Method of Analysis for Folic Acid

1. Description

Yellow to yellowish orange, crystalline powder; odorless or almost odorless.

2. Solubility

Practically insoluble in cold water and in most organic solvents; very slightly soluble in boiling water; soluble in dilute acids and in alkaline solutions.

3. Identification

A. By UV

Reagent required
0.1M Sodium hydroxide solution
Sample solution: Dissolve 100 mg sample with 0.1 M Sodium hydroxide solution. And further dilute 1 ml of above solution with 100 ml with 0.1 M Sodium hydroxide solution.
Procedure: The light absorbance in the range between 230 nm to 380 nm
Three maxima at about 256 nm, 283 nm, and 365 nm; absorbance at about 256 nm, about 0.59,at about 283 nm, about 0.575 and at about 365 nm, about 0.206.

B. By thin layer chromatography

Stationary phase: Silica gel G
Mobile phase: 60 volume of Ethanol (95%), 20 volume of Strong ammonia solution and 20 volume of 1 Propanol.
Apply 2 ml of each of two solutions.
Diluents: 9 volumes of methanol and 2 volume of Strong ammonia solution.
Sample solution: Dissolve 50 mg sample 100 ml with diluents.
Standard solution: Dissolve 50 mg Folic acid WRS 100 ml with diluents.
After removal the plate, allow it to dry in air and examine under ultra-violet light (365 nm). The principle spot in the chromatogram obtained with sample solution corresponds to that in the chromatogram obtained with standard solution.

4. Specific optical rotation

Limit: About + 20°
Procedure: Dissolve 1.0 g of sample with 100 ml of 0.1 M Sodium hydroxide.and test using a calibrated polarimeter.

5. Free amines

The absorbance of the unreduced solution as determined in the assay is not more than one-sixth of the absorbance of the reduced solution.

6. Sulfated ash

Limit: Not more than 0.2%
Procedure: Heat a silica crucible to redness for 10 min., allow cooling in desiccators and weighing. Place about 1 g of accurately weighed substance being examined in the silica crucible, moisten with sulphuric acid, ignite gently, again moisten with sulphuric acid and ignite at about 800°C, cool, weigh again, ignite for 15 min. and repeat this procedure until two successive weighing do not differ by more than 0.5 mg.
Calculation
                                     W3 – W1
% Sulphated ash = --------------------- X 100
                                     W2 – W1
Where:
W1 = Weight of empty platinum crucible.
W2 = Weight of crucible + sample.
W3 = Weight of crucible + residue. (After ignition)

7. Water

Limit: Between 5.0 and 8.5% w/v
Reagent required
Anhydrous methanol AR
KF reagent Pyridine free single solution
Procedure: Take about 40 ml of anhydrous methanol in the titration vessel, neutralize with KF reagent and find out the factor of KF reagent in mg H2O/ 5 ml in triplicate and enter mean value.
Add accurately about 150 mg. of the sample to the titration vessel. Allow to titrate with KF reagent to the electrometric end point. Record the percentage of water content obtained by Karl Fischer titrator. Find out water content of the sample in triplicate and take mean value.

8. Assay

Limit: Not less than 95.0% and Not more than 102% calculate on anhydrous basis.
Reagent required
0.1M Sodium hydroxide
2.0M Hydrochloric acid
Zinc powder
0.1%w/v Sodium nitrite solution
0.5%w/v Ammonia sulphamate
1%w/v N- (1-naphthyl) ethylenediamine dihydrochloride solution
Solution (A): Dissolve accurately about 50 mg of sample in 100 ml volumetric flask with 0.1M Sodium hydroxide solution and dilute up to 50 ml with 0.1M Sodium hydroxide.
Solution (B): Dissolve accurately about 50 mg of Folic acid WRS in 100 ml volumetric flask with 0.1M Sodium hydroxide solution and dilute up to 50 ml with 0.1M Sodium hydroxide.
Sample preparation: To 3.0 ml of solution (A) add 20 ml of 2.0M Hydrochloric acid and dilute 100 ml with water. To 50 ml of sample preparation add 0.5 g Zinc powder, allow to stand protected from light for 20 minutes with frequent shaking and filter. Dilute 10 ml of the filtrate to 25 ml with water.
Standard preparation: To 3.0 ml of solution (B) add 20 ml of 2.0M Hydrochloric acid and dilute 100 ml with water. To 50 ml of standard preparation add 0.5 g Zinc powder, allow to stand protected from light for 20 minutes with frequent shaking and filter. Dilute 10 ml of the filtrate to 25 ml with water.
Blank preparation: Take 25 ml of water in place of sample.
Unreduced solution: Take 30 ml of solution (A) and solution (B) add 20 ml of 2M hydrochloric acid and sufficient water to produced 100 ml and mix. To 10 ml of the each of solution add 15 ml of water.
Procedure: To each of Sample, Standard, Blank preparation and Unreduced solution add 5 ml of 2M hydrochloric acid and 5 ml of a 0.1%w/v Sodium nitrite solution, mix and allow to stand for 2 minutes. Add 5 ml of 0.5%w/v Ammonia sulphamate, mix and allow to stand for 2 minutes. Add 5 ml of 1%w/v N- (1-naphthyl) ethylenediamine dihydrochloride solution mix and allow standing for 10 minutes. Add sufficient water to produced 50 ml with water. And measure the absorbance at about 550 nm.
Subtract one-tenth of the absorbance of the unreduced solution from that of the reduced solution and from the result calculate the amount of C19H19N7O6 on anhydrous basis.
Calculation
% Assay of Folic acid =
         Sample. Abs area x Wt. of std. x 5 x 50 x 50 x Purity x 100
= ------------------------------------------------------------------------------------
          Standard Area x.50 x 50 x Sample wt x 5 x (100- %LOD)