Cod Liver Oil
» Cod Liver Oil is the partially destearinated fixed oil obtained from fresh livers of Gadus morrhua Linné and other species of Fam. Gadidae. Cod Liver Oil contains, in each g, not less than 180 µg (600 USP Units) and not more than 750 µg (2500 USP Units) of vitamin A and not less than 1.5 µg (60 USP Units) and not more than 6.25 µg (250 USP Units) of vitamin D.
Cod Liver Oil may be flavored by the addition of not more than 1 percent of a suitable flavor or a mixture of flavors. A suitable antioxidant may be added.
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 per g of oil. The potencies may be expressed also in metric units, on the basis that 1 USP Vitamin A Unit = 0.3 µg and 40 USP Vitamin D Units = 1 µg. Where the content of docosahexaenoic acid or eicosapentaenoic acid are claimed, state their concentrations in mg per g.
A: Presence of vitamin ATo 1 mL of a 1 in 40 solution of it in chloroform, add 10 mL of antimony trichloride TS: a blue color results immediately.
B: Fatty acid profile
Antioxidant solution Dissolve an accurately weighed quantity of butylated hydroxytoluene in hexanes to obtain a solution having a concentration of 0.05 mg per mL.
Standard solution Transfer 0.450 g of USP Cod Liver Oil RS, accurately weighed, into a 10-mL volumetric flask, and dissolve in and dilute with Antioxidant solution to volume. Transfer 2.0 mL of this 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 minutes. Cool, add 2 mL of a solution of 120 g of boron trichloride in 1000 mL of methanol, cover with nitrogen, cap tightly, mix, and heat in a water bath for 30 minutes. Cool to 40 to 50, add 1 mL of isooctane, cap, and mix in a vortex mixer or shake vigorously for at least 30 seconds. 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 seconds. 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.
System suitability mixture Prepare a mixture containing accurately weighed and equal amounts of methyl palmitate, methyl stearate, methyl arachidate, and methyl behenate. [noteA suitable mixture is available from Supelco, Bellefonte, PA, as GLC-40 cat. number 1895-1AMP.]
Test solution Proceed as directed for the Standard solution, except to use an accurately weighed quantity of Cod Liver Oil.
Chromatographic system (see Chromatography 621) The gas chromatograph is equipped with a flame-ionization detector and a 0.25-mm × 30-m fused silica capillary column coated with a 0.25-µm film of G16. The temperature of the detector is maintained at 280 and that of the injection port at 250. Initially the temperature of the column is equilibrated at 170, then the temperature is increased at a rate of 1 per minute to 225, and maintained at 225 for 20 minutes. The carrier gas is helium, with a split flow ratio of 1:200. Chromatograph the Standard solution, the System suitability mixture, and the Test solution, and record the peak responses as directed for Procedure: the chromatogram obtained 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. The resolution, R, between the peaks in the Standard solution due to methyl oleate and to methyl cis-vaccinate is not less than 1.3, and that between methyl gadoleate and methyl gondoate is sufficient for purposes of identification and area measurement; the theoretical area percentages for methyl palmitate, methyl stearate, methyl arachidate, and methyl behenate in the System suitability mixture are 24.4, 24.8, 25.2, and 25.6, respectively. In a suitable instrument, the area percentages from the System suitability mixture are within 1% of the theoretical values. The number of fatty acid methyl ester peaks exceeding 0.05% of the total area 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 n-7, 16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, and 22:6 n-3.)
Procedure Separately inject equal volumes (about 1 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Identify the retention times of the relevant fatty acid methyl esters in the Test solution by comparing the chromatogram of the Test solution with that of the Standard solution. Calculate the area percentage for each fatty acid methyl ester taken by the formula:
100(rA / rB)in which rA is the average peak area of each individual fatty acid; and rB is the total peak area from all peaks in the chromatogram, except the solvent front and butylated hydroxytoluene. The fatty acids observed in the Test solution should meet the limits described in the table.
Color When viewed transversely in a tall, cylindrical, standard oil-specimen bottle of colorless glass of about 120-mL capacity, the color is not more intense than that of a mixture of 11 mL of cobaltous chloride CS, 76 mL of ferric chloride CS, and 33 mL of water, in a similar bottle of the same internal diameter.
Specific gravity 841: between 0.918 and 0.927.
Nondestearinated cod liver oil Fill a tall, cylindrical, standard oil-specimen bottle of about 120-mL capacity with Oil at a temperature between 23 and 28, insert the stopper, and immerse the bottle in a mixture of ice and water for 3 hours: the oil remains clear and does not deposit stearin.
Unsaponifiable matter 401: not more than 1.30%.
Acid value 401 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 Oil in the mixture, and boil the oil solution gently under a reflux condenser for 10 minutes. 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 seconds: not more than 1.0 mL of 0.10 N sodium hydroxide is required.
Iodine value 401: between 145 and 180.
Saponification value 401 Its saponification value is between 180 and 192. If carbon dioxide has been used as a preservative, expose the Oil in a shallow dish in a vacuum desiccator for 24 hours before weighing the specimen for determination of the saponification value.
Anisidine value 401: not more than 30.
Assay for vitamin A Using 500 mg to 1 g, accurately weighed, of Oil, proceed as directed under Vitamin A Assay 571.
Assay for vitamin D
Butylated hydroxytoluene solution Dissolve a quantity of butylated hydroxytoluene in chromatographic hexane to obtain a solution containing 10 mg per mL.
Aqueous potassium hydroxide solution Dissolve 800 g 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, dilute with freshly boiled water to 100 mL, and mix. [notePrepare this solution fresh daily.]
Ascorbic acid solution Dissolve 10 g of ascorbic acid in 100 mL of water. [notePrepare this solution fresh daily.]
Mobile phase A Prepare a 3 in 1000 mixture of n-amyl alcohol in dehydrated hexane.
Mobile phase B Prepare a mixture of acetonitrile, water, and phosphoric acid (96:3.8:0.2). Make adjustments if necessary (see System Suitability under Chromatography 621).
Internal standard solution Prepare a solution of USP Ergocalciferol RS in alcohol having a concentration of about 0.5 mg per 100 mL.
Standard preparation Prepare a solution of USP Cholecalciferol RS in ethyl alcohol, having a known concentration of about 5 µg per mL. Transfer 2.0 mL of this solution and 2.0 mL of the Internal standard solution to a round-bottomed flask. Proceed as directed for the Assay preparation 1 beginning with Add 5 mL of .
Assay preparation 1 Transfer an accurately weighed quantity of about 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 minutes. Add 100 mL of a sodium chloride solution (1 in 100). Cool rapidly under running water, and transfer the saponified mixture to a 500-mL separator, rinsing the saponification flask with 75 mL of a sodium chloride solution (1 in 100) and then with 150 mL of a mixture of ether and hexane (1:1). Shake the combined saponified mixture and rinsings vigorously for 30 seconds, 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 sodium chloride solution (1 in 100). 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 Mobile phase A. [noteGentle heating in an ultrasonic bath may be required. A large fraction of the white residue is cholesterol.]
Assay preparation 2 To 4.00 g of Cod Liver Oil, add 2.0 mL of Internal standard solution, and proceed as directed for Assay preparation 1 beginning with Add 5 mL of .
Chromatographic system Use a chromatograph, operated at room temperature, fitted with an UV detector that monitors absorption at 265 nm; a 25-cm × 4.6-mm stainless steel cleanup column packed with column packing L10 and using Mobile phase A; and a 15-cm × 4.6-mm stainless steel analytical column with 5-µm packing L1, and using Mobile phase B. Chromatograph five injections of the Standard preparation, and measure the peak responses as directed for Procedure: the resolution, R, between cholecalciferol and ergocalciferol is not less than 1.4; and the relative standard deviation for the cholecalciferol peak response is not more than 2.0%.
Procedure Separately inject equal volumes (about 350 µL) of the Standard preparation, Assay preparation 1, and Assay preparation 2 into the clean-up chromatographic system. Collect separately the eluates from 2 minutes before until 2 minutes 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. Inject equal volumes, not exceeding 200 µL, into the analytical chromatographic system, and measure the peak responses at the retention times corresponding to cholecalciferol and ergocalciferol. Calculate the content of vitamin D, in µg, in the Cod Liver Oil taken by the formula:
2C(RU / RS)in which C is the concentration, in µg per mL, of USP Cholecalciferol RS in the Standard preparation; RS is the response of the cholecalciferol relative to the internal standard in the Standard preparation; and RU is the corrected relative response of Assay preparation 2 calculated by the formula:
rU2 / [rIS2 (rIS1 × rU2 / rU1)]in which, rU2 and rU1 are the peak responses for cholecalciferol in the Assay preparation 1 and 2, respectively; and rIS1 and rIS2 are the peak responses for ergocalciferol in the Assay preparation 1 and 2, respectively.
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