2A.

  Assay for Total Calcium contents : (In-house Specification)

Chemicals/Reagent            : Sodium Hydroxide 

: Calcon Carboxylic Acid Triturate.
: Disodium Edetate                        
: distilled water
Procedure:

Weigh 1.8 g of content of a Sachet and transfer to a 500 ml conical

flask and dilute up to 300 ml with distilled water. Add 6ml of 10 M
NaOH and about 10 mg of Calcon Carboxylic Acid triturate. Titrate with
0.1M Di-Sodium Edetate VS until the colour changes from violet to blue.
Each ml of 0.1M EDTA is equivalent to 4.008 mg of Calcium.     

Calculation:-
                                    Consumed Vol. of EDTA x Factor of EDTA x 4.008 x 10. 5
mg of total calcium / sachet  =  -----------------------------------------------------------------------------
                                                      Weight of sample in gm

                                                            mg of total calcium / sachet 

% Assay =  ------------------------------------------------ x 100
                                        Label Claim

2B.   Assay for Ascorbic acid: (In-house Specification)

Chemicals/Reagent          : HCl 1% v/v.

: Starch Mucilage
: boiled distilled water).
: Iodine solution
: K-iodide solution
: Distilled water             

Procedure:

Transfer a quantity of powder equivalent to 100 mg of ascorbic acid in

a 250 ml of conical flask. Add 50 ml of distilled water and 20 ml of
dilute HCl. Mix well to dissolve the contents.
Titrate the resulting solution with 0.1 N Iodine VS, using starch
mucilage as indicator. 
Each ml of 0.1 N Iodine solution consumed is equivalent to 0.00881 g of
Ascorbic acid.

Calculation :

              Consumed Vol. of Iodine x Factor of Iodine x 8.81 x 10. 5

mg of Ascorbic acid / sachet  =  -----------------------------------------------------------------------------
                                                          Weight of sample in gm

                                                         mg of Ascorbic acid / sachet 

% Assay =  ------------------------------------------------ x 100
                                        Label Claim

2C.  Assay for Folic Acid: -  (In-house Specification)

Method of analysis  : Microbiological (By Plates diffusion Method) 

Apparatus :

 Petri Plate 
 Bulb and Graduated Pipettes 
 Volumetric Flask 
 No.5 (8mm) Cork Borer
 Micro Pipettes or Syringes (Disposable)
 Incubator (get at 37°C) 
 Water bath (50°C) 
 Vernier Caliper
 Bench Centrifuge
 Level Detector  
 Wire Loop

Standard Preparation:

Test Organism:

 Streptococcus faecium ATCCR 8043 (formerly Streptococcus faecalis

is    ATCC.8043 is used in the microbiological assay of Folic Acid.

Stock Culture :  

 Lactobacilli Agar is used for carrying stock culture of

streptococcus faecalis is ATCC.8043.      

Preparation of Inoculum :

Using 20 hours subculture of Streptococcus faecal is ATCC 8043 on

Lactobacilli Agar plates and incubate at 37°C for 24hrs. After the
incubation period wash the surface growth of three plates with 50ml
sterile saline in conical flask and store in refrigerator for one week.

Assay Medium for Folic Acid:

Prepare Folic assay medium according to manufacturer’s instructions.

Add 1.5 gm of agar agar in 100 ml and heat to boiling 2-3 minutes.
Agitate to disperse the slight precipitate that form and sterilize for
10 minutes at 15 Ibs /inch2 & 121°C.

Preparation of plates:-

 Melt the folic assay medium in water bath. Cool to about 40-45°C &
add 1 ml Inoculum of test organism of Streptococcus fecal is
ATCC.8043 and mix well.
 Pour 40ml of assay medium in each petri plate allow solidifying for
half to one hour.
  After the solidifying make the wells in solid agar with sterile
borer No.5 (8mm diameter). After 30 minutes fill the two well with
0.1ml sample and two wells with standard solution (High & Law).
 Incubate the plate in upright position for 18-24 hours at 35-37°C.   

Potassium Phosphate Buffer preparation (pH 8.00):

Weigh accurately 8.15 g of dibasic potassium phosphate and 0.372g of
monobasic potassium phosphate. Dissolve both in 1000 ml of distilled
water. Adjust pH to 8.00 with 0.01 N NaOH or 0.01 N hydrochloric acid.
Preparation of sample and Standard Solution:

Stock Standard Solution: (100 mcg / ml solution).

Accurately weigh 10 mg of 100 % pure folic acid USP reference standard

is dissolved in buffer 100ml of volumetric flask. (100 mcg/ml
solution).
Store stock solution in dark less than 10-15°C.  

Working Standard : 

Take 0.5ml from 110mcg/ml solution in 100ml volumetric flask make up

the volume with buffer (0.50 mcg/ml solution) high concentration. Take
25ml from (0.50mcg/ml solution) in 50ml volumetric flask and make up
the volume with buffer (0.25 mcg/ml solution) low concentration.

Sample Preparation : 

 Weigh the accurately 10.5gms of sample in 100 ml volumetric flask

add 30 ml buffer solution. dissolve the Sample and make up the
volume with buffer up to mark (10mcg/ml solution)         
 Take the 5ml of (10 mcg/ml solution) in 100ml volumetric flask, make
up the volume with buffer up to mark (0.5mcg/ml solution) high
concentration.
 Take 25ml of (0.5mcg/ml solution) in 50 ml volumetric flask make up
the volume with buffer up to the mark (0.25mcg/ml solution) low
concentration.

Measuring of the zone:-   

 After incubation measure the zone of exhibition with the Vernier
Caliper or Zone reader from the backside of the petri plate on the
dark shaded paper.

Calculation :

        Add the reading of the 4 concentrations and calculate the potency as

follows:
a = (Sample High + Sample low) - (Standard High + Standard low)
            (Sample High - Sample low) + (Standard High - Standard low)

I= Concentration of high dilution

           Concentration of low dilution

Log I = % Potency = Antilog + (a x log I)

  
Content =  % x claimed content   
          100

2D.  Assay for Vitamin B : - (In-house Specification)

12

There are two methods of analysis of Vitamin B   

12

Method - I : Method of Analysis: Microbiological (By Agar plate

diffusion method)

Apparatus :

1. 04 Pettri plates 
2. Bulb and graduated pipettes
3. Volumetric Flasks
4. No.5 Cork borer
5. Micro Pippette or syringes (disposable)
6. Incubator (get at 37C)
7. Water bath (50 C)
8. Vernier Caliper
9. Bench Centrifuge
10. Level Detector
11. Autoclave apparatus
12. Wire loop

All glassware must be thoroughly cleaned and rinsed with distilled

water and dried in a drying oven.    Plates and pipettes are dry heat
sterilized before use but aseptic precautions are unnecessary with this
method of assay.

Standard :
 
Cyanocobalamin (Vitamin B12) pure 100 % 

Culture : Escherichia.coli NC1B 9270, ATCC 14169

          

Media : 

Bactomicro Inoculum Broth (Difco) 

Prepare Bactomicro Inoculum Broth (Difco) in distilled water then
distribute 10 ml in test tube & autoclave at 15 Ibs/sq inch for 10
minutes.

Peptone --------------------------------------------------- 0.5gm

Yeast extrat----------------------------------------------- 2.0gm
Glucose --------------------------------------------------- 1.0gm
KH2 PO4---------------------------------------------------- 6.2gm
Tween 80--------------------------------------------------- 0.01gm

In 100 ml distilled water and autoclave it.

Bactomicro Assay Culture Agar (Difco)

Prepare Bactomicro Assay culture agar (Difco) in distilled water then

distribute 10 ml in test tube and autoclave at 15 Ibs/sq inch for 10
minutes and allow to set as slopes.

E. Coli Vitamin B Assay Agar

12

Prepare the medium in 3 parts A, B & C

Solution ‘A’ :

Part-I :

14 gm K2HPO4 Dipotassium hydrogen phosphate 6gms K H2PO4 Potassium

Dihydrogen phosphate dissolve in a small amount of distilled water.

Part-II :
 
i.               1.0gm Trisodium Citrate Dihydrate
ii.              100mg Sodium Chloride (NaCl)
iii.             200mg MgSO4 7HO2O 
iv.              2.0gm Ammonium Sulphate
  
Mix the above parts of the two solutions & make up to 1.0 litre with
distilled water. Add 3.0gm of Asparagine (0.3 % w/v ) & adjust to pH
7.0 distribute this medium in 150 ml amounts in bottles &   sterilize
by autoclave at 15 Ibs/sq. inch for 15 minutes.

Solution ‘B’: 

4.5gm Agar, Agar in 120ml distilled water, distribute this medium in

500 ml flask & autoclave at 15 Ibs/ sq. inch for 15 minutes.

Solution ‘C’: 

21% W/V solution of D-Glucose 

First sterilize the 100 ml distilled water in autoclave at 15 Ibs/ sq.
inch for 15 minutes after sterilization, cool it at room temperature &
then add 21 gm D-Glucose aseptically and store in the refrigerator.

Buffer Solution (Acetate Buffer 0.02 M, PH 4.6) :

 
1. Glacial Acetic Acid ---------------------------------------- 1.2 
gms
2. Sodium Acetate 3H2O ---------------------------------------- 2.72
gms
3. Saline solution (0.85 % - 0.90%) --------------------------- 1000 ml

Procedure:

Preparation of Culture :
 
Maintain working stock culture of E coli (NCIB 9270) on medium (2) as a
slope culture: making monthly subculture from a 20 hours Micro Inoculum
broth culture. Incubate the slope for 12-16 hours at 37C.
 Preparation of Inoculum :

Using a 20 hours subculture of E.coli (NCIB 9270 ATCC14169), which has

been prepared one day prior to experiment), inoculate loopful to micro
inoculum broth and incubate at 37C for 20 hours (1 x 10ml broth per
large plate assay). Mix and centrifuge the 20 hrs broth culture. Wash
twice with saline and re-suspend in 10 ml saline. Use the suspension
from one tube to inoculate large assay plate. 

Preparation of Plates: -
Place the assay medium solution A & B in a water bath at 50C. Mix
solution A 150 ml of solution B 120 ml of solution & 25 ml of solution
‘C’. Add 5 ml of wash E.coli Suspension to this mixture. Mix thoroughly
and pour 40 ml into a assay plate, allow to set and dry the surface for
upto 1/2 to one hour in a warm air stream or on room atmosphere.
Preparation of wells & its filling. 
Cut wells in the agar using No.5 (8mm) cork borer. Fill the wells with
0.1 ml of standard of test solutions using the same pipettes. Incubate
the assay plate for 16-24 hours.
Preparation of Solution and Standard:

Stock Standard Solution :

Dissolve 25mg crystalline Cyanocobalamin (100% pure) in 25ml methanol

in 100 ml volumetric flask, make up the volume with distilled water
(250 mcg/ml sol.) This is the stock solutions store it in the
refrigerator.
Working standard solution:-
 Take 0.4 ml of 250 mcg. Solution of vitamin B in 100ml volumetric
12

flask, make up the volume with distilled water. (1 mcg/ml Sol.)

 Take 5ml of 1mcg. / ml sol. In 100ml volumetric flask and make up
with acetate buffer. (0.05mcg./ml sol.) high concentration.
 Take 25ml of 0.05mcg./ml solution in 50ml volumetric flask and make
up with acetate buffer (0.025mcg/ml sol.) Low concentration.

Sample Solution :
 
Sample is diluted with Acetate buffer to the 0.05mcg./ml sol. (high
concentration) and 0.025 mcg/ml sol. (Low concentration) by the
following method.
 Weigh the accurately 10.5gms of sample in 100ml volumetric flask add
30ml buffer dissolve the Sample and make up the volume with buffer
up to mark (2.5mcg/ml solution).         
 Take the 1ml of (2.5mcg/ml solution) in 50ml volumetric flask, make
up the volume with Buffer up to mark (0.05mcg/ml solution) high
concentration.
 Take 25ml of (0.05mcg/ml solution) in 50 ml volumetric flask make up
the volume with buffer up to the mark (0.025mcg/ml solution) low
concentration.

Calculation: -

Add the reading of the 4 concentrations and calculate the potency as

follows:
 a =   (Sample High + Sample low) - (Standard High + Standard low)
      (Sample High - Sample low) + (Standard High - Standard low)

 
Spl. H is high concentration for sample
Spl. L is low concentration for sample.
Std. H is High concentration for standard
Std. L is low concentration for standard.

I= Concentration of high dilution

    Concentration of low dilution

% Potency = Antilog + (a x log I)

 

Content =     % x claimed content   

                  100

Method –II : (In-house Specification)

Method of Analysis : (By Agar plate diffusion method)

Apparatus :
 
 1) Petri plates 
2) Bulb and graduated pipettes
3) Volumetric Flasks
4) No.5 Cork borer
5) Micro Pipette or syringes (disposable)
6) Incubator (get at 37C)
7) Water bath (50 C)
8) Vernier Caliper
9) Bench Centrifuge
10) Level Detector
11) Autoclave apparatus
12) Wire Loop

All glassware must be thoroughly cleaned and rinsed with distilled water and
dried in a drying oven. Plates and pipettes are dry heat sterilized before
use but aseptic precautions are unnecessary with this method of assay.

Standard: -  Cyanocobalamin (Vitamin B12) pure 100 %

            
Culture: - Escherichia. coli M200
                             
E.coli Vitamin B Assay Agar
12

Prepare the medium in two parts A, B

Solution “A”: 

1) Agar Agar        ---------------------------------------- 5.5gms

2) D-glucose        ---------------------------------------- 0.66gms
3) Distilled water  ---------------------------------------- 200 ml

Solution "B" :
1) Potassium dihydrogen Phosphate.    ---------------------- 6gms 
2) DiPotassium hydrogen Phosphate.    ---------------------- 14gms 
3) Magnessium Sulphate Pentahydrate.  ---------------------- 200mg
4) Ammonium Sulphate.                 ---------------------- 2gms
5) Sodium Chloride                    ---------------------- 100mg
6) Sodium Citrate.                    ---------------------- 1gm
7) 2% 2,3,5 triphenyl tetrazolium Chloride. ---------------- 20ml
8) Distilled water ----------------------------------------- 1 litre.

 Note- 2% of 2,3,5 triphenyl tetrazolium Chloride prepare in Methanol.

Mix above Chemicals in distilled water and make up to 1 litre. Distribute
this medium in 130 ml amounts in conical flask and sterilize solution" A"
and "B" by autoclave at 121C 15lbs/sq. inch for 20 minutes.

Preparation of Inoculum: -
Using 20 hours old subculture of E. coli M200 on TSA plates and incubate at
37C for 24 hrs. After the incubation period wash the surface growth of three
plates with 200 ml of sterile saline in conical flask and store in
refrigerator for one month.

Preparation of plates:-
Place the assay medium solution" A" and "B" at 50C mix solution "A" 200ml
and solution "B"130ml. Add the 1.5ml of wash E.coli M200 inoculum to this
mixture. Mix thoroughly and pour the 40 to 45ml in to assay plates, allow to
plates solidify for half to one hour make the wells in agar medium using
sterile cork borer No-5 (8mm). Fill the wells with 0.1ml standard and
sample, low and high using the sterile pipettes.
           
Incubation:- 
Incubate the assay plates at 37°C for over night (16 hrs). Read zone
diameters and calculate the results accordingly.

Buffer Solution_:

1. Sodium Phosphate dibasic -----------------------------12.9gms

2. Citric Acid (Anhydrous) ----------------------------- 11.6gms
3. Sodium Metabisulphite ------------------------------- 10gms
4. Distilled water ------------------------------------- 1 liter

Dissolve the above chemicals in 1 liter of the distilled water.

Preparation of Solution and Standard:

Stock Standard Solution:

 
Dissolve 25mg crystalline Cyanocobalamin (100% pure) in 25ml methanol in 100
ml volumetric flask, make up the volume with   distilled water (250 mcg/ml
sol.) This is the stock solutions store it in the refrigerator after the
assay.
Working Standard: -
Take 0.4 ml of 250 mcg/ml Solution of vitamin B in 100ml volumetric flask,
12

make up the Volume with Buffer (1 mcg./ml Solution).

Take 5ml of 1mcg./ml solution in 100ml volumetric flask and make up with
buffer.  (0.05mcg./ml solution) high concentration.
 Take 25ml of (0.05mcg./ml solution) in 50ml volumetric flask and make up
with buffer. (0.025mcg/ml sol.) Low concentration.

Sample Solution :
 
Sample is diluted with buffer to the (0.05mcg./ml solution) high
concentration, and (0.025 mcg/ml solution).low concentration by the
following method.
 Weigh the accurately 10.5gms of sample in 100ml volumetric flask add 30ml
buffer 
   dissolve the sample and make up the volume with buffer up to mark (2.5mcg/ml
solution).         
 Take the 1ml of (2.5mcg/ml solution) in 50ml volumetric flask, make up
the volume 
   with buffer up to mark (0.05mcg/ml solution) high concentration.
 Take 25ml of (0.05mcg/ml solution) in 50 ml volumetric flask make up the
volume with buffer up to the mark (0.025mcg/ml solution) low
concentration.       

Calculation: -
After incubation period measure the zone of exhibition with Vernier Caliper
or Zone reader. Add the reading of the 4 concentrations and calculate the
potency as follows:
     
     a = (Sample High + Sample low) - (Standard High + Standard low)
         (Sample High - Sample low) + (Standard High - Standard low)

Where:-  
Spl. H is high concentration for sample
Spl. L is low concentration for sample.
Std. H is High concentration for standard.
Std. L is low concentration for standard.

I= Concentration of high dilution

    Concentration of low dilution

% Potency = Antilog + (a x log I)

 
Content =  % x claimed content   
        100
6A.   Assay For Ferric Ammonium Citrate : (In-house Specification)

Reagents : HCl(Conc.)
: KI (Solid)
: Sodium Thiosulphate (0.1N)
: Starch as indicator.
Procedure:-

1. Transfer 10 ml (By weight) of sample in a 250 ml glass-Stoppered

flask. 
2. Dissolve in 25 ml of distilled water and shake well.
3. Add 5 ml of HCl (Conc.) and 4 gm of potassium iodide.
4. Insert the stopper in the flask & allow standing in the dark for 15
minutes.
5. Add 100 ml of water.
 6. Titrate the librated iodine with 0.1 N Sodium thiosulphate, adding
3 ml of starch TS as the end point is approached.
7. Perform a blank determination & make necessary correction.
8. Each ml of 0.1 N Sodium Thiosulphate is equivalent to 5.585
of Fe.
 
Calculation :
                    Consumed Vol. of titrant x Factor of titrant x 5.585 x Sp. gravity x 100
mg of Fe / 100 ml  =  ------------------------------------------------------------------------------------------
                                                              Weight of sample in gm

                                                   mg of Fe / 100 ml 

% Assay =  --------------------------- x 100
                           Label Claim

6B. Assay for Thiamine HCl, Pyridoxine HCl, Nicotinamide & Folic acid By
HPLC Method :   (In-house  Specification):

REAGENTS:

Water Distilled water  

Methanol             HPLC Grade
1-hexasulphonic acid               Reagent Grade
Acetic acid                     Reagent Grade

A. Acetic Acid Solution (0.5%).

Transfer 5 ml acetic acid in 1000 ml volumetric flask. Make the volume

upto 1000 ml with distilled water.

Mobile Phase.

Dissolve 1.4 gm of 1-hexasulphonic acid in 730 ml of water & 10 ml of

acetic acid solution. Add 270 ml of methanol. Mix well and filter with
0.45 micron filter and degas.

Apparatus:

HPLC Pump Agilent 1100 Series

Degasser Agilent 1100 Series
Auto Sampler       Agilent 1100 Series
Column oven       Agilent 1100 Series            
Or Equivalent
Detector Agilent 1100 Series
Reporting System       Agilent 1100 Series
Reversed Phase, Stainless steel column, (Bondapak C , 5)      
18

4.6 mm ID x 250 mm

Injection Volume:       20 l
Flow Rate 1.0 ml/minute
Wavelength 280 nm
Temperature                          40C
 System Suitability:

1. Inject five replicate injections of the standard solution and

calculate the percent RSD (NMT 2%).
2. Or Set the operating condition and purge the mobile phase
until a steady base line is obtained.
3. Inject working standard solution of Thiamine HCl, Pyridoxine
HCl & Nicotinamide onto the column and obtain the
chromatogram.
4. From Chromatogram, calculate the tailing factor/or resolution
factor if required (Tailing factor: Ideally 1, or the value
that does not effect the precision).

Standard Preparation :

Transfer 20 mg of Thiamine HCl, 40 mg of Pyridoxine HCl, 50 mg of

Nicotinamide & 10 mg of Folic acid in each 100 ml volumetric flask
(In Folic acid flask add 1N NaOH to dissolve the contents). Add 50
ml of 0.5% acetic acid in each flask to dissolve the contents. Make
the volume up to the mark in all three flasks with the same
solvent. 
Transfer 5.0 ml of Thiamine HCl, 5.0 ml of Pyridoxine HCl, 20.0 ml
of Nicotinamide & 5.0 ml of Folic acid in 50 ml volumetric flask and
mix the following quantities of the above solutions. Dilute to 50 ml
with 0.5% acetic acid. Filter through 0.45 syringe filter & degas.

Sample Preparation :

1. Transfer 5 ml syrup in 50 ml volumetric flask.

2. Add 25 ml of 0.5% Acetic acid & mix to dissolve.
3. Make the volume with 0.5% Acetic Acid solution.
4. Filter in a test tube and then degas. Inject 20µl of sample (In
duplicate)

HPLC Procedure:

1. Flush the column with water at a flow rate of 1.0 ml for at

least 30   minutes.
2. Set the conditions outlined under parameters.
3. Equilibrate the column with mobile phase until a steady
baseline is    obtained.
4. After the column is equilibrated, inject the standard and
sample   solutions.
5.    At the end of the analysis, again flush the column with
mobile phase    for 20 minutes followed by water for 30
minutes and then with methanol for additional 30 minutes. 
 
Calculation:

a. For Thiamine HCl :

                                                          Spl. Peak Area    Std wt. (mg)      5                             50             
      mg of Thiamine HCl / 100ml =  ------------------ x ---------------- x ---- x %Purity x -------------- x Sp. Gravity x
100 
                                                     Std. Peak Area          100             50                       Spl wt. (g)             
                       
                       mg of Thiamine HCl / 100ml
% Assay =  ----------------------------------------- x 100
                                    Label Claim
b. For Pyridoxine HCl :

                                                             Spl. Peak Area    Std  wt. (mg)      5                            

50             
      mg of Pyridoxine HCl / 100ml =  ------------------ x ---------------- x ---- x %Purity x -------------- x Sp. Gravity
x 100 
                                                      Std. Peak Area           100             50                       Spl  wt. (g)             
                       
                       mg of Pyridoxine HCl / 100ml
% Assay  =  ----------------------------------------- x 100
                                    Label Claim

c. For Nicotinamide :
                                                          Spl. Peak Area    Std  wt. (mg)     20                            
50             
      mg of Nicotinamide / 100ml =  ------------------ x ---------------- x ---- x %Purity x -------------- x Sp. Gravity x
100 
                                                   Std. Peak Area           100             50                       Spl  wt. (g)             
                       
                       mg of Nicotinamide / 100ml
% Assay =  ----------------------------------------- x 100
                                    Label Claim

d. For Folic Acid :

                                                     Spl. Peak Area    Std  wt. (mg)      5                             50             
      mg of Folic Acid / 100ml =  ------------------- x ---------------- x ---- x %Purity x -------------- x Sp. Gravity x
100 
                                              Std. Peak Area           100             50                       Spl  wt. (g)             
                       
                       mg of Folic Acid / 100ml
% Assay =  ----------------------------------------- x 100
                                    Label Claim

6C.  Alternative method for assay for Folic Acid :(In-house Specification)

Method of analysis: - Microbiological (By Plates diffusion

Method) 

Limits: - Not less than 90 % not more than 110% of

label claim.

Apparatus: -

 Petri Plates 
 Bulb and Graduated Pipettes 
 Volumetric Flask 
 No.5 (8mm) Cork Borer
 Micro Pipettes or Syringes (Disposable)
 Incubator (get at 37°C) 
 Water bath (50°C) 
  Vernier Caliper
 Hot air oven
 Match box  
 Wire Loop.

Standard Preparation :-   (Folic Acid)

Test Organism: -
 Streptococcus faecalis ATCC. 8043 is used in the Microbiological
assay of Folic Acid.

Stock Culture:
 
 Lactobaccilli agar is used for carrying stock culture of
streptococcus faecalis ATCC.8043. 

Preparation of Media:- 

Prepare the 50ml of Lactobaccilli agar according to the manufacturer’s

instructions in conical flask close the mouth of the flask with cotton
plug or aluminum foil. Boil media using water bath or micro wave oven
until a clear solution forms. Autoclave the media at 121°C with
pressure 15lbs/inch for 15 minutes. After sterilization of media
2

maintained the temperature of media at 45ºC-50ºC and pour the media in

sterile Petri plates under laminar air flow hood. After the solidifying
the plates take a stock culture of Streptococcus faecalis ATCC. 8043
and streak the culture on Lactobaccilli agar plates in a aseptic
condition under laminar air flow hood. Incubate those plates in
incubator at 35-37°C for 24 hours.
  
Preparation of Inoculum:-

Using 24 hours subculture of Streptococcus faecalis ATCC 8043 on

Lactobacilli Agar plates.  After the incubation period wash the surface
growth of three plates with 50ml sterile saline in conical flask and
store in refrigerator for one week.

Folic Acid Assay Medium: - 

Prepare Folic assay medium according to manufacturer’s instructions.

Add 1.5 gm of agar agar in 100ml and heat to boiling 2-3 minutes.

 Agitate to disperse the slight precipitate that form and sterilize

for 10 minutes at 15 Ibs /inch2 (121°C).
Preparation of plates:-

 Cool down the folic acid medium to about 40-45°C after the
autoclaving & add 1ml Inoculum of test organism of Streptococcus
fecalis ATCC 8043 and mix well.
 Pour 40ml of assay medium in each petri plate allow solidifying
for half to one hour.
 After the solidifying make the wells in solid agar with sterile
borer No.5 (8mm diameter). After 30 minutes fill the two well with
0.1ml sample and two wells with standard solution (High & Law).
 Incubate the plate in upright position for 18-24 hours at 35-37°C.
 Potassium Phosphate Buffer preparation (pH 8.00):
 Weigh accurately 8.15g of dibasic potassium phosphate and 0.372g
of monobasic potassium phosphate.
 Dissolve both in 1000 ml of distilled water.
 Adjust pH to 8.00 with 5N NaOH or 5N hydrochloric acid.

Preparation of Solution and Standard:

Stock Standard Solution: (100 mcg / ml solution).

 Accurately weigh 10 mg of 100 % pure folic acid USP reference

standard is dissolved in buffer 100 ml of volumetric flask. (100
mcg/ml solution).
 Store stock solution in dark less than 10-15°C.  

Working Standard:- 

   Take 0.5 ml from 100 mcg/ml solution in 100 ml volumetric flask make up
the volume with buffer (0.50 mcg/ml solution) high concentration. 
Take 25ml from (0.50 mcg/ml solution) in 50 ml volumetric flask and
make up the volume with buffer (0.25 mcg/ml solution) low
concentration.

Working Sample:- 

   Sample is diluted with phosphate buffer pH 8.00 to the 0.50 mcg/ml

solution (high concentration) and 0.25 mcg/ml solution (low
concentration) by the following method.

 Take 0.5 ml of Viviron plus syrup in 100 ml volumetric flask.

 Add 50 ml phosphate buffer, shake for 10 minutes 
 After shaking, make up the volume with buffer to the mark. (0.50
mcg/ml sol.) high concentration.
 Take 25ml of (0.50 mcg/ml solution) in 50 ml volumetric flask make
up the volume with buffer up to the mark (0.25 mcg/ml solution)
low concentration.
Measuring of the zone:-
  
 After incubation measure the zone of exhibition with the Vernier
Caliper or Zone reader from the backside of the petri plate on the
dark shaded paper.

Calculation: -

   Calculate the potency of Folic acid as follows:

a = (Sample High + Sample low) - (Standard High + Standard low)

    (Sample High - Sample low) + (Standard High - Standard low)

I= Concentration of high dilution

    Concentration of low dilution

Log I =  
% Potency = Antilog + (a x log I)

Content =  % Potency x claimed content   

              100