SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

AOCS Official Method Ce 1b-89

Reapproved 1997 • Revised 2005

Fatty Acid Composition by GLC

Marine Oils

DEFINITION
This method determines the fatty acid composition of marine oils and marine oil esters by capillary
column gas–liquid chromatography.
SCOPE
This method is designed to determine the fatty acid composition of marine oils and marine oil esters
in relative (area %) values, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in
absolute (mg/g) values using a bonded polyglycol liquid phase in a flexible fused silica capillary col-
umn. C23:0 fatty acid is used as the internal standard. The method is applicable to the analysis of
marine oil or marine oil ethyl or methyl esters, capsules of EPA and DHA and minor naturally occur-
ring polyunsaturated fatty acids (see Notes, 1).

APPARATUS
1. Gas chromatograph—with capillary injection system
(split mode preferred, operated at a split ratio of 1:50;
see Notes, 2) and flame-ionization detector (FID; see
Notes, 3), capable of meeting the following minimum
requirements (see Notes, 4):
Injection port, 250°C
Detector, 270°C
Oven temperature profile:
Initial temperature, 170°C
Initial hold time, 0 min
Program rate, 1.0°C/min
Final temperature, 225°C
Final hold time, 0 min
2. The capillary GLC column should be of flexible fused
silica, 25 m or more in length and 0.20–0.35 mm i.d.
The liquid phase must be bonded Carbowax-20M or an
equivalent polyglycol. Examples of the latter are
Supelcowax-10, 30 m × 0.25 mm (or 0.32 mm) with
0.25 µm coating (Supelco, Inc., Bellefonte, PA, USA;
catalog no. 2-4079 or no. 2-4080) and Omegawax 320,
30 m × 0.32 mm i.d. with 0.25 µm coating (Supelco,
Inc., catalog no. 2-4152). No constraints are placed on
the column supplier; however, the manufacturer’s
claim for the upper temperature limit of the column
should be noted and verified.
3. Carrier gas—hydrogen or helium, 99.99% pure or bet-
ter, is required.
Note—An oxygen scrubber is mandatory with
Carbowax-20M and similar columns.
4. Any suitable amplifier, recorder, integrator or data
processor may be used.
5. Constant-temperature water bath or dry heating
block—maintained at 100°C.
6. Screw-cap tubes—16 × 125 mm, with leak-tight
Teflon™-lined caps.
7. Vial—screw- or crimp-cap, 12 mL.
8. Volumetric pipets—1 and 2 mL.
9. Pasteur-type pipets.
10. Volumetric flasks—25 and 100 mL.
11. Analytical balance—± 0.0001 g.
12. Source of dry nitrogen.
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REAGENTS
1. Sodium hydroxide—reagent grade.
2. Methyl alcohol—reagent grade (see Notes, Caution).
3. BF3—12% in methanol, 2-mL ampules (Supelco, Inc.,
catalog no. 3-3020, or equivalent).
4. Isooctane—reagent grade (see Notes, Caution).
5. Sodium chloride—reagent grade.
6. C23:0 methyl ester—see Notes, 5.
7. C23:0 ethyl ester—see Notes, 5.
8. Solutions
(a) Alcoholic sodium hydroxide (NaOH), 0.5 N–dis-
solve 2.0 g NaOH in methanol and make to 100
mL with methanol.
(b) Sodium chloride (NaCl)—saturated solution, dis-
solve 36 g NaCl in 100 mL distilled water.
PROCEDURE
1. Oils
(a) Accurately weigh (± 0.1 mg) approximately 25 mg
of C23:0 methyl ester internal standard (IS) into a
25-mL volumetric flask and make to volume with
isooctane.
(b) Pipet 1.0-mL aliquots of IS (see Notes, 6) into cul-
ture tubes and evaporate the solvent. Store the tubes
in a freezer if they will not be used immediately.
(c) Accurately weigh (± 0.1 mg) approximately 25 mg
of marine oil sample (may be from fish oil cap-
sule) into the culture tube containing the IS.
(d) Add 1.5 mL of 0.5 N NaOH. Blanket with nitro-
gen, cap tightly, mix and heat at 100°C for 5 min.
(e) Cool, add 2 mL BF3/methanol reagent, blanket
with nitrogen, cap tightly, mix and heat at 100°C
for 30 min.
(f) Cool to 30–40°C, add 1 mL of isooctane, blanket
with nitrogen, cap and vortex or shake vigorously
for 30 sec while still warm.
(g) Immediately add 5 mL of saturated NaCl solution,
blanket with nitrogen, cap and agitate thoroughly.
(h) Cool to room temperature. When isooctane layer
separates from the aqueous layer, transfer the
isooctane layer to a clean glass tube, blanket with
nitrogen, and cap.
SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1b-89 • Fatty Acid Composition by GLC

(i) Extract the methanol/water phase again with an of isooctane, blanket with nitrogen, cap, and mix
additional 1 mL of isooctane. thoroughly.
(j) Combine isooctane extracts (see Notes, 7) and (e) Inject 1–2 µL under appropriate gas chromato-
concentrate to approximately 1 mL with a stream graphic conditions (see Apparatus, 1).
of dry nitrogen. See Figures 1, 2 and 3 for chromatograms of
(k) Inject (see Notes, 8) 1–2 µL under appropriate gas methyl and ethyl esters of various marine oils; see
chromatographic conditions (see Apparatus, 1). Tables 1 and 2 for retention times of marine oil
2. Methyl or ethyl esters methyl esters.
(a) Accurately weigh (± 0.1 mg) approximately 25
CALCULATIONS
mg of C23:0 methyl or ethyl ester (appropriate to
1. Area percentages—Calculate the area percent for fatty
the substrate to be analyzed) IS into a 25-mL
acids by the formula:
volumetric flask and make to volume with
isooctane. 100 (Ax)
Area % fatty acids =
(b) Pipet a 1.0-mL aliquot (see Notes, 6) of the IS into At − AIS
a culture tube. Where—
(c) Evaporate the solvent. Ax = area counts for fatty acid x
(d) Accurately weigh (± 0.1 mg) no more than 15 mg At = total area counts for the chromatogram
of esters into the tube containing the IS, add 1 mL AIS = area counts of the internal standard

22:6n – 3
20:5n – 3
16:1n – 7

18:1n – 9
18:1n – 7

18:4n – 3
14:0

16:0

18:0

23:0 (IS)

22:5n – 3
18:2n – 6

20:1n – 9
18:3n – 3

20:4n – 3
Response

20:4n – 6

21:5n – 3
22:1

22:5n – 6
20:2n – 6

20:3n – 6

22:4n – 6
20:3n – 3
20:0

24:1
Time 24:0

Figure 1. Fatty acid methyl esters of soft-gelatin-encapsulated, steam-deodorized menhaden

oil (collaborative study sample 1) analyzed on a flexible fused silica capillary column (30 m
× 0.25 mm), coated with Supelcowax-10, under the conditions noted in the text.
20:5n – 3
20:1n – 9
18:1n – 9

22:6n – 3
23:0 (IS)
22:1n – 11 + 13
22:0 Me

24:0 Me
20:0 Me
14:0 Me

16:0 Me

18:0 Me

20:1n – 7
18:1n – 7

22:5n – 3
18:0
Response

20:4n – 3
20:4n – 6
16:0
14:0

Time

Figure 2. Fatty acid ethyl esters of a soft-gelatin-encapsulated commercial product,

SuperEPA 500 (collaborative study sample 2) analyzed on a flexible fused silica capillary
column (30 m × 0.25 mm), coated with Supelcowax-10, under the conditions noted in
the text. The location of added saturated methyl esters is indicated for reference.

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 SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1b-89 • Fatty Acid Composition by GLC

16:1n – 7

18:1n – 9

22:1n – 11 + 13
16:0
14:0

20:5n – 3
18:1n – 7

20:1n – 9

22:6n – 3
20:1n – 11

23:0 (IS)
18:2n – 6
18:0

18:4n – 3
Response

18:3n – 3

22:5m––33
22:1n – 9
20:2n – 6
20:3n – 6
20:4n – 6
20:3n – 3
20:4n – 3

22:5n – 6
22:4n – 6

22:5n
22:0

24:1
24:0
20:0
Time
Figure 3. Chromatogram of cod liver oil (collaborative study sample 3) methyl esters
analyzed on a flexible fused silica capillary column (30 m × 0.25 mm), coated with
Supelcowax-10, under the conditions noted in the text.

2. Calculation of mg of EPA and DHA per g of sample NOTES

(a) For marine oils and fish oil capsules (methyl
esters)—weight of EPA and DHA (see Notes, 9),
expressed as mg of EPA/DHA fatty acid per g of oil:
(Ax)(WIS)(CFx)
EPA or DHA, mg/g =
(AIS)(WS)(1.04)
(b) For ethyl esters—weight of EPA and DHA (see
Notes, 9), expressed as mg of EPA/DHA fatty acid
per g of sample:
(Ax)(WIS)(CFx)
EPA or DHA, mg/g =
(AIS)(WS)(1.08)
Where—
Ax = area counts for EPA (20:5n-3) or DHA
(22:6n-3)
AIS = area counts for internal standard
CFx = theoretical correction factor for EPA or
DHA
WIS = mass of internal standard added to the sam-
ple, in mg
WS = sample mass, in mg
3. Detector correction factors—Theoretical detector cor-
rection factors relative to C23:0 (the internal standard,
IS) for C20:5n-3 (0.99) and C22:6n-3 (0.97) should be
applied to the analytical data for optimal accuracy.
PRECISION
1. Collaborative study statistics, based on results from 19
laboratories that participated in the study, appear in
Table 3.
Caution
Methanol (methyl alcohol) is flammable and toxic. Avoid
contact with eyes. Avoid breathing vapors. Use effective
fume-removal device. Can react vigorously with sodium
hydroxide.
× 1000
Isooctane is highly flammable. An effective fume hood
should be used at all times when working with isooctane.
NUMBERED NOTES
1. It has been shown that the alkali-catalyzed methanoly-
sis step for the determination of long-chain marine oil
fatty acids produces dimethyl and mixed-alcohol esters
× 1000 from the plasticizer dioctyl [actually di(2-ethylhexyl)]
phthalate. An independent boron trifluoride-catalyzed
methanolysis step produced a lower level of artifacts,
but the official two-step process produced a higher
conversion than either catalyst did independently. The
retention times for the dimethyl, mixed-alcohol and
dioctyl phthalates are discussed (References, 1) in rela-
tion to methyl esters of common fatty acids on poly-
glycol wall-coated open-tubular GLC columns.
2. There is usually sufficient sample in marine oils analy-
sis to permit operation in the split mode.
3. The column end may be inserted directly in the detec-
tor jet, or there may be a connection at the base of the
jet, with or without makeup gas. If the FID requires
makeup gas, nitrogen is satisfactory.
4. These are suggested operating conditions for a 30-m
column.
5. Authentic standards of saturated fatty acid esters, if need-
ed for peak identification, are widely available. C21:5n-3
should be baseline separated from C23:0 (IS), and C24:0
should be baseline separated from C22:6n-3 (see Fig. 1).

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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1b-89 • Fatty Acid Composition by GLC

Table 1 Table 2
Retention times and area percent of fatty acid methyl esters Retention times and area percent of cod liver oil methyl
of steam-deodorized menhaden oil (collaborative study sam- esters (collaborative study sample 3). See Figure 3.
ple 1). See Figure 1.

Retention Relative Retention Relative

time retention Area time retention Area
Fatty acid (min) time percent Fatty acid (min) time percent
14:0 4.14 0.335 8.3 14:0 4.17 0.335 5.7
16:0 7.13 0.578 18.7 16:0 7.17 0.576 13.3
16:1n-7 7.66 0.621 12.1 16:1n-7 7.71 0.620 8.8
18:0 12.34 1.00 3.1 18:0 12.44 1.00 2.3
18:1n-9 12.97 1.05 18:1n-9 13.08 1.05
11.9 18.0
18:1n-7 13.22 1.07 18:1n-7 13.32 1.07
18:2n-6 14.57 1.18 1.2 18:2n-6 14.68 1.18 2.0
18:3n-3 17.08 1.38 0.8 18:3n-3 17.20 1.38 1.0
18:4n-3 18.38 1.40 2.9 18:4n-3 18.50 1.49 2.0
20:0 20.32 1.65 0.2 20:0 20.47 1.65 0.2
20:1n-11 20.91 1.69 20:1n-11 21.08 1.69
1.8 10.0
20:1n-9 21.12 1.71 20:1n-9 21.28 1.71
20:2n-6 23.37 1.89 0.1 20:2n-6 23.52 1.89 0.3
20:3n-6 24.66 2.00 0.2 20:3n-6 24.81 1.99 0.1
20:3n-3 26.66 2.16 0.2 20:3n-3 26.86 2.16 0.1
20:4n-6 25.74 2.09 0.8 20:4n-6 25.91 2.08 0.4
20:4n-3 28.07 2.27 1.3 20:4n-3 28.25 2.27 0.6
20:5n-3 29.29 2.37 13.8 20:5n-3 29.43 2.37 7.8
22:0 30.32 2.46 0.2 22:0 30.50 2.45 0.3
22:1n-11+13 31.27 2.53 22:1n-11+13 31.50 2.53
1.4 9.0
22:1n-9 31.63 2.56 22:1n-9 31.83 2.56
22:4n-6 37.17 3.03 0.2 22:4n-6 37.70 3.03 0.2
22:5n-6 38.83 3.14 0.2 22:5n-6 39.04 3.14 0.1
22:5n-3 41.11 3.33 2.1 22:5n-3 41.32 3.32 1.0
22:6n-3 42.86 3.47 7.9 22:6n-3 43.77 3.52 7.0
24:0 42.45 3.44 0.1 24:0 42.67 3.43 0.2
24:1n-9 43.54 3.52 0.3 24:1 43.77 3.52 0.9

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 SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1b-89 • Fatty Acid Composition by GLC

Table 3 6. If the peak height of the IS is ≤one-half that of the EPA

Statistical analysis of results for 6 fish oils, based on results or DHA peaks, repeat the analysis using 2.0 mL of IS.
received from 21 laboratories. 7. Optional—Wash isooctane with 2 mL of water and dry
over anhydrous sodium sulfate.
Samplesa 1 2 3 4 5 6 8. Manual injection with a microsyringe is preferred;
however, an autoinjection may be used.
14:0 8.3 0.4 5.7 7.0 5.4 8.3
9. Please note that this calculation gives results for fatty
16:0 18.7 0.9 13.3 15.0 7.8 19.0 acids and not for esters of fatty acids. The analyst can
18:0 3.1 2.1 2.3 2.9 0.8 3.1 confirm detector correction factors with pure reference
18:1 11.9 8.7 18.0 14.1 10.2 11.9 standards on individual instruments and capillary
columns. The weight % calculation should always be
x, %

18:4n-3 2.9 1.9 2.0 2.1 4.4 2.9 based on theoretical FID correction factors. In case of
20:1 1.8 14.2 10.0 3.2 1.3 1.8 strongly deviating experimental factors (>5% deviation
20:5n-3 13.8 26.4 7.8 17.0 27.5 13.4 from the theoretical factor), the analyst should locate
22:1 1.4 10.6 9.0 2.4 1.0 1.3
the cause of the deviation, rather than applying the
experimental factor.
22:5n-3 2.0 4.3 1.0 2.1 2.2 2.0
22:6n-3 7.9 18.7 7.0 12.3 12.5 7.8 REFERENCES
14:0 11.7 17.1 13.1 8.3 12.0 11.6 1. J. Chromatogr. 587:263 (1991).
16:0 7.0 12.2 7.5 6.9 5.7 10.0
OTHER REFERENCES
18:0 3.6 7.0 3.1 14.4 5.6 5.7 INFORM 1:987 (1990).
18:1 1.9 5.7 2.8 3.0 6.7 3.6 J. Am. Oil Chem. Soc. 64:499 (1987).
Ibid. 66:1822 (1989).
RSDR

18:4n-3 2.4 7.6 4.2 6.3 5.5 4.5

General referee report on oils and fats, J. Assoc. Off. Anal.
20:1 8.0 3.2 4.5 9.7 18.1 16.2 Chem. 73:105 (1990).
20:5n-3 5.8 5.6 5.5 7.4 7.5 9.8 J. Chromatogr. Biomed. Appl. 533:1 (1990).
22:1 17.2 4.7 8.9 10.0 13.4 16.1 J. AOAC Int. 75:488 (1992).
22:5n-3 14.6 8.5 9.7 12.2 8.8 16.2
AOAC International, 16th ed., Vol. II, Method 991.39,
Gaithersburg, MD, 1995, Chap. 41, p. 20.
22:6n-3 16.1 7.5 9.9 11.7 7.5 15.5
RSDR x, mg/g

20:5n-3 115.3 223.1 64.3 157.5 241.4 117.7

22:6n-3 66.0 157.9 56.4 105.3 105.4 67.2
20:5n-3 6.0 9.2 7.7 19.8 5.4 7.9
22:6n-3 4.2 9.0 8.3 12.6 5.2 8.6
x, % RSDr
14:0 8.3 5.8
RSDR—Blind duplicate pair

16:0 18.7 2.9

(Samples 1 and 6)

18:0 3.1 1.8

18:1 11.9 1.6
18:4n-3 2.9 1.5
20:1 1.8 7.3
20:5n-3 13.8 1.9
22:1 1.4 7.9
22:5n-3 2.0 6.7
22:6n-3 7.9 3.7
x, mg/g
x, mg/g

20:5n-3 116.5 5.9

22:6n-3 66.6 5.3
aAll samples were soft-gelatin encapsulated. 1. Steam-deodorized
menhaden oil; 2. commercial ethyl ester preparation; 3. cod liver
oil; 4. commercial fish oil; 5. commercial fish oil enriched in
EPA and DHA; 6. steam-deodorized menhaden oil (blind dupli-
cate of sample 1).

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