Analysis method of Suspension of Tribasic Calcium phosphate with vitamin D3 and Vitamin B12


Analysis method of Suspension of Tribasic Calcium phosphate with vitamin D3 and Vitamin B12


1.1 To lay down a procedure for analysis of Tribasic Calcium phosphate, vitamin D3 and VitaminB12 Suspension.


2.1 This procedure is applicable to the analysis of Tribasic Calcium phosphate, vitamin D3 & Vitamin B12 Suspension quality control laboratories.

3.1 Q.C- Chemist

4.1 Manager-Quality Assurance

5.1 Description: Pour 50ml 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.
5.5 ASSAY:
Each 5.0 ml contains:
Tribasic Calcium phosphate I.P. 210 mg.
Eq. to elemental calcium 82 mg.
Vitamin D3(Cholecalciferol) I.P 200IU
Vitamin B12 2.5 mcg.

Estimation of Tribasic Calcium phosphate, vitamin D3 & Vitamin B12
Method of Tribasic Calcium phosphate.
Weight Accurately equivalent to 1.0gm. sample weight dissolve in 10 ml of hydrochloric acid. Heat on water bath add 50 ml of water, cool and dilute to 250 ml with water. To 25 ml of the resulting solution add 30 ml of 0.05 m disodium edetate 10 ml of ammonia buffer pH. 10.9 and 100 ml of water and titrate the excess of disodium edetate with 0.05 m zinc chloride carry out a blank titration using mordant black II indicator
1 ml of 0.05 M Disodium edetate is equivalent to 0.00517 gm of Ca3(po4)2

V= Volume (in ml) of Zinc Chloride Solution consumed in titration.

N = Actual normality of Zinc Chloride Solution.

AW= Average weight.

Wt. = Weight of Sample.




Method of Cholecalciferol (vitamin D3)


Diluted solvent: Methanol

90: 10
Standard preparation: Weight accurately about 25 mg std wt. of vitamin D3 in 100 ml volumetric
Flask. Further dilution 1.0 ml to 250 ml volumetric flask with methanol.

Sample preparation: Weight accurately Equivalent to 0.1 mg of vitamin D3 and diluted to100 ml with methanol.

Chromatographic condition:

Wavelength -290nm
Flow rate – 1.0 ml /mint.
Column -c18 (150 x4.6) 5µm.
Temp – 350c
Retention time – about 5.70 mint
Injection volume – 100µl.


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



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 I.P. Indian Pharmacopeia
6.0 NM Nano meter



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