by For Vitamin A (As palmitate) content:
Transfer a sample equivalent to about 2500 IU of vitamin A in a round bottom flask, add 40 ml methanol and 40 ml water, 3 ml of potassium hydroxide solution (50%) and 10 ml of glycerol then reflux on water bath for 30 minutes. Cool and transfer the content into separating funnel and extract with 4 x 60 ml of diethyl ether. Wash the combined ether layer with 4 x 40 ml water and evaporate the ether extract on a water bath. Dissolve the residue in 100 ml isopropyl alcohol and transfer quantitatively to 250 ml volumetric flask and makeup to volume.
Measure the absorbance at 325nm and calculate the Vitamin A taking 1830 as the value of A (1%, 1cm.) at 325 nm.
For Cholecalciferol (Vitamin D3) content:
Standard Preparation: Prepare 0.5 mcg/ml of Vitamin D3 WS solution in methanol (90%).
Test Preparation: Prepare test solution in methanol (90%) to get concentration of vitamin D3 0.5 mcg/ml.
Temperature: Ambient
Chromatographic Conditions:
1. Mobile phase: Water + Methanol as (3:97).
2. Flow Rate: 1 ml/minute.
3. Column: Hypersil – 5 ODS (10cm × 4.6mm) (C18).
4. Volume Injected: 20 µl
5. Type of Detector: UV-264 nm
For Thiamine Hydrochloride (Vitamin B1) content:
Reagents: 1. 0.1% w/v solution p-amino phenol in ethyl alcohol.
2. 2% v/v ammonium hydroxide solution in water.
3. 0.02M potassium permagnet solution in water.
Standard Preparation: Prepare 20 mcg/ml solution of Thiamine Hydrochloride WS, in water and filter.
Test Solution: Weigh accurately the sample of the syrup equivalent to about 2.0 mg of Thiamine Hydrochloride dissolve with 50 ml to 60 ml water and neutralise by potassium permanganate solution (0.02 M) and make up to 100ml with water and filter (20mcg/ml).
Procedure: To 5 ml each of sample and standard solution, add 0.5 ml P-aminophenol and 5 ml of dilute ammonium hydroxide solution and stand for 10 minutes then extract the coloured complex with 10 ml of chloroform. Withdraw the chloroform layer and filter. Read the absorbance of sample and standard solution at about 430 nm against chloroform blank.
For Vitamin B2 (Riboflavin Sodium Phosphate) content:
Standard Preparation: Weigh accurately equivalent about 100 mg of Riboflavin Sodium Phosphate WS in to a 100 ml volumetric flask and dissolve in 50 ml 1M Sodium Hydroxide, after addition of 5 ml glacial acetic acid solution make up to volume with 1M Sodium Hydroxide. Transfer 2 ml of the solution into 200 ml volumetric flask add 5 ml 1M NaOH and 0.9 ml of glacial acetic acid and dilute to 200 ml with water and filter (10 mcg/ml).
Test Preparation: Weigh accurately sample of the syrup equivalent to about 1.0 mg of Riboflavin into a 100 ml volumetric flask, after addition 5 ml 1M Sodium Hydroxide and 0.5 ml of glacial acetic acid dilute to volume with water and filter (10 mcg/ml).
Read the absorbance of both the standard and test solution at about 444 nm using water as a blank.
For Pyridoxine Hydrochloride (Vitamin B6) content:
Reagents: 1. 4% w/v Boric acid in water.
2. 0.4% w/v chlorimide in isopropyl alcohol.
3. Ammonia-ammonium chloride buffer: in a 100 ml of volumetric flask take 16 g of ammonium chloride and dissolve in 70 ml of water, add 16 ml of strong ammonia (30%) w/v) dilute to 100 ml with water.
Standard solution: Take about 50.0 mg of Vitamin B6 working standard in 100 ml volumetric flask, dissolve in water and make up the volume to 100 ml with water. Dilute further 2 ml to 100 ml with water and filter.
Test solution: Weigh accurately sample of the syrup equivalent to 1.0 ml of Vitamin B6 in 100 ml volumetric flask dissolve with 50 ml water shake for 5 minutes and make up the volume to 100 ml with water, filter
Colour development (in test tube)
Standard Standard blank Test Test Blank
(in ml) (in ml) (in ml) (in ml)
Standard solution 2 2 x x
Test solution x x 2 2
Isopropyl alcohol 5 5 5 5
Water 1 x 1 x
Buffer 2 2 2 2
Boric acid x 1 x 1
Colour development: With chlorimide reagent – Add 1 ml chlorimide reagent rapidly and shake vigorously for 30 seconds accurately timed (after addition of reagent) determine the absorbance at 650 nm using water as blank. Similarly take the other reagent also.
Note: it is better to check the time for maximum intensity.
The reading may also be taken exactly after 60 seconds.
For Dexpanthenol content:
Standard Preparation: Dissolve an accurately weighed about 50 mg of DexpanthenolWS in to a 100 ml volumetric flask, add 50 ml of water and Sonicate for 5 minutes, after that make up the volume to 100 ml with water and filter.
Take 2 ml of the filtrate into a 50 ml volumetric flask and make up the volume to 50 ml with water.
Test Preparation: Weigh accurately sample of the syrup equivalent about 1 mg of Dexpanthenolin to a 50 ml volumetric flask, add 25 ml of water and Sonicate for 5-10 minutes, after that make up the volume to 50 ml with water and filter.
Chromatographic conditions:
(a) A stainless steel column C08 (25 cm × 4.6 mm) (5 µm).
(b) Use the mobile phase described below.
(c) A flow rate is 1.0 ml per minute.
(d) Column temperature: Ambient.
(e) A detection wavelength is 210 nm.
(f) Inject volume: 20 µl.
Preparation of buffer solution: Dissolve about 6.8 g of potassium dihydrogen phosphate in to a 1000 ml flask with water and adjust the pH to 3.5 ± 0.5 with orthophosphoric acid.
Mobile phase preparation: A mixture of 5 ml methanol and 995 ml of buffer solution.
Procedure: Separately inject (about 20 µl) five replicate injections for standard preparation and inject two replicate injections for test preparation and calculate the results by comparison, record the chromatograms and measure the responses for the major peaks.
For Nicotinamide content:
Reagents: 1.Cyanogen Bromide Solution 10% w/v.
2. Aniline Solution 1% v/v in Methanol.
Standard Preparation: Prepared Nicotinamide WS solution in water to get concentration of 25 mcg/ml of Nicotinamide.
Test Preparation: Weigh syrup equivalent to about 5.0 mg of Nicotinamide in a 250 volumetric flask and dilute with water to volume and filter (25 mcg/ml).
Procedure:
Pipette into four marked tubes to quantities of the appropriate standard and the assay preparations as indicated in the table. Add other constituents, respectively, as listed in table, according to the directions given herein.
CONSTITUENTS TUBE 1 TUBE 2 TUBE 3 TUBE 4
ml ml ml ml
Standard Preparation 2 2 – –
Test Preparation – – 2 2
Cyanogen Bromide 5 5 5 5
Solution Water – 1 – 1
Aniline Solution 1 – 1 –
Read the absorbance at about 430nm using water as a blank.
Weigh sample of the syrup equivalent to about 100.0 mg of Vitamin C in a conical flask add 10 ml of dilute H2SO4 and 50ml water and titrate with 0.05M iodine solution VS using starch solution as indicator until a violet blue colour is obtained. Each ml of 0.05M iodine VS is equivalent to 8.806mg of C6H8O6
