by ASSAY METHOD VALIDATION PROTOCOL FOR CALCIUM & VITAMIN D3 TABLETS
| Superseded Protocol No. | Nil |
| Effective Date |
TABLE OF CONTENTS:
| Sr. No. | Subject | Page No. |
| (A) CALCIUM CONTENT BY TITRAMETRIC | ||
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility | ||
| Product profile | ||
| Methodology | ||
| Precision | ||
| Accuracy | ||
| Range & Linearity | ||
| . | Robustness | |
| (B) VITAMIN D3 CONTENT BY HPLC | ||
| Methodology | ||
| Validation parameter (By HPLC) | ||
| Specificity | ||
| Precision | ||
| Force Degradation | ||
| Linearity | ||
| Accuracy | ||
| Range | ||
| Stability of Analytical solution | ||
| Filter paper Selection Study | ||
| Robustness | ||
| Incident/Deviation | ||
| Summary | ||
| Revision History |
- PROTOCOL APPROVAL:
Prepared By:
| Functional Area | Name | Designation | Signature/ Date |
| Quality Control |
Reviewed By:
| Functional Area | Name | Designation | Signature/Date |
| Quality Assurance | |||
| Head Quality Control |
Approved By:
| Functional Area | Name | Designation | Signature/Date |
| Head QA |
Authorized By:
| Functional Area | Name | Designation | Signature/Date |
| Head Quality |
- Objective:
The objective of this validation is to provide documentary evidence that analytical methodology used in the determination of Assay content of Calcium Content and Vitamin D3 in (Calcium & Vitamin D3 Tablets) will yield consistent and reliable results within the predetermined acceptance criteria.
Analytical method validation will be performed by considering thefollowing parameters:
| Parameters | Calcium Content | Vitamin D3 |
| Specificity | – | yes |
| Precision | ||
| System Precision | – | yes |
| Method Precision | yes | yes |
| Intermediate Precision (Ruggedness) | yes | yes |
| Forced Degradation | – | yes |
| Linearity | yes | yes |
| Accuracy | yes | yes |
| Range | yes | yes |
| Stability of Analytical Solution | – | yes |
| Filter Paper Selection Study | – | yes |
| Robustness | yes | yes |
- Scope :
The scope of this protocol is based on SOP and applicable for the validation of Assay method of Calcium & Vitamin D3 Tablets.
- Responsibility of Validation Team:
| Departments | Responsibilities |
| QC | Preparation and Review of Validation protocol. |
| Perform the validation as per approved protocol and recording of data. | |
| Compilation and checking of data. | |
| Preparation and review of Validation report. | |
| To impart training of protocol to concerned department/persons. | |
| QA | Review and approval of Validation protocol. |
| Co-ordination with QC to carry out Validation. | |
| Review and approval of Validation report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category | Calcium Supplement | |
| Reason for Validation | First Validation | |
| Active Ingredient | Calcium Carbonate and Vitamin D3 | |
| Strength | Calcium Content 500 mg per tablet | |
| Vitamin D3 250IU per tablet | ||
| Methodology | Non- compendia (In-House) | |
| Method Reference | Calcium | USP-38 |
| Vitamin D3 | In-House | |
| Specification Limits | Calcium | 95.0% -110.0% |
| Vitamin D3 | 170.%-320.0% |
- (A) Standard Testing Procedure:
Assay of Calcium Carbonate (Oyster Shell) Equivalent to Elemental Calcium
Methodology:
Method of analysis:
Required reagents
- Disodium EDTA (AR Grade)
- Hydrochloric Acid (AR Grade)
- Sodium Hydroxide (AR Grade)
- Hydroxy Nepthol Blue (Indicator) (AR Grade)
Blank titration
Take 3 mL of 3M Hydrochloric acid then add 100ml of water and 15ml of 1M Sodium Hydroxide solution in a titration vessel.
Titrate the solution with 0.05 M Disodium EDTA solutions using 1mg Hydroxy Nepthol Blue as indicator.
Preparation of 3M Hydrochloric Acid Solution:
Take 100ml volumetric flask containing 50ml Milli-Q water add 25.5ml of Concentrate
Hydrochloric acid add slowly and make up volume upto mark with Milli-Q water.
1M Sodium hydroxide solution:
Weigh and dissolve 4.2g of Sodium Hydroxide in 100 ml volumetric flask dissolve with Milli-Q
Water makeup volume upto mark with Milli-Q water.
Preparation of and standardization of 0.05M Disodium EDTA Solution:
Weigh and Dissolve 18.6g of EDTA disodium in 1000.0 ml volumetric flask dissolve with
Milli-Q Water makeup volume upto mark with Milli-Q water
Standardization of 0.05M Disodium EDTA Solution:
Weigh accurately about 200mg of primary standard calcium carbonate, previously dried at 100ºC for 2 hours and cooled in a desiccator, transfer to 400 ml beaker add 10ml water, and swirl to form a slurry. Cover the beaker with a watch glass, and introduce 2ml of diluted hydrochloric acid from a pipette inserted between the lip of the beaker and the edge of the watch glass. Swirl the contents of the beaker to dissolve the calcium carbonate. Wash down the sides of the beaker, the outer surface of the pipet, and the watch glass with water, and dilute with water to about 100.0ml.while stirring the solution, preferably with a magnetic stirrer, add about 30ml of the edetate disodium solution from a 50ml buret. Add 15ml of sodium hydroxide TS and 300mg of Hydroxy Napththol Blue, and continue the titration with the edetate disodium solution to a blue endpoint.
(g CaCO3) X 1000
M = ——————————————————–
100.09 X mL EDTA
Assay of Calcium Content
Procedure:
Weigh and powdered 20 tablets of Galcal 500. Weight accurately about 0.1g of sample in a conical flask dissolve in 3ml of 3M Hydrochloric acid and add about 100 ml of water. Then add 15 ml of 1MSodium Hydroxide solution. Titrate with 0.05 M Disodium EDTA using approx. 1 mg of Hydroxy Nepthol Blue as Indicator.
Endpoint detection: visual change to distinct blue colour.
Each ml of 0.05M Disodium EDTA is equivalent to 0.002004 g of Calcium.
Blank Reading =A
Test Reading =B
Actual Test Reading = (B – A) ml
Calculation:
(B – A) ml X 0.002004 X ‘Molarity’ of 0.05M EDTA X Av. Wt.
= ——————————————————————————-
Sample weight X 0.05
- Precision
- Method Precision:
The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatability to multiple samplings of homogenous sample. It is usually expressed as the standard deviation and the relative standard deviation.
Prepare the six samples of same batch of Calcium in Calcium & Vitamin D3 Tablets and analyze as per method of analysis. Defined in the protocol.
Analysis sequence
| Sample | Weight of sample | Direct titration |
| Blank Solution | 1 | |
| Sample Solution-1 | 0.1g | 1 |
| Sample Solution-2 | 0.1g | 1 |
| Sample Solution-3 | 0.1g | 1 |
| Sample Solution-4 | 0.1g | 1 |
| Sample Solution-5 | 0.1g | 1 |
| Sample Solution-6 | 0.1g | 1 |
Calculate the %Assay, mean, standard deviation and %relative standard deviation.
Acceptance criteria:
%RSD of Assay results in six sample preparations should not be more than 2.0%.
- Intermediate Precision ( Ruggedness ):
Intermediate precision expresses within laboratory variation with different analysts on different days using same batch of drug product as per Methodology.
Test Procedure:
The analysis of the same batch of Calcium in Calcium & Vitamin D3 Tablets will be done in six replicate analyses by using different analyst, on different day.
Analysis sequence
| Sample | Weight of sample | Direct titration |
| Blank Solution | 1 | |
| Sample Solution-1 | 0.1g | 1 |
| Sample Solution-2 | 0.1g | 1 |
| Sample Solution-3 | 0.1g | 1 |
| Sample Solution-4 | 0.1g | 1 |
| Sample Solution-5 | 0.1g | 1 |
| Sample Solution-6 | 0.1g | 1 |
Calculate the %Assay, mean, standard deviation and %relative standard deviation.
Acceptance criteria
% RSD should not be more than 2.0% of six replicates analysis and overall % RSD of (Method precision & Intermediate precision) should not be more than 2.0%.
- Accuracy :
The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy should be established across the specified range of the analytical procedure.
Test Procedure:
Prepare the sample solution of the Calcium in Calcium & Vitamin D3 Tablets (at Concentration 70%, 90%, 100%, 120% and 130% of test concentration level in triplicate in each level and analyze as per method of analysis. Further the % recovery for each preparation was calculated against the average % assay of method precision. Calculate the % RSD for recovery obtained at each level separately and overall %RSD.
| Sample | Weight of sample | Direct titration |
| Blank Solution | 1 | |
| Sample Solution-70%-1 | 0.07g | 1 |
| Sample Solution-70%-2 | 0.07g | 1 |
| Sample Solution-70%-3 | 0.07g | 1 |
| Sample Solution-90%-1 | 0.09g | 1 |
| Sample Solution-90%-2 | 0.09g | 1 |
| Sample Solution-90%-3 | 0.09g | 1 |
| Sample Solution-100%-1 | 0.1g | 1 |
| Sample Solution-100%-2 | 0.1g | 1 |
| Sample Solution-100%-3 | 0.1g | 1 |
| Sample Solution-120%-1 | 0.12g | 1 |
| Sample Solution-120%-2 | 0.12g | 1 |
| Sample Solution-120%-3 | 0.12g | 1 |
| Sample Solution-130%-1 | 0.13g | 1 |
| Sample Solution-130%-2 | 0.13g | 1 |
| Sample Solution-130%-3 | 0.13g | 1 |
Acceptance criteria
The mean recovery at each level should be between 98.0% to 102.0% of the theoretical value and the % RSD not more than 2.0%.
The overall average should be 98.0% to 102.0% and %RSD should not be more than 2.0%.
- Range & Linearity:
The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The range is normally expressed in the same units as test results obtained by the analytical method. Plot the calibration curve of concentration v/s weight variation of the sample.
Acceptance criteria
The correlation coefficient (r) should be not less than 0.999.
Robustness:
Robustness of the method shall be investigated by checking the following parameters by deliberately varying the condition such as
- Effect of Disodium EDTA concentration ±10%
- Effect of Hydrochloric concentration ±10%
- (B)- Vitamin D3 (ASSAY BY HPLC):
Methodology:
Method of analysis:
Reagent:
- Acetonitrile (HPLC grade)
- Methanol (HPLC grade)
- n-Hexane (HPLC grade)
Note: Perform the activity under low actinic light & in amber colour glass-ware.
Chromatographic conditions:
Column : C18, 250 X 4.6mm, 5µm (Nucleosil or equivalent)
Flow rate : 1.5ml/min
Detector : 265nm (UV detector)
Temperature : Ambient
Injection Volume : 50µl
Run Time : About 15 minutes
Mobile Phase:
Mixture of Acetonitrile, Methanol and n-Hexane in the ratio 47: 47:8), filter through 0.45 µm-membrane filter paper and degas before use.
Standard Preparation:
Weigh accurately 25 mg of Vitamin D3 WS and transfer in 100.0 ml volumetric flask. Dissolve with about 50 ml of methanol and sonicate for about 5 minutes in a cool medium, make-up the volume with Methanol up to 100.0 ml with methanol. Dilute 1.0 ml to100.0 ml with Methanol and further dilute 10.0 ml to 100.0 ml with 40ml of Methanol and make up with methanol. Filter through Whatmann no. 42 or equivalent filter paper.
Test Preparation:
Weigh and powdered 20 tablets. Weigh about 1000 mg of powder (equivalent to 500 IU of Vitamin D3 considering 200% overages) and transfer in 50.0 ml volumetric flask, dissolve with 25ml of Methanol and sonicate for about 5 minutes in cool medium, make up the volume to 50.0 ml with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Procedure:
Wash the column initially with Methanol at flow rate of 1.0 ml/min for not less than 15 minutes. And then run Mobile Phase for not less than 15 minutes.
Inject the solution as per following sequence.
Blank: 1 injection
Standard: 5 injections
Test: 2 injections
Standard (Bracketing): 1 injection
Evaluation of System suitability:
Inject 50 µl of the standard solution in five replicate injections and check the system suitability parameters as mentioned below:
| Sr. No. | Parameters | Limit |
| 1. | Tailing Factor | Not more than 2.0 |
| 2. | Theoretical plate | Not Less than 2000 |
| 3. | % RSD for peck area of Vitamin D3from five replicate injections of standard solution. | Not more than 2.0 |
Calculation:
At Ws. 1.0 10.0 50 P
——— x ———–x ——- x ———- x———- x———– x 40000 x Av. Wt.
As 100 100 100 (Tw) 100
Where:
At: Mean area of the peak due to Vitamin D3 obtained in the sample chromatogram.
AS: Mean area of the peak due Vitamin D3 obtained in the standard chromatogram.
Ws: Weight taken for Standard preparation in mg.
Wt: Weight taken for test preparation in mg.
P= Potency of Vitamin D3 working standard.
ASSAY PARAMETER FOR METHOD VALIDATION OF VITAMINE D3 (BY HPLC)
Specificity:
Specificity of analytical method is its ability to assess unequivocally the analyte in presence of components that may be expected to be present in the placebo.
Specificity of test method should be established by injecting blank, Placebo, Standard solution and test solution. Placebo solution will be prepared at specification level as per method of analysis. Standard and test solutions should be prepared individually, i.e. test solution to be prepared as per method of analysis.
Analysis sequence:
Blank – 1 injection
Placebo solution – 1 injection
Standard solution – 5 injections
Test solutions (each strength 250IU) – 2 injection
Acceptance criteria
The excipient compounds must not interfere with the analysis of the targeted analyte.
No any peak should elute at the retention time VitaminD3 in blank and placebo solution. Peak purity of the main peak should pass in test and standard solution.
Precision:
System Precision:
The system precision is the closeness of agreement between the responses of detector. It is usually expressed as the standard deviation (SD) or the relative standard deviation (RSD).
Standard solution will be prepared as per method of analysis and injected six replicate injections and recorded the area response of main analyte peak. Relative standard deviation will be calculated of response of VitaminD3for six replicate injections.
Analysis sequence
Blank – 1 injection
Standard solution – 6 injections
Acceptance criteria
The % Relative standard deviation for six replicate injections of standard solution should not be more than 2.0%.
Method Precision:
The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatability to multiple samplings of homogenous sample. It is usually expressed as the standard deviation and the relative standard deviation.
Prepare the six samples of same batch VitaminD3 250IU strength and analyze as per method of analysis. Record the area on data sheet and calculate the % Assay, mean, standard deviation and % relative standard deviation.
Acceptance criteria:
%RSD of Assay results in six sample preparations should be not more than 2.0%.
Intermediate Precision ( Ruggedness ):
Intermediate precision expresses within laboratory variation with different analysts or equipment or different column/same column on different days using same batch of drug product as per method of analysis.
Test Procedure:
The analysis of the same batch will be done in six replicate analyses by using different columns by different analyst, by different system on different day. The mean, standard deviation and relative standard deviation will be calculated.
Acceptance criteria
% RSD should be not more than 2.0%.
Forced Degradation:
Prepare Blank, Placebo, standard solution, untreated test solution and treated test solution (By acid, base, oxidation, thermal, humidity, Water Hydrolysis and Photolytic).
Inject and calculate the % Assay. Calculate the % difference between the untreated test solution and treated test solution result. Check the peak purity of main peak.
Stress the sample at the following conditions and evaluate the peak purity and all degradation study procedure should be recorded in data sheet and mentioned in method validation report.
- Degradation by Hydrochloric Acid
- Degradation by Sodium Hydroxide
- Degradation by Hydrogen peroxide 30% (Oxidative degradation)
- Degradation by Thermal ( at 80°C or below melting point)
- Degradation by Humidity ( at 25°C/90%RH)
- Degradation by water hydrolysis.
- Photolytic (ultraviolet light for 2-4 hours)
Acceptance criteria
The treated sample results should be compared with untreated sample and the difference should be not more than 15%.
Peak purity of main peak should be passed.
- Procedure:
- Acid Hydrolysis:
Test Stock Solution: Accurately weigh 1000mg of powder(equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask and add 10 ml of 1.0M hydrochloric acid Stand for 60minute at room temperature and neutralize with 1.0M NaOH add 25ml of methanol sonicate about 5 minutes in cool medium makeup volume with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Base Hydrolysis:
Test Stock Solution: Accurately weigh 1000mg of powder(equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask and add 10 ml of 1.0M NaOH Stand for 60minute at room temperature and neutralize with 1.0M hydrochloric acid add 25ml of methanol sonicate about 5 minutes in cool medium makeup volume with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Oxidative Degradation:
Accurately weigh 1000mg of powder(equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask add 10 ml of 30%H2O2 Stand for 60minute at room temperature and add 25ml of methanol sonicate about 5 minutes in cool medium makeup volume with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
.Thermal Degradation ( at 80°C/24Hrs)
Accurately weigh 1000mg of powder (equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask dissolve with 25ml of Methanol and sonicate for about 5 minutes in cool medium, make up the volume to 50.0 ml with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Humidity Degradation (at 25°C/90%RH)
Accurately weigh 1000mg of powder (equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask dissolve with 25ml of Methanol and sonicate for about 5 minutes in cool medium, make up the volume to 50.0 ml with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Water Hydrolysis:
Accurately weigh 1000mg of powder(equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask add 10 ml of water at 105ºC Stand for 60minutes and add with 25ml of Methanol and sonicate for about 5 minutes in cool medium, make up the volume to 50.0 ml with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Photolytic Degradation (ultraviolet light at 365nm for 2-4 hours)
Accurately weigh 1000mg of powder (equivalent to 500IU of vitamin D3 considering 200% overages), place them in a 50.0 ml volumetric flask dissolve with 25ml of Methanol and sonicate for about 5 minutes in cool medium, make up the volume to 50.0 ml with methanol. Shake vigorously for 2 minutes. Allow to stand for 5 minutes in dark. Filter through Whatmann no. 42 or equivalent filter paper.
Linearity:
The linearity of an analytical procedure is its ability (within a given range) to obtained test results which are directly proportional to the concentration levels should be prepared.
Determine the linearity by preparing and inject the standard solution in the range of 70% to 130% of concentration level and calculate the correlation coefficient “r”.
Test Procedure:
Prepare the standard solutions at five concentrations, typically 70%, 90%, 100%, 120% and 130% of target concentration following finished product testing procedure. Three individually prepared replicates at each concentration will be analyzed. Plot concentration (x-axis) versus mean response (y-axis) for each concentration. Calculate the correlation coefficient (r). Record these calculations on the datasheet.
Acceptance criteria
The correlation coefficient (r) should be not less than 0.999.
Accuracy:
The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy should be established across the specified range of the analytical procedure.
Test Procedure:
Prepare the sample solution by spiking the vitamin D3 (Drug substances) to the placebo at about 70%, 100% and 130% of test concentration level in triplicate in each level and analyze as per method of analysis. Further the % recovery for each preparation was calculated against the known added amount (drug concentration at each level) % assay. Calculate the % RSD for recovery obtained at each level separately and overall %RSD.
Acceptance criteria
The mean recovery at each level should be between 98.0% to 102.0 % of the theoretical value and the % RSD not more than 2.0%.The overall average should be between 98.0% to 102.0% with %RSD not more than 2.0%.
Range:
The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The range is normally expressed in the same units as test results obtained by the analytical method. (e.g. percent, parts per million).
Stability of Analytical solution:
It is essential when validating an analytical method to confirm that the analyte has adequate stability in both the standard and sample solution during analytical measurement stages of the testing.
Test Procedure:
Prepare the standard solution and test solution as method of analysis and analyze the solution at the different time intervals and calculate the % difference for the assay result.
Acceptance criteria
The difference in results should be not more than 2.0% from initial results.
Filter Paper Selection Study:
Test Procedure:
Prepare test solution in triplicate as per method of analysis. A portion of test solution should be other portion of test solution should be filtered with filters (e.g. Whatmann no. 42, PVDF and Nylon filter) inject all samples and calculate the % assay for each sample and calculate the % assay difference Method Precision vs. filtered samples.
Acceptance criteria
Difference between Method Precision vs. filtered samples should be not more than 2%.
Robustness:
The method should show reliability of an analysis with respect to deliberate variation in method parameters.
Following deliberate variations should be done in method parameters:
- By changing the flow rate by ±10%.
- By changing the Column oven temperature by ±5°C.
- By Changing the Organic Solvent ±5%.
System suitability parameters should be performed as per finish product testing procedure for each deliberate variation.
Test Procedure:
Prepare the standard solution and test solution in triplicate as per the method by deliberate variations made in the method for each condition as mentioned in protocol and analyze.
Acceptance criteria
Overall % RSD should be not more than 2% with method precision data.
Incident/Deviation:
Any Incident or Deviation observed during Analytical Method validation should be recoded and reported in Validation Report.
Summary/Conclusion:
Final Conclusion should be drawn from analytical method validation for its use to analyze the assay in Calcium & Vitamin D3 Tablets.
Summary of validation report should be prepared and accordingly STP to be updated.
- Revision History :
| Revision No. | Details of changes | Reason for change |
| 00 | Nil | New Document |
