Standard Stock Solution Preparation :
Dissolve 25 mg of USP Biotin RS, in 50% alcohol in 500 ml volumetric flask. Make the volume with 50% alcohol to 500 ml and mix. Each ml contains 50 mg of USP Biotin RS. Store this solution in a refrigerator.
Standard Preparation :
Prepare the dilutions of the standard solution on the day of assay. Dilute the stock standard solution of Biotin with sufficient water to give a final solution containing 0.1ng per ml.
1. 1 ml of Standard Stock Solution---------500 ml (100ng/ ml )---Soln.(a)
2. 1 ml of solution (a)------------------------- 1000 ml ( 0.1ng / ml )---Soln.(b)
Assay Preparation :
Take sample equivalent to 100 mg in a 200 ml of volumetric flask. Add 3 ml of 5% alcohol, shake and heat in a water bath at 60-70°C for 5 minutes. Sonicate for 5 minutes, dilute to volume with 5% alcohol. Mix well.
1. Take sample eq. to 100 mcg of Biotin ------------200 ml. (500ng/ml)---Soln(c)
2. 5..0 ml of Solution (c)--------250 ml. (10ng /ml)---Soln.(d)
3. 5.0 ml of solution (d)---------500 ml. (0.1ng/ml)---Soln.(e)
Acid-hydrolyzed casein solution :
Mix 100 g of vitamin-free casein with 500 ml of 6N hydrochloric acid, and reflux the mixture for 8 to 12 hours. Remove the hydrochloric acid from the mixture by distillation under the reduced pressure until a thick paste remains. Redissolve the resulting paste in water, adjust the solution with 1 N sodium hydroxide to a pH 3.5 ± 0.1, and add water to make 1000 ml. Add 20 g of activated charcoal, stir for 1 hour, and filter. Repeat the treatment with activated charcoal. Store under toluene in a refrigerator at a temperature not below 10°. Filter the solution if a precipitate forms during storage.Cystine-tryptophan solution :
Suspend 4 g of L-cysteine in 1.0 g of L-tryptophan ( or 2.0 g of D,L-tryptophan) in 700 to 800 ml of water, heat to 70° to 80°, and add dilute hydrochloric acid ( 1 in 2 ) dropwise, with stirring, until the solids are dissolved. Cool, and add water to make 1000 ml. Store under toluene in a refrigerator at a temperature not below 10°.Adenine-guanine-uracil solution :
Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 ml of 4 N hydrochloric acid, cool, and add water to make 200 ml. Store under toluene in a refrigerator.Polysorbate-80 solution :
Dissolve 25 g of polysorbate 80 in alcohol to make 250 ml.Calcium Pantothenate solution :
Prepare a solution of calcium Pantothenate in 50% alcohol containing 10 mcg per ml. Store in a refrigerator.Riboflavine-thiamine hydrochloride solution:
Prepare a solution of riboflavine and thiamine hydrochloride in 0.02 N acetic acid containing 20 mcg of riboflavin and 10 mcg of thiamine hydrochloride per ml. Store under toluene, protected from light, in a refrigerator.p-Aminobenzoic acid-niacin-pyridoxine hydrochloride solution :
Prepare a solution in a mixture of water and neutralized alcohol (3:1) containing 10 mcg of p- aminobenzoic acid, 50 mcg of niacin, and 40 mcg of pyridoxine hydrochloride per ml. Store in a refrigerator.
Salt solution 1: Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 ml. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Salt solution 2: Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 ml. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Basal Medium Stock Solution :
Sr.No.
|
Solution
|
Quantity
|
1
|
Acid-hydrolyzed casein solution
|
25 ml
|
2
|
Cystine–tryptophan solution
|
25 ml
|
3
|
Polysorbate 80 solution
|
0.25 ml
|
4
|
Dextrose, anhydrous
|
10 g
|
5
|
Sodium acetate, anhydrous
|
5 g
|
6
|
Adenine-guanine-uracil solution
|
5 ml
|
7
|
Calcium Pantothenate solution
|
5 ml
|
8
|
Riboflavine-thiamine hydrochloride solution
|
5 ml
|
9
|
p-Aminobenzoic acid-niacin-pyridoxine hydrochloride solution
|
5 ml
|
10
|
Salt solution 1
|
5 ml
|
11
|
Salt solution 2
|
5 ml
|
Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed, and adjust to a pH of 6.8 with 1 N sodium hydroxide. Dilute with water to 250 ml, and mix. (Difco Biotin Assay Medium can also be used as basal medium, Cat.no.241910, 100g. pack)
Preparation of Stock culture of Lactobacillus Plantarum (ATCC 8014.) :
Dissolve 2.0 g of yeast extract in 100 ml of water, add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Add 10 ml portion of the hot solution to the test tube, close or cover the tubes, sterilize in an autoclave at 121°, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum, ATCC No.8014 incubating for 16 to 24 hours at a temperature between 30° to 37° held constant to within ± 0.5°. Store in refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for inoculum if the culture is more than 1 week old.
Preparation of Culture Medium :
To each series of test tubes containing 5.0 ml of basal medium stock solution, add 5.0 ml of water containing 0.5 ng of biotin. Plug the tubes with cotton, sterilize in an autoclave at 121°, and cool.
Preparation of Inoculum :
[Note; A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an inoculum comparable to a fresh culture.] Make a transfer of cells from the stock culture of Lactobacillus plantarum to a sterile tube containing 10 ml of culture medium. Incubate this culture for 16 to 24 hours at a temperature between 30° and 37° held constant to within ± 0.5°. The cell suspension so obtained is the inoculum.Procedure :
To similar size test tubes add in duplicate 1, 2, 3, 4, and 5 ml respectively of the standard preparation. To each tube and to 4 similar tubes containing no standard preparation add 5.0 ml of Basal medium stock solution and sufficient water to make 10 ml. To similar test tubes add, in duplicate volumes of the assay preparation corresponding to 3 or more of the levels above for the standard preparation, including the levels of 2.0, 3.0, and 4.0 ml. To each tube add 5.0 ml of Basal medium stock solution and sufficient water to make 10 ml. Cap the all tubes to prevent contamination and heat in an autoclave at 121°C for 5 minutes. After autoclaving cool the tubes. Add one drop of inoculum to each tube except 2 of the 4 tubes containing no standard preparation (which serves as the uninoculated blanks), and mix. Incubate the tubes at a temperature between 30° to 37° held constant to within ± 0.5 for 16 to 24 hours. Determine the transmittance of the tubes as below: Mix the contents of each tube and measure the transmittance on UV spectrophotometer in between 540-660 nm wavelength. Read the transmittance when a steady state is reached. Allow approximately the same time interval for the reading on each tube. With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
Calculations :
Prepare a standard concentration-response curve as below:
For each level of standard, calculate the response from the sum of duplicate values of the transmittance as the difference,y=2.00-S (of transmittance). Plot this response on the ordinate of cross-section paper against the logarithm of the ml of standard preparation per tube on the abscissa, using for the ordinate either an arithmetic or logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points. Calculate the response, y, adding together the two transmittances for each level of the assay preparation in. Read from the standard curve the logarithm of the volume of the standard preparation corresponding to each of those values of y that falls within the range of the lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of volume, in ml, of the assay preparation to obtain the difference, x , for each dosage level. Average the values of x for each of three or more dosage levels to obtain x- =M, the log relative potency of the assay preparation. Determine the quantity, in mg, of USP Biotin RS corresponding to the Biotin in the portion of the sample taken for assay as antilog:
M= antilog (M` + log R ),
Where R is the number of mg of biotin that was assumed to be present in each mg of the material taken for assay.
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