Analysis method of Ferrous Ascorbate Cyanocobalamin and Folic Acid Suspension


Analysis method of  Ferrous Ascorbate Cyanocobalamin and Folic Acid Suspension

1.1 To lay down a procedure for analysis of Ferrous Ascorbate, Cyanocobalamin and Folic Acid Suspension.

2.1 This procedure is applicable to the analysis of Ferrous Ascorbate, Cyanocobalamin& Folic Acid Suspension in Quality control

3.1 Q.C- Chemist

4.1 Manager-Quality Assurance


5.1 Description: Pour 50 ml finish sample in beaker and observed visually.

5.2 pH: Taken 50 ml sample in beaker rinse the pH electrode first with purified water followed by sample dip the electrode in sample and observed the pH.

5.3 Volume variation: Measured the volume by measuring cylinder and determine the volume variation.

5.4 Identification:
5.4.1 In the assay, the principle peak in the chromatogram obtain with the test solution correspond to the peak in the chromatogram obtained with the reference solution. and by chemical method.
5.5 ASSAY:
Each 5ml contains:
Ferrous Ascorbate Elemental Iron. 30mg.
Folic Acid I.P 0.5mg.
Cyanocobalamin I.P 2.5mcg.

Method of Ferrous Ascorbate with Folic Acid Suspension.
Estimation of Ferrous Ascorbate Iron.
Method of iron:
Weigh accurately equivalent to 50 mg of iron and 5 ml water. add 3ml sulphuric acid. heat and cool. Add potassium permanganate solution till. Change the colour pale yellow or disappear colour add 2 gm potassium iodide. After that 25 ml Hcl stay on 5 to 10 minute. Add starch solution as indicator. Titrate with 0.1 M sodium Thiosulphate.
Factor-5.85 mg
V= Volume (in ml) of Sodium Thiosulphate. Solution consumed in titration.

N = Actual Normality of Sodium Thiosulphate Solution.

AW= Average weight

Wt. = Weight of Sample.




Estimation of Folic Acid.

Buffer: 680 mg potassium dihydrogen orthophosphate dissolve in 100 ml water. Adjust to ph-6.0 with
1N sodium hydroxide.

Mobile phase: Buffer : methanol
90 : 10

Standard preparation: weigh accurately 50 mg of folic acid in 100 ml volumetric with 0.1N Sodium hydroxide. Further dilution 1.0ml to 50 ml with mobile phase.

Sample preparation: weight accurately equivalent to 1.0mg of sample weight add 10 ml of 0.1 N Sodium hydroxide shake for 5 minute. And diluted to 100 ml volumetric flask
With mobile phase.

Chromatographic condition:
Wavelength -280 nm
Column -C18 (250 x4.6) mm
Flow -1.2ml per minute.
Injection volume -10 micro liter.
Retention time about 15 mint.
Temperature – Ambient


Estimation of Cyanocobalamin (Vitamin B12)
CULTURE: E. coli mutant NCIM 2567 as a test organism. The culture is maintained by sub-culturing. It on a slant of E. coli maintenance Agar medium, once a week.

1. B12 Culture agar (E. coli maintenance medium)-Hi-media M-185
2. B12 Assay agar using (E. coli maintenance medium)-Hi-media M-110.

Weigh accurately about 100mg Vitamin B12 reference standard and dissolve in sufficient water to produce 100ml.
This solution may be preserved in a refrigerator for one year.

Dilute 2 ml of the stock solution to 100ml with water. I.e., 20mcg/ml dilution. This stock solution can be preserved for one month in a refrigerator.

This stock standard is allowed to come to room temperature on the day of the assay.
0.5ml of stock standard is further diluted to 100ml with water to get 100 Nanograms vitamin b12.
One ml of the 100 Nanograms solution is diluted to 4ml to get 25 Nanograms per ml. use 25 Nanograms per ml and 100 Nanograms per ml in assay.

Prepare the Sample suitably diluted with water to produce 25 Nanograms per ml and 100 Nanograms per ml of vitamin b12., filter the solution if necessary.

To 18-24 hours’ culture of E. coli mutant, add 10ml of sterile water or normal saline to make suspension. B12 assay agar is weighed and autoclaved for 15min at 15 pounds, cool to 45-degree cent. Add 1.5 ml to 2ml of culture suspension to 100ml of assay agar and mix well. Add 20 to 25ml of this agar is poured in to sterile Petri dishes of size 100mm in diameter. Allow to solidify. Make four cups is agar at proper distance to avoid overlapping of zones of inhibition. Sterilized borer of 5mm to 10mm in diameter is used to bore cups. Add 0.05 or 0.1 ml of standard solution of 100 Nanograms per ml (SH) and 25 Nanograms per ml (SL) dilutions to each agar cup.
Similarity add 0.05 or 0.1ml of test solution of 100 Nanograms per ml (TH) and 25 Nanograms per ml (TL) dilution to each agar cup labeled as test high and test low.

Cover the Petri dishes, invert and incubate them at 320C to 350C for 18 to 24 hours.

Measure the zones and calculate vitamin b12 content accordingly to the following formula:
%Assay = Antilog (2± a Log I)
Where: a= ( TH + TL) – ( SH + SL)
(TH- TL) + (SH – SL)

I = Ratio of dilution i.e. 10/25

TH= Zone of test sample with sample 100 Nanograms per ml

Tl = Zone of test sample with sample 25 Nanograms per ml

SH= Zone of standard with sample 100 Nanograms per ml

SL= Zone of standard with sample 25 Nanograms per ml



6.0 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


Sr. No. Reference Title
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