by ASSAY METHOD VALIDATION PROTOCOL OF TRYPSIN – CHYMOTRYPSIN TABLETS
| Superseded Protocol No. | Nil |
| Effective Date |
- 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 |
TABLE OF CONTENTS :
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility of Validation Team | ||
| Product Profile | ||
| Methodology | ||
| Specificity | ||
| Precision | ||
| Linearity | ||
| Accuracy/Recovery | ||
| Range | ||
| Robustness | ||
| Stability of Analytical Solution | ||
| Filter Paper Study | ||
| Incident/Deviation | ||
| Final Summary/Conclusion | ||
| Revision History |
- Objective:
The objective of this method validation is to provide documentary evidence that analytical methodology used in determination of the Trypsin Chymotrypsin in Trypsin Chymotrypsin tablets 40mg will yield consistent and reliable results within the predetermined acceptance criteria.
Analytical method validation will be performed by considering thefollowing parameters:
| Parameters | 40mg |
| Specificity | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision(Ruggedness) | |
| Linearity | |
| Accuracy | |
| Range | |
| Stability in Analytical Solutions | |
| Robustness | |
| Filter Paper Selection Study |
- Scope :
The scope of this method validation protocol is based on SOP and limited to analytical method validation for determination of Trypsin Chymotrypsin in Trypsin Chymotrypsin tablets.
- Responsibility of Validation Team:
| Departments | Responsibilities |
| QC | Preparation and Review of Validation protocol. |
| Analysis of samples 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 carryout Validation. | |
| Review and approval of Validation report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category | Proteolytic Enzyme |
| Reason for Verification | First Validation |
| Active Ingredient | Trypsin Chymotrypsin |
| Strength | Each Enteric Coated tablet contains: Trypsin Chymotrypsin 40 mg (eq. to 1.0 lac Armour units of enzymatic activity) |
| Methodology | In- House |
| Method Reference | In- House |
| Specification Limits (At release) | 100 % -140 % ( 40 mg – 56 mg) |
- Methodology:
Assay:
Regents:
Follins-ciocalteau stock solution
0.01M Hydrochloric acid.
Preparation of Reagents :
Follins ciocalteau reagent: Dilute 10.0 ml of stock solution to 30 ml with distilled water (Milli-Q water).
0.01MHydrochloric Acid:
Dilute 8.5 ml of Conc. Hydrochloric acid (36%) to 1000.0 ml water (Milli-Q water) and further dilute 100 ml to 1000 ml water (Milli-Q water).
Standard Preparation:
Weigh accurately about 400 mg of Trypsin Chymotrypsin WS in a 100.0 ml volumetric flask add 60 ml 0.01 M hydrochloric acid and shake for 5 minutes to dissolve. Add sufficient 0.01 M hydrochloric acid to make the volume, mix and filter through filter paper No. 41 or equivalent. Further dilute 5.0 ml to 50.0 ml with same solvent.
Sample Preparation:
Take 20 tablets and crushed up to fine powder and weigh tablet powder eq. to 40 mg of Trypsin Chymotrypsin in a 100.0 ml volumetric flask add 60 ml 0.01 M hydrochloric acid and shake for 15 minutes to dissolve. Add sufficient 0.01 M hydrochloric acid to make the volume, mix and filter through filter paper No. 41 or equivalent.
Procedure:
Take 3 Stoppard test tubes marked as Std., Test & blank. To Test tube marked as ‘Std’ add 5.0 ml of standard solution and to test tubes marked ‘Test’ add 5.0 ml of test solution and to test tube marked as ‘blank’ add 5.0 ml of 0.01 M Hydrochloric acid.
To all the test tubes add 5.0 ml of 6 % w/v Sodium carbonate solution, mix. To all the test tubes add 2.0 ml folin calteau reagent (1ml of folin calteau solution is diluted to 3.0 ml just before use), mix and allow to stand for 10 minutes. Read the colour intensity of both the solutions at about 660 nm against blank and calculate the result by comparison as given below:-
Calculations:
Trypsin Chymotrypsin Content:
AT WTS 5 100
————-x ———x ——-x———x P x Avg. Wt. = units
AS 100 50 WTT
Where:
AT – Absorbance of Test Solution
AS – Absorbance of Standard Solution
WTS – Weight of Working Standard
WTT – Weight of Test Substance
P – Potency of Working Standard / Reference standard
Sample Preparation Table:
| Blank (ml) | Placebo (ml) | Standard (ml) | Test (ml) | |
| 0.01MHCl | 5.0 | – | – | – |
| Placebo | – | 5.0 | – | – |
| Standard | – | – | 5.0 | – |
| Test | – | – | – | 5.0 |
| 6%Sodium Carbonate | 5.0 | 5 | 5.0 | 5.0 |
| Folin Calteau Reagent | 2.0 | 2.0 | 2.0 | 2.0 |
- 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 blank & placebo.
Test Procedure:
Prepare and take the absorbance of blank, Placebo, Standard solution & test solution as per defined above procedure scan from 200nm to 800nm.
Blank-1
Placebo-1
Standard Solution-1
Test Solution-1
Acceptance criteria
The interference from blank and placebo shall not be more than 2% of Standard absorbance. If the absorbance of placebo solution is more than 2% of standard solution then subtracts the placebo absorbance from the sample 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).
Prepare and take the absorbance of separately Blank and Standard solution as per assay methodology defined above and record the absorbance of Trypsin Chymotrypsin in the standard solution. Calculate the % RSD of response for six replicate absorbance.
Blank-1
Standard Solution-6
Acceptance criteria
The relative standard deviation for absorbance of Trypsin Chymotrypsin should not be more than 2%.
- Method Precision (Repeatability):
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 six individual sample of same batch and analyze as per assay methodology defined above.
Blank-1
Placebo-1
Standard Solution-2
Test Solution-6
Acceptance criteria
The relative standard deviation for six samples assay results should not be more than 2%.
- Intermediate Precision ( Ruggedness ):
Intermediate precision expresses within laboratory variation with different analysts or equipment within the same laboratory using same batch of drug product as specified under precision.
Test Procedure:
The analysis of the same batch will be done in six replicate analyses by using different analyst on different day. The mean, standard deviation and relative standard deviation will be calculated.
Prepare six individual sample of same batch and analyze as per assay methodology defined above.
Blank-1
Placebo-1
Standard Solution-2
Test Solution-6
Acceptance criteria
The relative standard (RSD) of six analyses should not be more than 2% and overall relative standard deviation should not be more than 2% compared with method precision results.
- Linearity and range:
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 shall be prepared.
Determine the linearity by preparing and take the absorbance of standard solution in the range of 80% to 150% of concentration level and calculate the correlation coefficient “r”.
Test Procedure:
Prepare the standard solutions at five concentrations, typically 80%, 90%, 100%, 110%, 120%, 130% and150% of target concentration as per above testing procedure.
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 not be 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 shall be established across the specified range of the analytical procedure.
Test Procedure:
Prepare the sample solution by spiking the Trypsin Chymotrypsin (Drug substances) to the placebo at about 80%, 100% and 150% of test concentration level in triplicate in each level and analyze as per finished product testing procedure. Calculate the %recovery, % RSD for recovery obtained at each level separately and overall %RSD.
For each sample, record the theoretical value, assay value, and percent recovery on test data sheet. Calculate the mean, standard deviation, % RSD, and percent recovery for all samples. Record results on the datasheet.
Acceptance criteria
The mean recovery at each level should be between 98% to 102 % of the theoretical value and the % RSD not more than 2.The overall average should be between 98% to 102% with %RSD should not be more than 2%.
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 verify 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 per assay methodology and analyze the solution at the different time intervals and calculate the difference for the assay result.
Stability of Analytical Solution:
| Duration | %Assay | % Deviation |
Acceptance criteria:
The difference in results shall be not more than 2% from initial results.
Robustness:
The method shall show reliability of an analysis with respect to deliberate variation in method parameters.
Following deliberate variations shall be done in method parameters:
- By changing the wavelength by ± 2 nm.
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.
Calculate the % assay. Calculate the % RSD for % assay for individual above experiment with method precision results.
Acceptance criteria:
Overall % RSD should not be more than 2 with method precision data.
Filter Paper Selection Study:
Test Procedure:
Prepare test solution in triplicate as per finish product testing procedure. A portion of test solution shall be centrifuge and other portion of test solution shall be filtered with whatmen filter paper No. 41and 0.45µm Nylon filter take the absorbance of all samples and calculate the % assay for each sample and calculate the % assay difference between centrifuges vs. filtered samples.
Acceptance criteria
Difference between centrifuges vs. filtered samples shall be not more than 2%.
Incident/Deviation:
Any Incident or Deviation shall be observed during Analytical Method validation should be recorded and reported in Validation Report.
Final Summary/Conclusion:
Final Conclusion shall be drawn regarding analysis method validation for its use to analyze the assay test in Trypsin-Chymotrypsin tablets by UV.
Revision History:
| Revision No. | Details of changes | Reason for changes |
| 00 | New Document | Nil |
