Analysis of vitamin B1, B6, B2 Nicotinamide and sodium pentothenate Injection

1.0 OBJECTIVE
1.1 To lay down a procedure for Analysis of vitamin B1, B6, B2 Nicotinamide and sodium pentothenate Injection.

2.0 SCOPE
2.1 This procedure is applicable to the Analysis of vitamin B1, B6, B2 Nicotinamide & sodium pentothenate Injection. in QC lab
.

3.0 RESPONSIBILITY
3.1 Q.C- Chemist

4.0 ACCOUNTABILITY
4.1 Head-Quality Assurance

5.0 Identification: – In the assay the principal peak in the chromatogram obtained with the test solution

corresponds to the peak in the chromatogram obtained with the reference solution.
6.0 Description: – Pour 10ml finish sample in Beaker and observed Visually.

7.0 pH: – Taken 40ml sample in beaker the pH. electrode first with purified water followed by sample

dip the electrode in sample and observe the pH.

8.0 Extractable volume: – Measured the volume by 10 ml Measuring cylinder and determine the volume.

9.0 Particulate matter: – Injections that are solutions, when examine under suitable conditions

of visibility are clear and practically free from particles that can be observed on visual inspection by the unaided eye.
10.0Sterility: Injection comply with the test for sterility as per SOP
11.0Bacterial endotoxins: – Injection comply with the test Endotoxins unit/mg of as per SOP

12.0 Composition: –
Each 2ml contains:
Thiamin Hydrochloride I.P. 20 mg.
Riboflavin phosphate I.P. 2.74 mg.
Pyridoxine Hydrochloride I.P. 2 mg.
Nicotinamide I.P. 30 mg.
Sodium pentothenate I.P. 1.0 mg.
Benzyl Alcohol I.P. 2.0 % v/v
(As preservative)

13.0 Assay: – Limit: – NLT 90.0 % and NMT 110.0%
Method of pyridoxine Hydrochloride and Niacinamide: – by liquid chromatography.
Diluent: – 50ml Acetonitrile: 10ml Glacial Acetic acid: 940ml water.
Mobile Phase: – Weigh 1.4 gm 1-Hexane sulphonate add 270ml Methanol, 10ml Glacial Acetic acid, 730ml water

and dilute to 1000ml with water.

Standard preparation: –Weight accurately about 100mg Working standard of Niacinamide and 100 mg

Working standard of Pyridoxine Hydrochloride diluted to 100 ml with Diluent. Further

dilution 5.0 ml to 50 ml volumetric flask with Diluent.

Sample preparation: – Take sample equivalent to 100 mg. Niacinamide & 100 mg. of

Pyridoxine Hydrochloride diluted to 100 ml volumetric flask with Diluent. Further dilution 5.0 ml to 50 ml

volumetric flask with Diluent.

Chromatographic condition: –
Wavelength- 280 nm
Flow rate- 1.0 ml /mint.
Column c18 (250 x4.6) bonded to porous silica 5µm.
Injection Volume: 10µl
Record the Chromatogram and measure the peak response of the measure peaks. Calculate the contents of Niacinamide & Pyridoxine hydrochloride Respectively. Retention time of the Niacinamide & Pyridoxine hydrochloride Principal peak is about 5.0 and 9.0 Minutes Respectively.

Calculation of Niacinamide: –

Area of spl Wt. of std
…………… x …………… X 100X % Potency of Standard X 1
Area of std Wt. of spl

Calculation of Pyridoxine hydrochloride: –

Area of spl Wt. of std
…………… x …………… X 100 X % Potency of Standard X 1
Area of std Wt. of spl

Procedure: – Inject Blank in single injection, Standard preparation in Replicates of three test preparation in duplicate injection.

System suitability: – % RSD of three replicate injections should not be more than 2.0.

Method of Thiamine Hydrochloride: –

Assay: – Limit: – NLT 90.0 % and NMT 110.0%

Mobile Phase: -Dissolve 1 gm. of Sodium heptanesulphonate in a mixture of 180 ml of methanol and 10 ml

of triethylamine diluting to 1000ml with water adjusting to pH 3.2 with Orthophoshoric Acid.

Standard preparation: -Weight accurately about 100 mg Working standard of Thiamine hydrochloride in

100 ml volumetric flask with 0.1 M hydrochloric acid. Further dilution 5.0 ml to 100 ml volumetric flask with water.

Sample preparation: –Take sample accurately equivalent to 100 mg of thiamine hydrochloride in 100 ml

volumetric 0.1 M hydrochloric acid. Further dilution 5.0 ml to 100 ml volumetric flask with water.

Chromatographic condition: –
Wavelength- 244 nm
Flow rate- 2.0 ml /mint.
column C18 (100 x4.6) bonded to porous silica 5µm.
Injection Volume: 20µl
Record the Chromatogram and measure the peak response of the measure peaks. Calculate the content of Thiamine Hydrochloride Respectively.

Method of Thiamine Hydrochloride: –

Area of spl Wt. of std
…………… x …………… X 100 X % Potency of Standard X 1
Area of std Wt. of spl

Method of Vitamin B2:

Assay: – Limit: – NLT 90.0 % and NMT 110.0%

Reagent required:
(a) Hydrochloric Acid

Standard Preparation: Weigh accurately 30mg of Vitamin B2 WS and transfer to 100ml volumetric flask, add 10ml hydrochloric acid, shake to dissolve then make up to volume with water. Dilute 5ml of this solution and add 8 ml Hcl then make up to volume 50ml with water.

Sample Preparation: Taken equivalent to 3mg of vitamin B2 in 100ml volumetric flask & add 10ml Hcl, shake to dissolve then make up to 100ml with water, filter. Measure the absorbance at 444nm of standard and sample against water as a reference blank

Calculation

Area of spl Wt. of std
…………… x …………… X 100 X % Potency of Standard X 1
Area of std Wt. of spl

Method of Benzyl Alcohol: –

Buffer: – 28.4 g of Sodium sulphate dilute in 1000ml water adjusted to pH 2.3 with Orthophoshoric Acid.

Mobile phase: – Buffer: Acetonitrile

55 : 45

Standard preparation: –Weight accurately 500 mg Working standard of Benzyl Alcohol in 100 ml volumetric flask with water. Further dilution 1 ml to 100 ml volumetric flask with water. (concentration 50 ppm)

Sample preparation: – Take sample accurately equivalent to500 mg Benzyl Alcohol Working standard diluted to 100 ml volumetric flask with water. Further dilution 1 ml to 100 ml volumetric flask with water. (concentration 50 ppm)

Chromatographic condition: –

Wavelength: – 214 nm

Flow rate: – 1.0 ml /mint.

Column c18 (250 x4.6) bonded to porous silica 5µm.

Injection Volume: – 20µl

Column temp. 40⁰C

Run time: – 30.0 minutes.

Record the Chromatogram and measure the peak response of the measure peaks. Calculate the content of Benzyl Alcohol Respectively.

Calculation: –

Area of spl Wt. of std

………………… X……………………. X 100 X % Potency of Standard x 1

Area of std Wt. of spl

Procedure: – Inject Blank in single injection, Standard preparation in Replicates of three test preparation in duplicate injection.

System suitability: – % RSD of three replicate injections should not be more than 2.0.

16.0 ABBREVIATION

Sr. No.	Abbreviation used	Full form of abbreviation used
1.0	STP	Standard Testing Procedure
2.0	QA	Quality assurance
3.0	STD	Standard
4.0	SPL	Sample
5.0	NM	Nano Meter