Oil- and Water-Soluble Vitamins with Minerals Oral Solution Oil- and ...

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of water and 20 mL of dehydrated alcohol. Add 150.0 mL of solvent hexane, insert the stopper, and shake for 1 minute. Add another 150 mL of solvent hexane, insert the stopper, shake, and allow the layers to separate. Discard the aqueous layer. Drain the solvent hexane extract through anhydrous sodium sulfate into a 500-mL round-bottom flask. Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65 . Immediately add 10.0 mL of Diluting solution, swirl to dissolve the residue, and filter. Chromatographic system— Proceed as directed for Chromatographic system in the Assay for vitamin A. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 5.0%. Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak heights for vitamin D. Calculate the quantity, in µg, of cholecalciferol (C 27 H 44 O) or of ergocalciferol (C 28 H 44 O) in each mL of the Oral Solution taken by the formula: 1.09(10C/V)(r U / r S ) in which 1.09 is a correction factor to account for the average amount of previtamin D in the Oral Solution; C is the concentration, in µg per mL, of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard preparation; V is the volume, in mL, of Oral Solution taken for the Assay preparation; and rU and rS are the vitamin D peak heights obtained from the Assay preparation and the Standard preparation, respectively. Assay for vitamin E— [note—Use low-actinic glassware throughout this procedure.] Diluting solution— Prepare a mixture of acetonitrile and ethyl acetate (1:1). Potassium hydroxide solution— Transfer 90 g of potassium hydroxide pellets to a 100-mL volumetric flask containing about 60 mL of water. Mix to dissolve, cool, and dilute with water to volume. Mobile phase— Prepare a filtered and degassed mixture of methanol, acetonitrile, and n-hexane (46.5:46.5:7.0). Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Tocopherol RS in Diluting solution, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.3 mg per mL. Assay preparation— Transfer an accurately measured volume of Oral Solution, equivalent to about 1.5 mg of alpha tocopherol, to a 125-mL conical flask fitted with a ground-glass joint, and add 25.0 mL of dehydrated alcohol. Attach a reflux condenser, and reflux in a boiling water bath for 1 minute. Cautiously add 3 mL of Potassium hydroxide solution through the condenser, and continue to reflux for 30 minutes. Remove the flask from the bath, and rinse the condenser with about 15 mL of water. Cool, and transfer with a minimum volume of water to a 250-mL separatory funnel. Rinse the flask with 50 mL of n-hexane, and add the rinsings to the separatory funnel. Insert the stopper, shake vigorously for 1 minute, and allow the layers to separate. Drain the aqueous layer into a second 250-mL separatory funnel, and repeat the extraction with 50 mL of n-hexane. Discard the aqueous layer, and combine the hexane extracts. Wash the combined extracts with 25 mL of water, allow the layers to separate, and discard the aqueous layer. Add 3 drops of glacial acetic acid, and repeat the washing procedure two more times. Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mL round-bottom flask. Rinse the funnel and sodium sulfate with n-hexane, and add the rinsing to the hexane solution in the flask. Evaporate the hexane solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 50 . Immediately add 5.0 mL of Diluting solution, and swirl to dissolve the residue. Chromatographic system— The liquid chromatograph is equipped with a 291-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at about 40 , and the flow rate is about 3 mL per minute. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the relative standard deviation for replicate injections is not more than 5%. Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak heights. Calculate the quantity, in mg, of alpha tocopherol (C 29 H 50 O 2 ) in each mL of the Oral Solution taken by the formula:
Page 1 and 2: Oil- and Water-Soluble Vitamins wit
Page 3: into a 500-mL round-bottom flask. E
Page 7 and 8: Assay preparation— Transfer an ac
Page 9 and 10: Standard preparation— Dissolve an
Page 11 and 12: Diluting solution, Mobile phase, an
Page 13 and 14: Transfer 10.0 mL of this solution t
Page 15 and 16: Standard stock solution— Dissolve
Page 17 and 18: the concentration, in µg per mL, o
Page 19 and 20: Standard preparations— Quantitati
Page 21: volume, and mix to obtain the solut

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