Cod Liver Oil
»Cod Liver Oil is the partially destearinated fixed oil obtained from fresh livers of Gadus morrhuaLinnéand other species of Fam.Gadidae.Cod Liver Oil contains,in each g,not less than 180µg (600USP Units)and not more than 750µg (2500USP Units)of vitamin Aand not less than 1.5µg (60USP Units)and not more than 6.25µg (250USP Units)of vitamin D.
Cod Liver Oil may be flavored by the addition of not more than 1percent of a suitable flavor or a mixture of flavors.Asuitable 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 Apotency and vitamin Dpotency,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 1USP Vitamin A Unit =0.3µg and 40USP Vitamin D Units =1µg.Where the content of docosahexaenoic acid or eicosapentaenoic acid are claimed,state their concentrations in mg per g.
A:Presence of vitamin A To 1mLof a 1in 40solution in chloroform add 10mLof 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 0.05mg per mL.
Standard solution Transfer 0.450g of USP Cod Liver Oil RS,accurately weighed,into a 10-mLvolumetric flask,and dissolve in and dilute with Antioxidant solutionto volume.Transfer 2.0mLof this solution into a quartz tube,and evaporate with gentle stream of nitrogen.Add 1.5mLof a 2%solution of sodium hydroxide in methanol,cap tightly with a polytetrafluoroethylene-lined cap,mix,and heat in a water bath for 7minutes.Cool,add 2mLof boron trichloridemethanol solution,cover with nitrogen,cap tightly,mix,and heat in a water bath for 30minutes.Cool to 40to 50,add 1mLof isooctane,cap,and mix in a vortex mixer or shake vigorously for at least 30seconds.Immediately add 5mLof saturated sodium chloride solution,cover with nitrogen,cap,and mix in a vortex mixer or shake thoroughly for at least 15seconds.Allow the upper layer to become clear,and transfer to a separate tube.Shake the methanol layer once more with 1mLof isooctane,and combine the isooctane extracts.Wash the combine extracts twice with 1mLof 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.[NOTEAsuitable mixture is available from Supelco,Bellefonte,PA,as GLC-40cat.number 1985-1AMP.]
Test solution Proceed as directed for the Standard solution,except to use an accurately weighed quantity of Cod Liver Oil.
Chromatographic system (see Chromatography á621ñ) 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 280and 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 1per minute to 225,and maintained at 225for 20minutes.The carrier gas is helium with a split flow ratio of 1:200.Chromatograph the Standard solution,System suitability mixture,and Test solution,and record the peak responses as directed for Procedure:the resolution,R,between the peaks in the Standard solution due to methyl oleate and methyl cis-vaccinate is not less than 1.3,and between methyl gadoleate and methyl gondoate is sufficient for purposes of identification and area measurement;the theoretical area percentages for methyl palmitate,metyl stearate,methyl arachidate,and methyl behenate are 24.4,24.8,25.2,and 25.6,respectively.In a suitable instrument,the area percentages from the System suitability mixtureare 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 24largest peaks of the methyl esters account for more than 90%of the total area.(These correspond to the following,in common elution order:14:0,15:0,16:0,16:1n-7,16:4n-1,18:0,18:1n-9,18:1n-7,18:2n-6,18:3n-3,18:4n-3,20:1n-11,20:1n-9,20:1n-7,20:2n-6,20:4n-6,20:3n-3,20:4n-3,20:5n-3,22:1n-11,22:1n-9,21:5n-3,22:5n-3,and 22:6n-3.)
Procedure Separately inject by equal volumes (about 1µL)of the Standard solutionand the Test solution into the chromatograph,record the chromatograms,and measure the peak responses.Calculate the area percent for each fatty acid methyl ester taken by the formula:
100(ra/rb),in which rais the average peak area of each individual fatty acid;and rbis the total peak area from all peaks in the chromatogram,excepting the solvent front and butylated hydroxytoluene.
Color When viewed transversely in a tall,cylindrical,standard oil-specimen bottle of colorless glass of about 120-mLcapacity,the color is not more intense than that of a mixture of 11mLof cobaltous chloride CS,76mLof ferric chloride CS,and 33mLof water,in a similar bottle of the same internal diameter.
Specific gravity á841ñ: between 0.918and 0.927.
Nondestearinated cod liver oil Fill a tall,cylindrical,standard oil-specimen bottle of about 120-mLcapacity with Oil at a temperature between 23and 28,insert the stopper,and immerse the bottle in a mixture of ice and water for 3hours:the oil remains clear and does not deposit stearin.
Unsaponifiable matter á401ñ: not more than 1.30%.
Acid value á401ñ Mix 15mLof alcohol with 15mLof ether,add 5drops of phenolphthalein TS,and neutralize with 0.1Nsodium hydroxide.Dissolve 2.0g of Oil in the mixture,and boil the oil solution gently under a reflux condenser for 10minutes.Cool,and titrate the mixture with 0.1Nsodium hydroxide VSto the production of a pink color that persists after shaking for 30seconds:not more than 1.0mLof 0.10Nsodium hydroxide is required.
Iodine value á401ñ: between 145and 180.
Saponification value á401ñ Its saponification value is between 180and 192.If carbon dioxide has been used as a preservative,expose the Oil in a shallow dish in a vacuum desiccator for 24hours before weighing the specimen for determination of the saponification value.
Anisidine value á401ñ: not more than 30.
Assay for vitamin A Using 500mg to 1g,accurately weighed,of Oil,proceed as directed under Vitamin A Assay á571ñ.
Assay for vitamin D
Butylated hydroxytoluene solution Dissolve a quantity of butylated hydroxytoluene in chromatographic hexane to obtain a solution containing 10mg per mL.
Aqueous potassium hydroxide solution Dissolve 800g of potassium hydroxide in 1000mLof freshly boiled water,mix,and cool.[NOTEPrepare this solution fresh daily.]
Alcoholic potassium hydroxide solution Dissolve 3g of potassium hydroxide in 50mLof freshly boiled water,add 10mLof alcohol,dilute with freshly boiled water to 100mL,and mix.[NOTEPrepare this solution fresh daily.]
Ascorbic acid solution Dissolve 10g of ascorbic acid in 100mLof water.[NOTEPrepare this solution fresh daily.]
Mobile phase A Prepare a 3in 1000mixture 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 Suitabilityunder Chromatography á621ñ).
Internal standard solution Prepare a solution of USP Ergocalciferol RSin alcohol having a concentration of about 0.5mg per 100mL.
Standard preparation Prepare a solution ofUSP Cholecalciferol RSin ethyl alcohol,having a known concentration of about 5µg per mL.Transfer 2.0mLof this solution and 2.0mLof the Internal standard solutionto a round-bottomed flask.Proceed as directed for the Assay preparation 1beginning with Add 5mLof .
Assay preparation 1 Transfer an accurately weighed quantity of about 4.00g of Cod Liver Oil,to a round-bottomed flask.Add 5mLof Ascorbic acid solution,100mLof alcohol,and 10mLof Aqueous potassium hydroxide solution,and mix.Reflux the mixture on a steam bath for 30minutes.Add 100mLof a sodium chloride solution (1in 100).Cool rapidly under running water,and transfer the saponified mixture to a 500-mLseparator,rinsing the saponification flask with 75mLof a sodium chloride solution (1in 100)and then with 150mLof a mixture of ether and hexane (1:1).Shake the combined saponified mixture and rinsings vigorously for 30seconds,and allow to stand until both layers are clear.Discard the lower layer.Wash the ether-hexane extracts by shaking vigorously with 50mLof Alcoholic potassium hydroxide solution,and then washing with three 50-mLportions of a sodium chloride solution (1in 100).Filter the upper layer through 5g of anhydrous sodium sulfate on a fast filter paper into a 250-mLflask suitable for a rotary evaporator.Wash the filter with 10mLof 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 1.5mLof Mobile phase A.[NOTEGentle heating in an ultrasonic bath may be required.Alarge fraction of the white residue is cholesterol.]
Assay preparation 2 To 4.00g of Cod Liver Oil,add 2.0mLof Internal standard solution,and proceed as directed for Assay preparation 1beginning with Add 5mLof .
Chromatographic system Use a chromatograph,operated at room temperature,fitted with an UVdetector that monitors absorption at 265nm;a 25-cm ×4.6-mm stainless steel cleanup column packed with column packing L10and 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 350µL)of the Standard preparation,Assay preparation 1,and Assay preparation 2into the clean-up chromatographic system.Collect separately the eluates from 2minutes before until 2minutes after the retention time of cholecalciferol in a glass tube,containing 1mLof Butylated hydroxytoluene solutionand fitted with a hermetic closure.Evaporate each tube under a stream of nitrogen at a temperature not exceeding 30.Dissolve each residue in 1.5mLof acetonitrile.Inject equal volumes,not exceeding 200µL,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 Cis the concentration,in µg per mL,of USP Cholecalciferol RSin theStandard preparation;RSis the response of the cholecalciferol relative to the internal standard in the Standard preparation;and RUis the corrected relative response of Assay preparation 2calculated by the formula:
rU2/[rIS2(rIS1×rU2/rU1)],in which,rU2and rU1are the peak responses for cholecalciferol in the Assay preparation 1and 2,respectively;and rIS1and rIS2are the peak responses for ergocalciferol in the Assay preparation 1and 2,respectively.
Auxiliary Information Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(DSN)Dietary Supplements:Non-Botanicals
USP28NF23Page 533Pharmacopeial Forum:Volume No.29(4)Page 1019