Cod Liver Oil
(kod liv' er oyl).
Cod Liver Oil is the partially destearinated fixed oil obtained from fresh livers of Gadus morrhua L. and other species of Fam. Gadidae. Cod Liver Oil contains, in each g, NLT 180 µg (600 USP Units) and NMT 750 µg (2500 USP Units) of vitamin A and NLT 1.5 µg (60 USP Units) and NMT 6.25 µg (250 USP Units) of vitamin D.
Cod Liver Oil may be flavored by the addition of NMT 1% of a suitable flavor or a mixture of flavors. A suitable antioxidant may be added.
• A. Presence of Vitamin A
Sample solution: 25 mg/mL of Cod Liver Oil in chloroform
Analysis: To 1 mL of the Sample solution add 10 mL of antimony trichloride TS.
Acceptance criteria: A blue color results immediately.
• B. Fatty Acid Profile
Antioxidant solution: 0.05 mg/mL of butylated hydroxytoluene in hexanes
System suitability solution: Prepare a mixture containing equal amounts of methyl palmitate, methyl stearate, methyl arachidate, and methyl behenate in Antioxidant solution.
Standard stock solution: 45 mg/mL of USP Cod Liver Oil RS in Antioxidant solution
Standard solution: Transfer 2.0 mL of the Standard stock solution into a quartz tube, and evaporate with a gentle stream of nitrogen. Add 1.5 mL of a 2% solution of sodium hydroxide in methanol, cap tightly with a polytetrafluoroethylene-lined cap, mix, and heat in a water bath for 7 min. Cool, add 2 mL of a 120 mg/mL solution of boron trichloride in methanol, cover with nitrogen, cap tightly, mix, and heat in a water bath for 30 min. Cool to 4050, add 1 mL of isooctane, cap, and mix in a vortex mixer or shake vigorously for at least 30 s. Immediately add 5 mL of saturated sodium chloride solution, cover with nitrogen, cap, and mix in a vortex mixer or shake thoroughly for at least 15 s. Allow the upper layer to become clear, and transfer to a separate tube. Shake the methanol layer once more with 1 mL of isooctane, and combine the isooctane extracts. Wash the combined extracts twice with 1 mL of water, and dry over anhydrous sodium sulfate.
Sample solution: Proceed as directed for the Standard solution, except replace USP Cod Liver Oil RS with Cod Liver Oil.
Detector: Flame ionization
Column: 0.25-mm × 30-m fused silica capillary column bonded with a 0.25-µm film of phase G16
Column: See Table 1.
Carrier gas: Helium
Split flow ratio: 200:1
Injection size: 1 µL
Samples: Standard solution and System suitability solution
Chromatogram similarity: The chromatogram from the Standard solution is similar to the reference chromatogram supplied with USP Cod Liver Oil RS. Identify the retention times of the relevant fatty acid methyl esters by comparing the chromatogram of the Standard solution with the reference chromatogram supplied with USP Cod Liver Oil RS.
Resolution: NLT 1.3 between methyl oleate and methyl cis-vaccinate, and that between methyl gadoleate and methyl gondoate is sufficient for purposes of identification and area measurement, Standard solution
Theoretical area percentages: 24.4 ± 1 for methyl palmitate, 24.8 ± 1 for methyl stearate, 25.2 ± 1 for methyl arachidate, and 25.6 ± 1 for methyl behenate, System suitability solution
Samples: Standard solution and Sample solution
Identify the retention times of the relevant fatty acid methyl esters in the Sample solution by comparing the chromatogram of the Sample solution with that of the Standard solution.
Determine the number of fatty acid methyl esters in the Sample solution. The number of fatty acid methyl ester peaks exceeding 0.05% of the total area of fatty acid methyl esters is at least 24, and the 24 largest peaks of the methyl esters account for more than 90% of the total area. (These correspond to the following, in common elution order: 14:0, 15:0, 16:0, 16:1 n7, 16:4 n1, 18:0, 18:1 n9, 18:1 n7, 18:2 n6, 18:3 n3, 18:4 n3, 20:1 n11, 20:1 n9, 20:1 n7, 20:2 n6, 20:4 n6, 20:3 n3, 20:4 n3, 20:5 n3, 22:1 n11, 22:1 n9, 21:5 n3, 22:5 n3, and 22:6 n3.)
Calculate the area percentage for each fatty acid methyl ester in the portion of Cod Liver Oil taken:
Result = (rA/rB) × 100
Acceptance criteria: The Sample solution meets the limits described in Table 2.
• Vitamin A
Sample: 500 mg to 1 g of Cod Liver Oil
Analysis: Proceed as directed under Vitamin A Assay 571.
Acceptance criteria: 180 µg (600 USP Units) to 750 µg (2500 USP Units) of vitamin A per g of Cod Liver Oil
• Vitamin D
Solution A: n-Amyl alcohol and dehydrated hexane (3:997)
Solution B: Acetonitrile, water, and phosphoric acid (96:3.8:0.2)
Butylated hydroxytoluene solution: 10 mg/mL of butylated hydroxytoluene in chromatographic hexane
Aqueous potassium hydroxide solution: Dissolve 800 mg of potassium hydroxide in 1000 mL of freshly boiled water, mix, and cool. [NotePrepare this solution fresh daily. ]
Alcoholic potassium hydroxide solution: Dissolve 3 g of potassium hydroxide in 50 mL of freshly boiled water, add 10 mL of alcohol, and dilute with freshly boiled water to 100 mL. [NotePrepare this solution fresh daily. ]
Ascorbic acid solution: 100 mg/mL of ascorbic acid in water. [NotePrepare this solution fresh daily. ]
Internal standard solution: 5 µg/mL of USP Ergocalciferol RS in alcohol
Standard stock solution: 5 µg/mL of USP Cholecalciferol RS in alcohol
Standard solution: Transfer 2.0 mL of the Standard stock solution and 2.0 mL of the Internal standard solution to a round-bottomed flask. Proceed as directed for Sample solution 1 beginning with Add 5 mL of .
Sample solution 1: Transfer 4.00 g of Cod Liver Oil to a round-bottomed flask. Add 5 mL of Ascorbic acid solution, 100 mL of alcohol, and 10 mL of Aqueous potassium hydroxide solution, and mix. Reflux the mixture on a steam bath for 30 min. Add 100 mL of a 10 mg/mL sodium chloride solution. Cool rapidly under running water, and transfer the saponified mixture to a 500-mL separator, rinsing the saponification flask with 75 mL of a 10 mg/mL sodium chloride solution, and then with 150 mL of a mixture of ether and hexane (1:1). Shake the combined saponified mixture and rinsings vigorously for 30 s, and allow to stand until both layers are clear. Discard the lower layer. Wash the ether-hexane extracts by shaking vigorously with 50 mL of Alcoholic potassium hydroxide solution, and then washing with three 50-mL portions of a 10 mg/mL sodium chloride solution. Filter the upper layer through 5 g of anhydrous sodium sulfate on a fast filter paper into a 250-mL flask suitable for a rotary evaporator. Wash the filter with 10 mL of a mixture of ether and hexane (1:1), and combine with the extract. Evaporate the solvent at reduced pressure at a temperature not exceeding 30, and fill with nitrogen when the evaporation is complete. Alternatively evaporate the solvent under a gentle stream of nitrogen at a temperature not exceeding 30. Dissolve the residue in 1.5 mL of Butylated hydroxytoluene solution. [NoteGentle heating in an ultrasonic bath may be required. A large fraction of the white residue is cholesterol. ]
Sample solution 2: To 4.00 g of Cod Liver Oil add 2.0 mL of Internal standard solution, and proceed as directed for Sample solution 1 beginning with Add 5 mL of .
Clean-up chromatographic system
Detector: UV 265 nm
Mobile phase: Solution A
Clean-up column: 4.6-mm × 25-cm stainless steel; packing L10
Injection size: 350 µL
Samples: Standard solution, Sample solution 1, and Sample solution 2
Collect separately the eluates from 2 min before to 2 min after the retention time of cholecalciferol in a glass tube containing 1 mL of Butylated hydroxytoluene solution and fitted with a hermetic closure. Evaporate each tube under a stream of nitrogen at a temperature not exceeding 30. Dissolve each residue in 1.5 mL of acetonitrile, and inject into the analytical chromatographic system below.
Analytical chromatographic system
Detector: UV 265 nm
Mobile phase: Solution B
Analytical column: 4.6-mm × 15-cm stainless steel; 5-µm packing L1
Injection size: 200 µL
Sample: Standard solution (after the clean-up)
Resolution: NLT 1.4 between cholecalciferol and ergocalciferol
Relative standard deviation: NMT 2.0% for the cholecalciferol peak from replicate injections
Samples: Standard solution, Sample solution 1, and Sample solution 2 (after the clean-up)
Calculate the content of vitamin D, in µg/g, in the portion of Cod Liver Oil taken:
Result = (RU/RS) × (CS/CU)
RU = rU2/[rIS2 (rIS1 × rU2/rU1)]
Acceptance criteria: 1.5 µg (60 USP Units) to 6.25 µg (250 USP Units) of vitamin D per g of Cod Liver Oil
• Specific Gravity 841: 0.9180.927
• Color: When viewed transversely in a tall, cylindrical, standard oil-specimen bottle of colorless glass of about 120-mL capacity, the color of the Cod Liver Oil is not more intense than that of a mixture of cobaltous chloride CS, ferric chloride CS, and water (11:76:33), in a similar bottle of the same internal diameter.
• Nondestearinated Cod Liver Oil
Sample: Cod Liver Oil
Analysis: Fill a tall, cylindrical, standard oil-specimen bottle of 120-mL capacity with the Sample at a temperature between 23 and 28, insert the stopper, and immerse the bottle in a mixture of ice and water for 3 h.
Acceptance criteria: The oil remains clear and does not deposit stearin.
• Fats and Fixed Oils, Unsaponifiable Matter 401: NMT 1.30%
• Fats and Fixed Oils, Acid Value 401
Sample solution: Mix 15 mL of alcohol with 15 mL of ether, add 5 drops of phenolphthalein TS, and neutralize with 0.1 N sodium hydroxide. Dissolve 2.0 g of Cod Liver Oil in the mixture, and boil the oil solution gently under a reflux condenser for 10 min.
Analysis: Cool, and titrate the mixture with 0.1 N sodium hydroxide VS to the production of a pink color that persists after shaking for 30 s.
Acceptance criteria: NMT 1.0 mL of 0.1 N sodium hydroxide is required.
• Fats and Fixed Oils, Iodine Value 401: 145180
• Fats and Fixed Oils, Saponification Value 401: 180192
[NoteIf carbon dioxide has been used as a preservative, expose the Cod Liver Oil in a shallow dish in a vacuum desiccator for 24 h before weighing the specimen for determination of the saponification value. ]
• Fats and Fixed Oils, Anisidine Value 401: NMT 30
• Packaging and Storage: Preserve in tight containers. It may be bottled or otherwise packaged in containers from which air has been expelled by the production of a vacuum or by an inert gas.
• Labeling: The vitamin A potency and vitamin D potency, when designated on the label, are expressed in USP Units/g of oil. The potencies may be expressed also in metric units, on the basis that 1 USP Vitamin A Unit equals 0.3 µg and 40 USP Vitamin D Units equals 1 µg. Where the content of docosahexaenoic acid or eicosapentaenoic acid is claimed, state the concentration in mg/g.
• USP Reference Standards 11
USP Cod Liver Oil RS
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USP35NF30 Page 2756Pharmacopeial Forum: Volume No. 32(5) Page 1443