Multivitamin Tablets contain the following Active Ingredients:
Ascorbic Acid BP 15mg
Cholecalciferol BP 300IU
Niacinamide BP 7.5mg
Vitamin-A Acetate BP 2500IU
Riboflavin BP 0.5mg
Thiamine Mononitrate BP 1mg
Assay of Thiamine Mononitrate, Riboflavin, Niacinamide, Ascorbic acid
Chromatographic Conditions:
Column : 150 mm × 4.6 mm, C18, 5 µm
Flow Rate : 1 mL/minute
Column Temperature : 30°C
Detection Wavelength : 280 nm
Injection Volume : 20 µL
Mobile Phase:
Water : Methanol : Acetonitrile : Glacial Acetic Acid ( 75 : 16.5 : 7 : 0.8, v/v) containing 140 mg of Sodium Hexane Sulphonate per 100 ml.
Diluent:
Water: Acetonitrile: Glacial Acetic Acid (94 : 5 : 1, v/v) containing 140 mg of Sodium Hexane Sulphonate per 100 ml.
Standard Preparation:
Weigh accurately 20mg of Thiamine Mononitrate working standard, 10mg of Riboflavin working standard, 150mg Niacinamide working standard and 300mg of Ascorbic acid working standard & transferred in to 100ml volumetric flask, add 80 ml diluent & mix well. Immerse the volumetric flask in a hot water bath maintained at 65°C for 10 minutes with regular shaking until the material is dissolved. Chill rapidly & dilute 100 ml with diluent & mix. Dilute 3ml of this solution with 25 ml of mobile phase.
Sample Preparation:
Weigh & powder 50 tablets, weigh tablet powder equivalent to 20mg of Thiamine Mononitrate & transferred in to 100ml volumetric flask, add 80 ml diluent & mix well. Immerse the volumetric flask in a hot water bath maintained at 65°C for about 20 minutes with regular shaking until the material is dissolved. Chill rapidly and dilute with diluent to volume & mix. Filter with 0.45 µ filter. Dilute 3ml of this solution with 25 ml of mobile phase.
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Procedure:
Inject standard preparation in five replicate & record the chromatogram. Inject sample preparation in duplicate, record the chromatogram. The relative standard deviation for replicate injections is not more than 3%.
Calculation:
Where,
At = Average Area of Sample solution
As = Average Area of Standard solution
Ws = Weight of Standard
Wt= Weight of Sample
V = Volume of Medium
P = Potency of Standard
L = Label Claim
Assay of Cholecalciferol:
Chromatographic Conditions:
Column : 150 mm × 4.6 mm, C18, 5 µm
Flow Rate : 1.5 mL/minute
Column Temperature : 30°C
Detection Wavelength : 266 nm
Injection Volume : 20 µL
Mobile Phase : Methanol: Acetonitrile : Water (60 : 35 : 5, v/v)
Standard Preparation I:
Weigh accurately about 50mg of Cholecalciferol working standard (1mg = 40000IU) to a 50 ml volumetric flask, dissolve in and dilute to volume with Methanol. Mix well.
Standard Preparation II:
Dilute 1ml of standard I in to 100 ml with methanol (400 IU/mL)
Standard Preparation III:
Dilute 5ml of standard II in to 50 ml with methanol (40 IU/mL)
Sample Preparation:
Accurately weigh Tablet powder equivalent to 400IU of Cholecalciferol to 10 mL of volumetric flask. Add 7mL of methanol sonicate for 10 minutes and make up the volume to 10 mL with methanol so as to obtain about 40 IU/ml of cholecalciferol (Vitamin D3). Filter the standard and test preparation through 0.45 µm filter.
Procedure:
Inject standard preparation in five replicate & record the chromatogram. Inject sample preparation in duplicate, record the chromatogram. The relative standard deviation for replicate injections is not more than 2%.
Calculation:
Where,
At = Average Area of Sample solution
As = Average Area of Standard solution
Ws = Weight of Standard
Wt= Weight of Sample
ATW = Average Tablet Weight
Assay of Vitamin A Acetate (Retinol):
Weigh & transfer 700mg of tablet powder into a 250ml round bottom flask. Add 50ml of ethyl alcohol and 5ml of 50% w/v Potassium hydroxide solution and 50mg of hydroquinone reflux the content in a water bath for one hour in oxygen-free nitrogen. Cool, transfer the contents quantitatively into a 250ml separator, wash the flask with 25ml of water and transfer the washing into the above 250ml separator.
Extract with 5 × 40 ml of solvent ether, pooling the solvent ether into another 500ml separator. Wash the combined solvent ether extract with water till the ether layer is almost colorless. Pass the solvent ether through sodium sulfate anhydrous. Collect the solvent ether into a 250ml volumetric flask. Dilute to volume with solvent ether; mix well.
Pipette 5ml of the above solution into a dry 50 ml volumetric flask; and dilute to volume with isopropanol. Mix well. Measure the absorbance of the solution at 325 nm using blank solution as reference.
Blank solution: 5 ml solvent ether → 50ml with Isopropyl alcohol
Calculation:
Where,
At = Absorbance of Sample solution
Wt= Weight of Sample in g
ATW = Average Tablet Weight
NOTE: Analysts have to perform Method Validation as per Regulatory Guidelines.
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