EUROPEAN PHARMACOPOEIA 11.

0 Cod-liver oil

Relative density (2.2.5): 0.917 to 0.930.

Refractive index (2.2.6) : 1.477 to 1.484.
Acid value (2.5.1) : maximum 2.0, determined on 20.00 g.
Anisidine value (2.5.36) : maximum 30.0.
Iodine value (2.5.4, Method B): 150 to 180, determined on
G. 6-methylergoline-8β-carboxamide (dihydroergine), 0.14 g to 0.16 g.
Use starch solution R2.
Peroxide value (2.5.5, Method B) : maximum 10.0.
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
determined on 5.0 g.
Stearin. Heat at least 10 mL to 60-90 °C and cool to room
H. 6-methylergoline-8β-carboxylic acid (dihydrolysergic temperature. Immerse for 3 h in a bath of iced water or a
acid). thermostatically controlled bath at 0 ± 0.5 °C. If necessary, to
eliminate insoluble matter, filter the sample after heating. The
07/2022:1192 sample remains clear.
Composition of fatty acids. Gas chromatography (2.2.28).
Trivial name of fatty Nomenclature Lower Upper
acid limit area limit area
(per cent) (per cent)
COD-LIVER OIL Saturated fatty acids :

Myristic acid 14:0 2.0 6.0

Iecoris aselli oleum Palmitic acid 16:0 7.0 14.0
Stearic acid 18:0 1.0 4.0
DEFINITION Mono-unsaturated fatty acids :
Purified fatty oil obtained from the fresh livers of wild
cod, Gadus morhua L. and other species of Gadidae, solid Palmitoleic acid 16:1 n-7 4.5 11.5
substances being removed by cooling and filtering. A suitable cis-Vaccenic acid 18:1 n-7 2.0 7.0
antioxidant may be added. Oleic acid 18:1 n-9 12.0 21.0
Gadoleic acid 20:1 n-11 1.0 5.5
Content :
Gondoic acid 20:1 n-9 5.0 17.0
– vitamin A : 600 IU (180 μg) to 2500 IU (750 μg) per gram ; Erucic acid 22:1 n-9 0 1.5
– vitamin D3 : 60 IU (1.5 μg) to 250 IU (6.25 μg) per gram. Cetoleic 22:1 n-11+13 5.0 12.0
acid (22:1 n-11)
PRODUCTION
Poly-unsaturated fatty acids :
The content of dioxins and dioxin-like PCBs (polychlorinated
biphenyls) is controlled using methods and limits in Linoleic acid 18:2 n-6 0.5 3.0
accordance with the requirements set in the European Union α-Linolenic acid 18:3 n-3 0 2.0
or other applicable regulations. Moroctic acid 18:4 n-3 0.5 4.5
Timnodonic 20:5 n-3 7.0 16.0
CHARACTERS (eicosapentaenoic)
acid (EPA)
Appearance : clear, yellowish liquid.
Cervonic 22:6 n-3 6.0 18.0
Solubility : practically insoluble in water, miscible with light (docosahexaenoic)
petroleum, slightly soluble in ethanol (96 per cent). acid (DHA)

IDENTIFICATION Test solution. Dilute about 0.45 g of the substance to

First identification : A, B, C. be examined to 10.0 mL with a 50 mg/L solution of
butylhydroxytoluene R in trimethylpentane R. Transfer 2.0 mL
Second identification : C, D. of this solution to a quartz tube and evaporate the solvent
A. In the assay for vitamin A using method A, the test solution with a gentle current of nitrogen R. Add 1.5 mL of a 20 g/L
shows an absorption maximum (2.2.25) at 325 ± 2 nm. In solution of sodium hydroxide R in methanol R, cover with
the assay for vitamin A using method B, the chromatogram nitrogen R, cap tightly with a polytetrafluoroethylene-lined
obtained with the test solution shows a peak corresponding cap, mix and heat on a water-bath for 7 min. Cool, add
to the peak due to all-trans-retinol in the chromatogram 2 mL of boron trichloride-methanol solution R, cover with
obtained with the reference solution. nitrogen R, cap tightly, mix and heat on a water-bath for
B. In the assay for vitamin D3, the chromatogram obtained 30 min. Cool to 40-50 °C, add 1 mL of trimethylpentane R, cap
with test solution (a) shows a peak corresponding to the and vortex or shake vigorously for at least 30 s. Immediately
peak due to cholecalciferol in the chromatogram obtained add 5 mL of saturated sodium chloride solution R, cover with
with reference solution (b). nitrogen R, cap and vortex or shake vigorously for at least 15 s.
C. Composition of fatty acids (see Tests). Allow the upper layer to become clear and transfer it to a
D. To 0.1 g add 0.5 mL of methylene chloride R and 1 mL of separate tube. Shake the methanol layer once more with 1 mL
antimony trichloride solution R. Mix. A deep blue colour of trimethylpentane R and combine the trimethylpentane
develops in about 10 s. extracts. Wash the combined extracts with 2 quantities, each
of 1 mL, of water R and dry over anhydrous sodium sulfate R.
TESTS Prepare 2 solutions for each sample.
Appearance. The substance to be examined is not more Reference solution. Dissolve 0.300 g of methyl arachidate R,
intensely coloured than a reference solution prepared as 0.300 g of methyl behenate R, 0.300 g of methyl palmitate R
follows : to 3.0 mL of red primary solution add 25.0 mL of and 0.300 g of methyl stearate R in a 50 mg/L solution of
yellow primary solution and dilute to 50.0 mL with a 10 g/L butylhydroxytoluene R in trimethylpentane R and dilute to
solution of hydrochloric acid R (2.2.2, Method II). 10.0 mL with the same solution.

General Notices (1) apply to all monographs and other texts 2413
Cod-liver oil EUROPEAN PHARMACOPOEIA 11.0

Column : – resolution : in the chromatogram obtained with the test

solution :
– material : fused silica ;
– minimum 1.3 between the peaks due to methyl oleate
– size : l = 30 m, Ø = 0.25 mm ; and methyl cis-vaccenate ; the resolution between the
– stationary phase : macrogol 20 000 R (film thickness peaks due to methyl gadoleate and methyl gondoate
0.25 μm). is sufficient for purposes of identification and area
measurement.
Carrier gas : hydrogen for chromatography R or helium for Calculate the area per cent for each fatty acid methyl ester
chromatography R, with an oxygen scrubber. using the following expression :
Split ratio : 1:200. Ax
´ 100
Temperature : At
Time Temperature Ax = peak area of fatty acid x ;
(min) (°C)
Column 0 - 55 170 → 225 At = sum of the peak areas (up to C22:6 n-3).
55 - 75 225 The calculation is not valid unless :
– the total area is based only on peaks due solely to fatty acid
Injection port 250
methyl esters ;
Detector 280 – the number of fatty acid methyl ester peaks exceeding
0.05 per cent of the total area is at least 24 ;
Detection : flame ionisation. – the 24 largest peaks of the methyl esters account for more
Injection : 1 μL, twice. than 90 per cent of the total area (these correspond to, in
common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
System suitability : 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3,
– the 15 fatty acids to be tested are satisfactorily identified 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3,
from the chromatogram shown in Figure 1192.-1 ; 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3,
22:6 n-3).
– in the chromatogram obtained with the reference solution,
multiply the areas of the peaks due to methyl palmitate, ASSAY
methyl stearate, methyl arachidate and methyl behenate Vitamin A. Carry out the test as rapidly as possible, avoiding
by the corresponding response factors in Table 1192.-1 ; exposure to actinic light and air, oxidising agents, oxidation
normalise the corrected areas of the peaks due to the fatty catalysts (for example, copper and iron) and acids.
acid methyl esters to a sum of 100 per cent ; the normalised Use method A. If method A is found not to be valid, use
area percentage of each fatty acid methyl ester is to be method B.
within ± 1.0 percentage unit of the corresponding weight
percentage ; METHOD A
Ultraviolet absorption spectrophotometry (2.2.25).
Table 1192.-1. Test solution. To 1.00 g in a round-bottomed flask, add
Fatty acid methyl ester Theoretical response factor
3 mL of a freshly prepared 50 per cent m/m solution of
potassium hydroxide R and 30 mL of anhydrous ethanol R.
Methyl palmitate 1.049 Boil under reflux in a current of nitrogen R for 30 min. Cool
rapidly and add 30 mL of water R. Extract with 50 mL of
Methyl stearate 1.029 ether R. Repeat the extraction 3 times and discard the lower
Methyl arachidate 1.013 layer after complete separation. Wash the combined upper
Methyl behenate 1.000 layers with 4 quantities, each of 50 mL, of water R, and
evaporate to dryness under a gentle current of nitrogen R

Figure 1192.-1. – Chromatogram for the test for composition of fatty acids of cod-liver oil

2414 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 11.0 Cod-liver oil

at a temperature not exceeding 30 °C or by other suitable Reference solution (a). Prepare a solution of retinol acetate CRS
means at a temperature not exceeding 30 °C under reduced in 2-propanol R1 so that 1 mL contains about 1000 IU of
pressure. Dissolve the residue in sufficient 2-propanol R1 to all-trans-retinol.
give an expected concentration of vitamin A equivalent to The exact concentration of reference solution (a) is assessed
10-15 IU/mL. by ultraviolet absorption spectrophotometry (2.2.25). Dilute
Measure the absorbances of the solution at 300 nm, 310 nm, reference solution (a) with 2-propanol R1 to a presumed
325 nm and 334 nm and at the wavelength of maximum concentration of 10-15 IU/mL and measure the absorbance
absorption with a suitable spectrophotometer in specially at 326 nm in matched 1 cm cells using 2-propanol R1 as the
matched 1 cm cells, using 2-propanol R1 as the compensation compensation liquid.
liquid.
Calculate the content of vitamin A in International Units
Calculate the content of vitamin A, as all-trans-retinol, in per millilitre of reference solution (a) using the following
International Units per gram, using the following expression : expression, taking into account the assigned content of retinol
acetate CRS :
1821
A325 ´ ´V
100m 1900 ´ V2
A326 ´
A325 = absorbance at 325 nm ; 100 ´ V1
m = mass of the substance to be examined, in grams ; A326 = absorbance at 326 nm ;
V = total volume of solution containing 10-15 IU of V1 = volume of reference solution (a) used ;
vitamin A per millilitre ; V2 = volume of the diluted solution ;
1821 = conversion factor for the specific absorbance of
all-trans-retinol, in International Units. 1900 = conversion factor for the specific absorbance of
retinol acetate CRS, in International Units.
The above expression can be used only if A325 has a value
not greater than A325,corr/0.970, where A325,corr is the corrected Reference solution (b). Proceed as described for the test
absorbance at 325 nm and is given by the following equation : solution but using 2.00 mL of reference solution (a) in place of
the substance to be examined.
A325,corr = 6.815A325 - 2.555A310 - 4.260A334
The exact concentration of reference solution (b) is assessed
A designates the absorbance at the wavelength indicated by by ultraviolet absorption spectrophotometry (2.2.25). Dilute
the subscript. reference solution (b) with 2-propanol R1 to a presumed
If A325 has a value greater than A325,corr/0.970, calculate the all-trans-retinol concentration of 10-15 IU/mL and measure
content of vitamin A using the following expression : the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
1821 Calculate the content of all-trans-retinol in International Units
A325,corr ´ ´V
100m per millilitre of reference solution (b), using the following
The assay is not valid unless : expression :
– the wavelength of the maximum absorption lies between 1821 ´ V3
323 nm and 327 nm ; A325 ´
100 ´ V4
– the absorbance at 300 nm relative to that at 325 nm is at
most 0.73. A325 = absorbance at 325 nm ;
METHOD B V3 = volume of the diluted solution ;
Liquid chromatography (2.2.29).
V4 = volume of reference solution (b) used ;
Test solution. Prepare duplicates. To 2.00 g of the substance to
be examined in a round-bottomed flask, add 5 mL of a freshly 1821 = conversion factor for the specific absorbance of
prepared 100 g/L solution of ascorbic acid R, 10 mL of a freshly all-trans-retinol, in International Units.
prepared 800 g/L solution of potassium hydroxide R and Column :
100 mL of anhydrous ethanol R. Boil under a reflux condenser
on a water-bath for 15 min. Add 100 mL of a 10 g/L solution – size : l = 0.25 m, Ø = 4.6 mm ;
of sodium chloride R and cool. Transfer the solution to a – stationary phase : octadecylsilyl silica gel for
500 mL separating funnel, rinsing the round-bottomed flask chromatography R (5-10 μm).
with about 75 mL of a 10 g/L solution of sodium chloride R
and then with 150 mL of a mixture of equal volumes of ether R Mobile phase : water for chromatography R, methanol R
and light petroleum R1. Shake for 1 min. When the layers have (3:97 V/V).
separated completely, discard the lower layer and wash the Flow rate : 1 mL/min.
upper layer, first with 50 mL of a 30 g/L solution of potassium Detection : spectrophotometer at 325 nm.
hydroxide R in a 10 per cent V/V solution of anhydrous
ethanol R and then with 3 quantities, each of 50 mL, of a 10 g/L Injection : 10 μL ; inject in triplicate the test solution and
solution of sodium chloride R. Filter the upper layer through reference solution (b).
5 g of anhydrous sodium sulfate R on a fast filter paper into a Retention time : all-trans-retinol = 5 ± 1 min.
250 mL flask. Wash the funnel with 10 mL of fresh extraction
mixture, filter and combine the upper layers. Distil them at System suitability :
a temperature not exceeding 30 °C under reduced pressure – the chromatogram obtained with the test solution shows a
and fill with nitrogen R when evaporation is completed. peak corresponding to the peak due to all-trans-retinol in
Alternatively, evaporate the solvent under a gentle current of the chromatogram obtained with reference solution (b) ;
nitrogen R at a temperature not exceeding 30 °C. Dissolve
the residue in 2-propanol R1, transfer to a 25 mL volumetric – the results obtained with the duplicate test solutions do not
flask and dilute to 25 mL with 2-propanol R1. Gentle heating differ by more than 5 per cent ;
in an ultrasonic bath may be required. A large fraction of the – the recovery of all-trans-retinol in reference solution (b) as
white residue is cholesterol, constituting approximately 50 per assessed by direct absorption spectrophotometry is greater
cent m/m of the unsaponifiable matter of cod-liver oil. than 95 per cent.

General Notices (1) apply to all monographs and other texts 2415
Cod-liver oil EUROPEAN PHARMACOPOEIA 11.0

Calculate the content of vitamin A using the following Injection : 350 μL of reference solution (b) and test
expression : solutions (a) and (b). Collect each eluate from 2 min before
until 2 min after the retention time of cholecalciferol, in
C´V 1
A1 ´ ´ a ground-glass-stoppered tube containing 1 mL of a 1 g/L
A2 m solution of butylhydroxytoluene R in hexane R. Evaporate
A1 = area of the peak due to all-trans-retinol in the separately to dryness at a temperature not exceeding 30 °C
chromatogram obtained with the test solution ; under a gentle current of nitrogen R. Dissolve each residue in
1.5 mL of acetonitrile R.
A2 = area of the peak due to all-trans-retinol in
the chromatogram obtained with reference DETERMINATION
solution (b) ; Column :
C = concentration of retinol acetate CRS in – size : l = 0.15 m, Ø = 4.6 mm ;
reference solution (a) as assessed prior to the – stationary phase : octadecylsilyl silica gel for
saponification, in International Units per millilitre chromatography R (5 μm).
(= 1000 IU/mL) ; Mobile phase : phosphoric acid R, 96 per cent V/V solution of
V = volume of reference solution (a) treated (2.00 mL); acetonitrile R (0.2:99.8 V/V).
m = mass of the substance to be examined in the test Flow rate : 1.0 mL/min.
solution (2.00 g). Detection : spectrophotometer at 265 nm.
Vitamin D3. Liquid chromatography (2.2.29). Carry out the Injection : 2 quantities not exceeding 200 μL of each of the
assay as rapidly as possible, avoiding exposure to actinic light 3 solutions obtained under Purification.
and air. System suitability :
Internal standard solution. Dissolve 0.50 mg of – resolution : minimum 1.4 between the peaks due to
ergocalciferol CRS in anhydrous ethanol R and dilute to ergocalciferol and cholecalciferol in the chromatogram
100.0 mL with the same solvent. obtained with reference solution (b) ;
Test solution (a). To 4.00 g of the substance to be examined – the results obtained with the test solution (b) duplicates do
in a round-bottomed flask, add 5 mL of a freshly prepared not differ by more than 5 per cent.
100 g/L solution of ascorbic acid R, 10 mL of a freshly prepared Calculate the content of vitamin D in International Units per
3
800 g/L solution of potassium hydroxide R and 100 mL of gram using the following expression, taking into account the
anhydrous ethanol R. Boil under a reflux condenser on a assigned content of cholecalciferol CRS :
water-bath for 30 min. Add 100 mL of a 10 g/L solution of
sodium chloride R and cool the solution to room temperature. A2 A3 m V
´ ´ 2 ´ 2 ´ 40
Transfer the solution to a 500 mL separating funnel, rinsing the A6 é A5 ù m1 V1
round-bottomed flask with about 75 mL of a 10 g/L solution A4 - ê ú ´ A 2
êAú
of sodium chloride R and then with 150 mL of a mixture of ë 1û
equal volumes of ether R and light petroleum R1. Shake for m1 = mass of the substance to be examined in test
1 min. When the layers have separated completely, discard the solution (b), in grams ;
lower layer and wash the upper layer, first with 50 mL of a m2 = total mass of cholecalciferol CRS used for
30 g/L solution of potassium hydroxide R in a 10 per cent V/V the preparation of reference solution (a), in
solution of anhydrous ethanol R, and then with 3 quantities, micrograms (500 μg) ;
each of 50 mL, of a 10 g/L solution of sodium chloride R. Filter A = area (or height) of the peak due to cholecalciferol in
1
the upper layer through 5 g of anhydrous sodium sulfate R the chromatogram obtained with test solution (a) ;
on a fast filter paper into a 250 mL flask. Wash the funnel
with 10 mL of fresh extraction mixture, filter and combine A 2 = area (or height) of the peak due to cholecalciferol in
the upper layers. Distil them at a temperature not exceeding the chromatogram obtained with test solution (b) ;
30 °C under reduced pressure and fill with nitrogen R when A3 = area (or height) of the peak due to ergocalciferol
evaporation is completed. Alternatively, evaporate the solvent in the chromatogram obtained with reference
under a gentle current of nitrogen R at a temperature not solution (b) ;
exceeding 30 °C. Dissolve the residue in 1.5 mL of the mobile A 4 = area (or height) of the peak due to ergocalciferol in
phase described under Purification. Gentle heating in an the chromatogram obtained with test solution (b) ;
ultrasonic bath may be required. A large fraction of the A 5 = area (or height) of a possible peak in the
white residue is cholesterol, constituting approximately 50 per chromatogram obtained with test solution (a) with
cent m/m of the unsaponifiable matter of cod-liver oil. the same retention time as the peak co-eluting with
Test solution (b). Prepare duplicates. To 4.00 g of the substance ergocalciferol in test solution (b) ;
to be examined add 2.0 mL of the internal standard solution A6 = area (or height) of the peak due to cholecalciferol
and proceed as described for test solution (a). in the chromatogram obtained with reference
Reference solution (a). Dissolve 0.50 mg of cholecalciferol CRS solution (b) ;
in anhydrous ethanol R and dilute to 100.0 mL with the same V 1 = total volume of reference solution (a) (100 mL) ;
solvent. V2 = volume of reference solution (a) used to prepare
Reference solution (b). Into a round-bottomed flask, reference solution (b) (2.0 mL).
add 2.0 mL of reference solution (a) and 2.0 mL of the
internal standard solution and proceed as described for test STORAGE
solution (a). In an airtight and well-filled container, protected from light. If
PURIFICATION no antioxidant is added, store under an inert gas.
Column : Once the container has been opened, its contents are used as
soon as possible and any part of the contents not used at once
– size : l = 0.25 m, Ø = 4.6 mm ;
is protected by an atmosphere of inert gas.
– stationary phase : cyanosilyl silica gel for chromatography R
(10 μm). LABELLING
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V). The label states :
Flow rate : 1.1 mL/min. – the number of International Units of vitamin A per gram ;
Detection : spectrophotometer at 265 nm. – the number of International Units of vitamin D3 per gram.

2416 See the information section on general monographs (cover pages)