Method of Analysis for Riboflavin

1. Description

Yellow to orange-yellow, crystalline powder.

2. Solubility

Very slightly soluble in water, more soluble in saline solution than in water, practically insoluble in ethanol (95%), chloroform and in the ether.

3. Identification

A. The IR absorption spectrum of the sample is concordant with the Riboflavine reference spectrum or with the spectrum obtained from Riboflavin WRS.
B. Dissolve about 1.0 mg sample with 100 ml water. The solution has a pale greenish yellow color by transmitted light and an intense yellowish green fluorescence by reflected light, which disappears on the addition of mineral acids or alkalis.

4. pH

Limit: Between 5.5 and 7.2
Procedure: Use saturated solution and filter.

5. Light absorbance

Limit: Maxima at 223, 267, 373 & 444nm.
The ratio between 373 nm to that at about 267 nm: 0.31 and 0.33
The ratio between 444 nm to that at about 267 nm: 0.36 and 0.39
Reagent required
2M Sodium hydroxide
Glacial acetic acid
1.4% w/v Sodium acetate solution
Procedure: Weigh accurately about 65 mg sample in 500 ml amber glass volumetric flask, and add 5 ml of water and dissolve in 5 ml of 2 M Sodium hydroxide.
As soon as dissolution is complete add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. To 20 ml of this solution add 3.5 ml of 1.4% w/v Sodium acetate solution and dilute with 200 ml of water. Take 50 ml of above solution in 100 ml of volumetric flask and dilute up to 100 ml with water.
Measure the light absorbance in the range 210 nm to 460 nm of the resulting solution exhibits maxima at about 223, 263, 373 and 444 nm.

6. Specific optical rotation

Limit: Between –115° and –135°
Reagent required
0.05M Sodium hydroxide
Procedure: Dissolve 500 mg sample with 100 ml 0.05 M Sodium hydroxide and measure the angle of rotation within 30 minutes of preparing the solution.

7. Lumiflavin

Limit: Solution is not more intensely colored than reference solution BYS6
Procedure: Weigh accurately 25 mg sample and shake with 10 ml of chloroform for 5 minutes.
To each of the Nessler cylinders, add 10 ml of freshly prepared hydrogen sulfide solution, mix and dilute to 50 ml with water. Allow to stand for 5 min. and view downwards over a white surface. The color produced with test solution is not more intense than produced with the standard solution.

8. Sulfated ash

Limit: Not more than 0.1%
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)

9. Loss on Drying

Limit: Not more than 1.5% of its weight.
Procedure: Weigh 1.000 g of substance in a clean and dried pre-weighed LOD Bottle. Cover the stopper and gently shake to distribute material to not more than 10 MM height. Place the LOD Bottle in the oven and remove the cover and leave it also inside the oven. Dry the sample at 105° C for 2 hr. On opening the chamber, immediately close the LOD Bottle, transfer it to desiccators and bring it to room temperature. Weigh up to constant weight.
Calculation
                                   W2 – W1
% Loss on Drying = --------------- X 100
                                   W2 – W3
Where,
W1 = Weight of empty clean and dried LOD Bottle.
W2 = Weight of LOD Bottle + sample.
W3 = Weight of LOD Bottle + sample. (After drying)

10. Assay

Limit: 98.0 to 101.0% on dried basis.
Procedure: Carry out the procedure in subdued light. Weigh accurately about 65mg and transfer to an amber-glass 500 ml volumetric flask, suspend in 5 ml of water, ensuring that it is completely wetted. Dissolve in 5 ml of 2M sodium hydroxide. As soon as dissolution is complete add 100ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. To 20.0 ml of this solution add 3.5 ml of a 1.4%w/v solution of sodium acetate and dilute to 200.0ml with water. Measure the absorbance of the resulting solution at the maximum at about 444 nm. Calculate the content of C₁₇H₂₀N₄O₆ taking 328 as the value of A (1%, 1 cm) at the maximum at about 444 nm.