Determine by microbiological method.
Test organism: E. Coli M200 (N.C.I.B)
Culture Maintenance medium: Himedia B12 Culture agar (M 185)
Reconstitute the dehydrated media’s as per the manufacturer’s instruction. Adjust the H. Boil to melt the agar. Mix thoroughly and dispense the medium in 15 – 18ml amounts in screw cap bottles. Sterilise at 15 p.s.i. At 121°C for 15 minutes. Allow the medium to set in an inclined position to give the maximum surface area. Incubate at 30°C – 35°C for 48 hours to check for sterility. Label and store in the fridge.
Seed inoculum of E. coli:
Using a bacteriological loop subculture E.coli onto fresh culture medium. Incubate at 37°C± 0.5°C for 24 hours. Harvest the growth with 5ml of sterile saline and shake well to obtain a uniform suspension.
Vitamin B12 assay agar M 110 (Himedia) is used. Medium to be prepared as directed on B12 assay agar bottle.
Preparation of assay plates
Reconstitute and sterilised the dehydrated media’s as per the manufacturer’s instructions. Allow to cool to about 45°C. Add 2ml of culture suspension per 100ml of assay medium and mix thoroughly but gently to avoid air bubbles. Distribute 25ml of the inoculated medium to each of the required number of sterile petri dishes. Allow to set and transfer to the refrigerator until use. Remove the plate 30 minutes before use and bore 6 cups of 8mm diameter with a sterile borer.
B12 Extraction Buffer: (To be prepared as 10 X)
Diabasic potassium phosphate K2HPO4 : 158 gms
Citric acid C6H8O7H2O : 120gms
Distilled water ; 1000ml
Dissolve the ingredients separately and mix well. Adjust pH to 4.6. Dispense in 500ml bottles. Sterilise by autoclaving at 121°C for 20 minutes. Incubate at 37°C for 48hours. After checking the sterility, label the bottles and store in cold room.
At the time of use dilute 100ml of 1000ml with distilled water. Add 2.5gms of sodium buffer for extraction of B12 from test sample and standard solution.
Preparation of stock standard solution
Weigh accurately 50mg of Cyanocobalamin AWS in a 100ml volumetric flask. Add 24% v/v of ethyl alcohol and make up the volume with distilled water (500mcg/ml). Transfer to clean glass bottles and keep in refrigerator. Label details of potency, date of preparation and use before date. This solution should be used within 30 days. Calculate the factor by dividing the actual strength obtained by the theoretical strength.
Working Standard Solution
On the day of the assay, prepare fresh working standard solution by diluting the primary standard solution using distilled water to the plating levels of 0.05 mcg/ml (SH) and 0.0125 mcg/ml (SL).
50mg --> 100ml (500 mcg/ml)
10ml -->500ml (10mcg/ml)
5ml --> 1000ml (0.05 mcg/ml)...........(SH)
2.5ml--> 10ml (0.0125 mcg/ml).......(SL)
Preparation of Sample solution
Weigh accurately about 1.0gm of sample into a 150ml conical flask and add 50ml of extraction buffer. Cover the flask with aluminium foil and autoclave at 15 p.s.i. at 121°C for 15 minutes. Cool and flask pH of the solution to 7.0 with 20% w/v sodium hydroxide. Transfer quantitatively to a 100ml volumetric flask and dilute to volume with distilled water (10 mcg/ml). Dilute 5ml of 1000ml with distilled water to give test high level solution (0.05mcg/ml). Further dilute 2.5ml to 10ml with distilled water to give test low-level solution (0.0125 mcg/ml.
1.0g --> 100ml (10mcg/ml)
5ml --> 1000ml (0.05mcg/ml)............(SH)
2.5 --> 10ml (0.0125 mcg/ml).......(SL)
Plating out solutions
Make 6 cups equidistant to each other in the plates and add equal amount (100Micro Liter) of standard as well as test solutions (High and low level) in the respective cups such that each level of standard and sample are plated into three cups. Maintain the plates at room temperature for one hour and then incubate at 37°C for 16-18 hours.
Measurement of zones
Measure the diameter of the zone of exhibition with the help of Vernier calipers.
Sum the diameters of the zones for each dilution and calculate the assay percent potency using the following formula.
Percent potency (%) = Antilog (2 ± a log i)
i = Ratio of high dose to low dose (10:1)
a =(TH + TL) – (SH + SL) / (TH-TL) + (SH – SL)
SH – Sum of standard high doses
SL – Sum of standard low dose
TH – Sum of test high dose
TL – Sum of test low dose
(Value of “a” may have a positive or negative value and should be used algebraically)
Calculate the Vitamin B12 content as mcg per g.
Mcg / g = % Pot. X Std wt (mg) X Std. Pot. (as is basis) / Sample wt. X
Fiducial limit: Fiducial limits of error must be between 95-105%
Validity of assay: If the determined potency is less than 50% or more than 150% of the standard, the assay is invalid and should be repeated using higher or lower dilution as the case may be.
Record the appearance of the tablets, paying attention to the presence of surface defects, visible impurities, discoloration and odour.
Weight 20 tablets together and determine the average weight as follows:
Av. Wt. = Weight of 20 tablets / 20
Uniformity of weight
Weigh 20 tablets individually selected at random and calculate the average weight. Not more than 2 of the individual tablets deviate from the average weight by more than 5% and none deviates by more than 10%.
Apparatus: The apparatus used for Disintegration test should comply with IP 1996 specifications. Reference IP 1996 Vol II,
Method : Ensure the temperature of water int he beaker is maintained at 37± 2°C. assembly in the beaker containing water at 37°C. Run the apparatus till all the tablets disintegrate and note the time taken using a stop watch.
If 1 or 2 tablets fail to disintegrate, repeat the test on 12 additional tablets. Not less than 16 of the total of 18 tablets tested disintegrate.
Loss on drying at 60°C under vacuum for 3 hours
Powder the tablets and determine the moisture content on 1 g of the sample.