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Title: Valid from: Replaces: Vitamin A …

Document:Code:Page:Analytical Method 10002-011 of 1 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis Eriksen1. PurposeThe purpose of this SOP is to update method Index1. Purpose .. 12. Index .. 13. Enclosures .. 14. Principle .. 25. 26. 27. Chromatographic 48. Standard solution:.. Test solution .. Saponification .. Extraction .. Final Tablets and Standard assay concentration determination .

Document: Code: Page: Analytical Method 10002-01 2 of 2 Title: Valid from: Replaces: Vitamin A determination by HPLC New Date of revision: September 2008

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1 Document:Code:Page:Analytical Method 10002-011 of 1 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis Eriksen1. PurposeThe purpose of this SOP is to update method Index1. Purpose .. 12. Index .. 13. Enclosures .. 14. Principle .. 25. 26. 27. Chromatographic 48. Standard solution:.. Test solution .. Saponification .. Extraction .. Final Tablets and Standard assay concentration determination .

2 Standard to HPLC .. 79. Calculations .. 710. 83. Enclosures1. Dokumentation2. Examples of chromatogramsDocument:Code:Page:Analytic al Method 10002-012 of 2 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis Eriksen4. PrincipleVitamin A (Retinol) is determined by high performance liquid chromatography withUV-detection after saponification and extraction. The method is in particular usablefor tablets containing large amounts of betacaroten and for products containing a littleamount of Vitamin concentration of the sample: About g of Vitamin .

3 Make two single determinations at two different ApparatusShimadzu High Pressure Liquid Chromatograph Autoinjector SIL 10 A XL CBM box 10 A UV- Vis detector SPD 10A Pump LC 10 AT FVC 10 ALor use similar HPLC equipmentGrinding mill, Krups 75 or similarPerkin Elmer model Lambda 20 UV/VIS Spectrophotometer or ReagentsPotassium hydroxide Merck art. 5021 Ascorbic acid Merck art. 127 Sodium sulphate Merck art. 106649 Butylhydroxytoluen (BHT), Fluka art. 34750 Document:Code:Page:Analytical Method 10002-013 of 3 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis EriksenEther Superfos Kemi, art.

4 1416064 Ethanol (99%), DDSFN itrogen, = n-amylalcohol, Merck art. 975 Heptane Ratburn art. 1004 Milli-Q WaterIsopropanol = 2-propanol-R1, Merck art. 101040 Vitamin A Fe standard (= mcg/g)Diluted Sodium Hydroxide solution (2M) Baker art. 7067 Phenolphthalein solution R1 Merck art. 7233 Potassium hydroxide solution (17M / 90%):Dissolve 180 g of potassium hydroxide in 126 ml of for three monthSodium sulphate solution (3%):Dissolve 30 g of anhydrous sodium sulphate in water and dilute with water to1000 for three monthBHT-solution ( ), alcoholic:Dissolve g of butylhydroxytoluen in ethanol and dilute with ethanol to for three monthSodium ascorbate solution (2%):Dissolve g of ascorbic acid in 10 ml of diluted sodium hydroxide solutionand dilute with water to 200 :Code:Page:Analytical Method 10002-014 of 4 Title: Valid from: Replaces.

5 Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis EriksenDo not store7. Chromatographic conditionsColumn:C18, 15 cm x mm, YMC 120 , 5 m, OdDMeSi - B- 564, phase: Heptane : 1-propanol (99:1)Flow rate: 2 ml/minuteDetection: UV-absorption 325 nmInjection volume: 100 lAttenuation: App. 28 (8)Chart Speed: App. 3 mm/minuteRun time: App. 12 minutesRetention time: App. 7 minutes for Retinol8.

6 Standard solution:Weigh out, in duplicate, about g of the standard in a conical flask. Use A-acetateconcentration, Fe standard (= mcg/g).Add 10 ml of sodium ascorbate solution (2%) and heat in a steam bath for 5 30 ml of BTH-solution ( ) and 3 ml of potassium hydroxide (17M).Document:Code:Page:Analytical Method 10002-015 of 5 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis EriksenConnect the flask of an air condenser and reflux for 30 minutes on a steam bath(shake frequently).

7 After cooling, transfer the solution by 30 ml of sodium sulphate 3% and 100 ml ofether to at separating funnel containing 100 ml of from the extraction Test solution1. Liquid, anhydrous solutions:Mix the sample and weigh out accurately the sample (= p g) (see table below) intoan Erlenmeyer flask. Dilute to 10 ml with sodium ascorbate solution (2%).2. Oily solutions:Weigh out accurately the amount of sample (=p g) (see table below) into a 100 mlErlenmeyer TabletsPulverise 20 tablets in a grinding out accurately the powder (=p g) (see table below) into a 100 ml Erlenmeyerflask.

8 Add 10 ml of sodium ascorbate solution (2%) and heat in a steam bath for 5minutes with frequently A conc. in the sample( g/ml, g/g or g/tabl.)Weigh an amount of the samplecontaining about:A 10 and < 8080 g of Vitamin AB 80 and < 400400 g of Vitamin AC 400 and < 20002000 g of Vitamin SaponificationAdd 30 ml of alcoholic BTH-solution ( ) and 3 ml of potassium hydroxide (17M).Connect the flask of an air condenser and reflux for 30 minutes on a steam bath(shake frequently).Document:Code:Page:Analytica l Method 10002-016 of 6 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis EriksenCool and transfer with 30 ml of sodium sulphate 3% and 100 ml of ether to a 500 mlseparating funnel containing 100 ml of ExtractionShake for 2 stand until the layers are clearly separated (about 30 minutes), and discharge thelower aqueous layer (if an emulsion is formed, add some drops of ethanol 99%).

9 Wash the ether extract with 4 x 50 ml of water; shake carefully in the beginning inorder to avoid emulsification. Afterwards pour a couple of ml of water phase into acentrifuge tube containing a few drops of it is red - continue washing until the washings are no longer coloured , transfer the ether layer to a 250 ml volumetric flask or a Final Tablets and solutionsA+B: Transfer through a cotton plug covered with anhydrous sodium sulphate byether to a round-bottom flask. Evaporate in vacuum and dilute with n-heptane until afinal concentration of 3-4 mcg/ml is :Transfer to a 250 ml volumetric flask and fill up to volume with ether.

10 Ml in a50 ml flask is evaporated under a steam of nitrogen until a rest of 2 ml is left. Dilutewith n-heptane to a final volume of 50 StandardTransfer by ether into a 250 ml volumetric flask (= STD A) and fill to :Code:Page:Analytical Method 10002-017 of 7 Title: Valid from: Replaces: Vitamin A determination by HPLCNewDate of revision: September 2008 Prepared by:Approved by:Put into force by:Dennis EriksenDennis EriksenDennis Standard assay concentration determinationDilute ml of STD A with 2-propanol to a final volume of 100 the absorbances at 310, 325 and 334 nm according to SOP the assay Standard to HPLCD ilute ml of STD A with n-heptane to a final volume of 100 ChromatographyTransfer sample and standard to vials and inject 100 l of the solutions (double).


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