.01 29 141.32 18.67
5.02 29 142.56 18.74
5.00 28.8 141.57 18.57
Mean ± standard deviation 28.95 ±0.1 141.78 ±0.54 18.66 ±0.069
Coefficient of variation (variability), % ±0.34 ±0.38 ±0.37
50 mL 2:1 chloroform-methanol was used to extract 5 g tissue.
for 10 and 20% lipid-containing mackerel and 1% lipid-con- To determine method accuracy, the method was compared Downloaded from https://academic.oup.com/jaoac/article/79/2/487/5684567 by guest on 14 September 2022
taining cod, respectively. with the Bligh-Dyer method (Table 7). The proposed method
Table 6 shows how time necessary for phase separation after consistently showed a higher yield. When calculation was
addition of 0.5% NaCl affected the amount of fat measured. As based on the measured volume of the chloroform layer, no sig-
indicated by low variability and statistical insignificance, the nificant difference in cod lipid contents was observed between
amount of lipid measured remained constant regardless of the the 2 methods. By contrast, significant differences in mackerel
time of sampling, as long as a visible separation had occurred. lipid contents were observed. Mackerel samples required a sol-
This finding indicates that there is no increase in lipid extrac- vent that is more nonpolar than that used in the Bligh-Dyer
tion with extended standing time for clear separation. Although method. The discrepancy may be due to differences in solvent-
opacity was due to an emulsion, which contained some protei- to-sample ratio and solvent makeup. This observation reaffirms
naceous substances, the proteinaceous matter had no effect on that solvent polarity and sol vent-to-sample ratio are the most
the amount of lipid measured. This conclusion is supported by critical factors for lipid extraction of fish tissues. Solvent polar-
a study of Erickson (6), which showed that less than 0.006% ity can be increased by adding water, as in the Bligh-Dyer
proteinaceous matter is found in the chloroform fraction. method for cod lipid extraction, which favors a polar solvent.
Therefore, to save time, the sample should be taken out as soon However, addition of water becomes unnecessary when the
as a visible separation occurs. Phase separation was delayed methanol fraction is increased, as in the proposed method.
when the amount of 0.5% NaCl added was increased. A volume Therefore, moisture adjustment is not necessary, as long as sol-
of 20 mL was the minimum amount that allows phase separa- vent polarity is correctly selected and the tissue is wet and eas-
tion for both 2:1 and 1:2 chloroform-methanol and that accom- ily breakable for lipid release upon solvent extraction.
modates most water-soluble fractions. The minimum amounts In the proposed method, extraction of 5 g tissue with 50 mL
of water needed for phase separation of 50 mL 2:1 and 1:2 chlo- solvent is done in a single step and requires no more than 2 h
roform-methanol are 10 and 20 mL, respectively, according to for the whole procedure, including sample preparation and
a chloroform-methanol-water ternary diagram (2). lipid measurement. Extractions with the Folch and the Bligh-
Table 4. Mass balance of solvent during extraction and filtration
Sample Measured weights of solvent and solubles in tissue
a
Mackerel 61.1 g (50 mL2:1 CHCl3-MeOH) + solubles from 5 g tissue = 65.15 g (10% oil + 71% moisture = 4.05 g)
Cod 50.9 g (50 mL 1:2 CHCl3-MeOH)b + solubles from 5 g tissue = 55.05 g (1% oil + 82% moisture = 4.15 g)
Mackerel Measured weights of solvent + solubles remaining in jar and filter paper = 6.22 g
Solvent + solubles remaining in jar (0.7 g in jar + 0.3 g in residual tissue) = 1.0 g
Solvent + solubles remaining in filter paper + tissue homogenate = 5.22 g
Cod Measured weights of solvent + solubles remaining in jar and filter paper = 5.91 g
Solvent + solubles remaining in jar (0.7 g in jar + 0.2 g in residual tissue) = 0.9 g
Solvent + solubles remaining in filter paper + tissue homogenate = 5.01 g
Mackerel Estimated weight of filtrate to be recovered: 65.15 - 6.22 = 58.93
Actual weight of filtrate measured: 58.6 ± 0.28 (n = 3)
Cod Estimated weight of filtrate to be recovered: 55.05 - 5.91 =49.14
Actual weight of filtrate measured: 49 ± 0.21 (n = 3)
Mackerel Difference of estimated value from measured value = 0.56%
Cod Difference of estimated value from measured value = 0.28%
Theoretical weight based on density = 62.76 g.
Theoretical weight based on density = 51.42 g.
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