Standard Testing Procedure Methylcobalamin Niacinamide and Pyridoxine Injection

1.0 OBJECTIVE
1.1 To lay down a procedure for Analysis of Methylcobalamin Niacinamide and Pyridoxine Injection.

2.0 SCOPE
2.1 This procedure is applicable to the Analysis of Methylcobalamin, Niacinamide& Pyridoxine injection.

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 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 STP
11.0 Bacterial endotoxins: –
NMT 0.25 Endotoxins unit/mg of Ondansetron as per STP
12.0 Composition: –
Each 2ml contains:
Methylcobalamin I.P. 1000 mcg.
Pyridoxine Hydrochloride I.P. 100 mg.
Niacinamide I.P. 100 mg.
Benzyl Alcohol I.P. 2.0 % v/v
(As preservative)
13.0 Assay: – Limit: – NLT 90.0%
Method of Methylcobalamin: – by liquid chromatography.

Buffer: – 7.8 g of NaH2PO4 dilute in 1000ml water adjusted to pH 3.5 with Orthophoshoric Acid.

Mobile phase: Buffer: Acetonitrile
80: 20

Standard preparation: -Weight accurately about 55 mg Working standard of Methylcobalamin in 100 ml volumetric flask.

Further dilution 5.0 ml to 50 ml volumetric flask with water.

Sample preparation: -Take sample accurately 5 mg Methylcobalamin. diluted to 100 ml volumetric flask with water.

Chromatographic condition: –
Wavelength- 266 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 content of Methylcobalamin Respectively.

Retention time of the Principal peak is about 6.5 Minutes.

Calculation: –

Area of spl Wt. of std

…………… x …………… X 100X % Potency of Standard X 2

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.

14.0 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 & 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 & 9.0 Minutes Respectively.
Calculation of Niacinamide: –

Area of spl Wt. of std

…………… x …………… X 100X % Potency of Standard X 2

Area of std Wt. of spl

Calculation of Pyridoxine hydrochloride: –

Area of spl Wt. of std

…………… x …………… X 100X % Potency of Standard X 2

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.

15.0Assay: – Limit: -NLT 90.0 % and NMT 110.0%
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 100X % Potency of Standard X 2

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