by ANALYTICAL METHOD VALIDATION PROTOCOL OF DISSOLUTION OF CETIRIZINE DIHYDROCHLORIDE TABLETS
| Superseded Protocol No. | Nil |
| Effective Date |
Table of contents :
| Sr. No. | Subject | Page No. |
| Protocol Approval | ||
| Objective | ||
| Scope | ||
| Responsibility | ||
| Product profile | ||
| Methodology | ||
| Validation parameters | ||
| Incident/Deviation | ||
| Summary/Final conclusion/Recommendation | ||
| Abbreviation | ||
| 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 protocol is to validate the suitability of dissolution of Cetirizine dihydrochloride tablets by considering thefollowing parameters:
| Parameters | Cetirizine Dihydrochloride |
| Specificity | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision | |
| Linearity | |
| Range | |
| Stability of Analytical Solution | |
| Filter Effect study | |
| Accuracy | |
| Robustness | |
| System Suitability |
- Scope :
This protocol is applicable for the Validation of dissolution of Cetirizine dihydrochloride tablets.
- Responsibility of Validation Team:
| Departments | Responsibilities |
| QC | Preparation & Review of Protocol. |
| Analysis of samples and recording of data. | |
| Compilation and checking of data | |
| Preparation of Summary Report. | |
| To impart training of protocol to concerned department/persons. | |
| QA | Review and approval of protocol. |
| Co-ordination with QC to carryout Validation. | |
| Review of data and summary report. | |
| Head Quality | Authorization of protocol. |
- Product Profile:
| Category | Antihistamine |
| Reason for Validation | First Validation |
| Active Ingredient | Cetirizine Dihydrochloride |
| Strength | Each coated tablet contains: Cetirizine Dihydrochloride 10 mg |
| Method Reference | In House |
| Specification Limits | Not less than 80% (Q) of the labeled amount is dissolved in 15 minutes. |
- Methodology:
Chemical, reagents and filters:
Table 1.0: Chemical, reagents and filters
| Sr. No | Material /Chemicals/Filters | Grade |
| 1.0 | Potassium dihydrogen phosphate (KH2PO4) | AR Grade |
| 2.0 | Ortho phosphoric acid | HPLC Grade |
| 3.0 | Water | HPLC Grade or Milli Q water |
| 4.0 | Methanol | HPLC Grade |
| 5.0 | Acetonitrile | HPLC Grade |
| 6.0 | 0.45 µm membrane filter | Millipore or Equivalent |
| 7.0 | 0.45 µm Nylon filter | Millipore or Equivalent |
| 8.0 | Hydrochloric Acid | AR Grade |
| 9.0 | Sodium Hydroxide Pellets | AR Grade |
| 10.0 | Hydrogen Peroxide | AR Grade |
Dissolution Parameters:
Apparatus : Paddle (II)
Dissolution Medium : Purified Water
Dissolution Medium volume : 900 ml
Speed : 50 rpm
Temperature : 37°C ± 0.5 °C
Time : 15 minutes for Release
Chromatographic conditions (By HPLC):
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : ACE 5C18, 150 mm X 4.6 mm, 5 μm
Flow rate : 1.5 ml/min
Detector : 230 nm
Column Temperature : 40ºC
Injection Volume : 20 µl
Run time : 6.0 minutes (Cetirizine RT Approximately 3.0 min)
Program : Isocratic Method
Mobile Phase : Mixture of Phosphate Buffer: Methanol: (350:650 V/V)
Diluent : Water: Methanol: (50:50) filtered through 0.45μ filter
Preparation of Phosphate Buffer:
Weigh accurately about 6.8 g of Potassium dihydrogen phosphate (KH2PO4) in 1000 ml of water. Adjust pH with dilute ortho-phosphoric acid to 2.6 ± 0.05.
Standard Preparation:
Weigh accurately about 56 mg of Cetirizine dihydrochloride working standard, transfer into a 100 mL volumetric flask. Add 60ml of diluent and sonicate till dissolve and dilute to volume with diluent.
Pipette out 10.0 ml of this solution and transfer into 50 ml of volumetric flask and make up the volume with dissolution media and mix. Further pipette 5.0 ml of this solution and transfer into 50 ml of volumetric flask, make up the volume with dissolution media and mix.
Sample Preparation:
Perform the test on six tablets. Place the 1 tablet in each dissolution vessels containing 900.0 ml of dissolution medium, previously maintained specified temperature (37°C ± 0.5 °C) and run the instrument for 15.0 minutes. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Evaluation of System Suitability:
- Area % RSD : Should not be more than 2.0 % for peak area due to Cetirizine dihydrochloride from 6 replicate injections of standard solution.
- Tailing factor : Should not be more than 2.0 for peak due to Cetirizine dihydrochloride.
- Theoretical plates : Should not be less than 2000 for peak due to Cetirizine dihydrochloride.
Procedure:
Equilibrate the HPLC Column with mobile phase. Inject as per the following sequence or as per requirement.
Sequence of Injections:
Table 2.0: Sequence of Injection of Dissolution
| Preparation | No. of Injections | Action |
| Blank Solution | 1 | Record the blank Chromatograms |
| Standard solution 1 | 6 | Check for system suitability Criteria (i), (ii) and (iii) |
| Sample solution 1 to 6 | 1 | Inject the individual solution, Maximum 6 in a set. |
| Standard solution 1 (Bracketing standard) | 1 | %, Area RSD of total 7 injections ≤ 2.0% (6 Injection of standard solution + 1 injection of bracketing standard) |
Calculation for Dissolution (% labeled amount):
AT WS 10 5 900 P
% Released Content = ——- x ——- x —— x —— x —— x —— x 100
AS 100 50 50 LC 100
Where,
AS = Average peak area due to Cetirizine Dihydrochloride obtained from six replicate
injections of standard solution.
AT = Peak area due to Cetirizine Dihydrochloride obtained from sample Solution
WS = Weight of Cetirizine Dihydrochloride RS/WS taken in mg.
LC = Label claim of Cetirizine Dihydrochloride in mg/tablet (10).
P = Potency of Cetirizine Dihydrochloride RS/WS in %, on as basis.
- Validation parameters:
The following parameters to be perform for the Validation activity.
Specificity
Precision
Linearity
Range
Stability of Analytical solution
Filter Paper Selection Study
Accuracy
Robustness
System Suitability
- 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 and placebo.
Specificity of test method should be established by separately injecting blank solution, Placebo, standard solution, sample solution and spiked sample solution. Sample solution to be preparing as per method of analysis.
Standard Preparation:
Weigh accurately about 56 mg of Cetirizine dihydrochloride working standard, transfer into a 100 mL volumetric flask. Add 60ml of diluent and sonicate till dissolve and dilute to volume with diluent.
Pipette out 10.0 ml of this solution and transfer into 50 ml of volumetric flask and make up the volume with dissolution media and mix. Further pipette 5.0 ml of this solution and transfer into 50 ml of volumetric flask, make up the volume with dissolution media and mix.
Preparation of specificity solutions for dissolution test:
In the set of 4 dissolution vessels add 900 ml of dissolution medium.
i) Blank solution : Only dissolution medium (Water).
ii) Placebo solution: Weight and transfer 143.75 mg of placebo into dissolution medium.
iii) Test Solution: Weight 1 tablet and transfer into dissolution medium.
iv) Spiked test solution: Weight 143.75 mg placebo + 10 mg Cetirizine dihydrochloride WS into dissolution medium.
Sample Preparation:
Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Table 3.0: Sequence of Injection
| Solution | No of Injection to be injected in Sequence |
| Blank solution | 1 |
| Placebo Solution | 1 |
| Standard solution | 6 |
| Test Solution | 1 |
| Spiked Test Solution | 1 |
| Standard Solution + Bracketing | 1 |
Table 4.0: Specificity data
| Sr. No | Sample | RT (min.) | Peak purity | |
| 1 | Blank | NA | ||
| 2 | Placebo | NA | ||
| 3 | Standard Solution | Cetirizine Dihydrochloride | ||
| 4 | Sample Solution | Cetirizine Dihydrochloride | ||
| 5 | Spiked Solution | Cetirizine Dihydrochloride |
Acceptance Criteria:
- There should be no interference of the diluent, placebo at the retention time of analyte peak,
- Blank peak and Placebo peak should be well resolved from active peak and each other,
- Analyte peak in standard solution, sample solution and spiked sample solution should be spectrally pure.
- 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 to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % area RSD and % RT RSD of main analyte peak.
Table 5.0: System Precision- Repeatability of Standard Injections
| Sr. No. | Cetirizine Dihydrochloride | |
| Peak Area | Retention time (min.) | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance criteria:
% RSD for peak area and retention time of replicate standard solution injections should be NMT 2.0% and 1.0% respectively.
- 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. Check for system suitability criteria.
Test Procedure for Dissolution:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % release, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 6.0: Method precision results for Dissolution
| Sr. No. | Cetirizine Dihydrochloride | |
| Peak Area | % Release | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance Criteria:
Calculate the % Released for each tablets and calculate the mean, SD and % RSD.
% RSD for released values should be NMT 10.0%.
- 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.
Standard solution will be prepared as per method of analysis and injected six replicate injections to be injected in sequence and recorded the area response and retention time of main analyte peak. Calculate the % area RSD and % RT RSD of main analyte peak.
Table 7.0: Repeatability of Standard Injections
| Sr. No. | Cetirizine Dihydrochloride | |
| Peak Area | Retention time (min.) | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance criteria:
% RSD for peak area and retention time of replicate standard solution injections should be NMT 2.0% and 1.0% respectively.
Test Procedure:
Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % released, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.
Table 8.0: Intermediate precision results for Dissolution
| Sr. No. | Cetirizine Dihydrochloride | |
| Peak Area | % Release | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD |
Acceptance Criteria:
Calculate the % released for each tablets and calculate the mean, SD and % RSD.
% RSD for released values should be NMT 10.0%.
Table 9.0: Pooled results of dissolution of analyst – I & analyst – II
| Sample No. | Peak Area | % Dissolution |
| Cetirizine Dihydrochloride | ||
| ANALYST I | ||
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| ANALYST II | ||
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| Pooled % RSD |
Acceptance Criteria:
The pooled % RSD for released values of Analyst I and Analyst II should be NMT 10.0%.
- 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 shall be prepared. Check for system suitability criteria.
Prepare the standard solution of Cetirizine Dihydrochloride at five different concentrations typically 80, 90, 100, 110 and 120 % to consider the linearity of Cetirizine Dihydrochloride.
Cetirizine Dihydrochloride linearity stock solution: (250.0 ppm)
Weigh accurately about 56 mg of Cetirizine dihydrochloride working standard, transfer into a 100 mL volumetric flask. Add 60ml of diluent and sonicate till dissolve and dilute to volume with diluent, Pipette out 10.0 ml of this solution and transfer into 50 ml of volumetric flask and make up the volume with dissolution media and mix.
Table 10.0: Linearity levels preparation
| Level % | Vol. (mL) of Stock | Volume to be diluted with diluent (ml) |
| Cetirizine Dihydrochloride | ||
| 80 | 4.0 | 50 |
| 90 | 4.5 | 50 |
| 100 | 5.0 | 50 |
| 110 | 5.5 | 50 |
| 120 | 6.0 | 50 |
Table 11.0: Linearity of Cetirizine Dihydrochloride
| Level (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| 80 | 8.96 | |||
| 90 | 10.08 | |||
| 100 | 11.20 | |||
| 110 | 12.32 | |||
| 120 | 13.44 | |||
| Slope | ||||
| Intercept | ||||
| Corr. coeff.(r) |
Acceptance criteria
The correlation coefficient (r) should be not less than 0.999.
- 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.
- Stability of Analytical solution :
It is essential when the validation an analytical method to confirm that the analyte has adequate stability in both the standard and sample solution at room temperature (ambient temperature) during analytical measurement stages of the testing.
Test Procedure:
Prepare the blank solution, standard solution and test solution as per method of analysis and analyze the solution at the different time intervals i.e. 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, and 48 hours. Calculate the % release at each time interval with respect to the initial replicate injection of standard solution. Calculate the standard area % RSD at different time interval.
Acceptance Criteria
The difference in results should be not more than 2.0 % from initial results and the area % RSD of standard solution should be not be more than 2.0 %.
- Filter Paper Selection Study
Prepare the 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 filter. Centrifuge the sample at 2000 rpm for 2 minutes and inject. Also filter a portion of the same sample through 0.45µ PVDF syringe filter, further analyse as per the method and inject. From the data calculate the % release.
Table 12.0: Effect of Filtration (Cetirizine Dihydrochloride)
| Sample | Filter Used | Mean Peak area | % Release | Mean % Release | Difference |
| Cetirizine Dihydrochloride | |||||
| Test 1 | After centrifuge | ||||
| Test 2 | |||||
| Test 3 | |||||
| Test 1 | 0.45µ Nylon syringe filter | ||||
| Test 2 | |||||
| Test 3 |
Acceptance Criteria:
The difference between the filtered and centrifuge sample for the % released values should be NMT 5.0%.
- 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. Check the system suitability criteria.
Test Procedure:
Prepare the sample solution by spiking the Cetirizine Dihydrochloride (Drug substances) with placebo to the place at about 80%, 100% and 120% of test concentration level in triplicate at each level and analyze as per finished product testing procedure. Further the % recovery for each preparation calculated by amount added and amount recovered and calculate the % RSD for recovery obtained at each level separately. Obtained results are report in tabular manner as given.
Standard Preparation:
Weigh accurately about 56 mg of Cetirizine dihydrochloride working standard, transfer into a 100 mL volumetric flask. Add 60ml of diluent and sonicate till dissolve and dilute to volume with diluent.
Pipette out 10.0 ml of this solution and transfer into 50 ml of volumetric flask and make up the volume with dissolution media and mix. Further pipette 5.0 ml of this solution and transfer into 50 ml of volumetric flask, make up the volume with dissolution media and mix.
Placebo Solution:
Weight accurately 143.75 mg of placebo powder and transfer into dissolution vessels containing 900 ml dissolution medium. Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Level I: 80%
Weight accurately 143.75 mg of placebo powder and 8.0 mg of Cetirizine dihydrochloride working standard and transfer into dissolution vessels containing 900 ml dissolution medium. Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Level II: 100%
Weight accurately 143.75 mg of placebo powder and 10.0 mg of Cetirizine dihydrochloride working standard and transfer into dissolution vessels containing 900 ml dissolution medium. Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Level II: 120%
Weight accurately 143.75 mg of placebo powder and 12.0 mg of Cetirizine dihydrochloride working standard and transfer into dissolution vessels containing 900 ml dissolution medium. Run the apparatus as per given condition. After specified time, withdraw specified volume of aliquot from each vessel and filter through 0.45 µm Nylon Syringe filter, discard first 2-3 ml of the filtrate.
Calculation:
Cetirizine Dihydrochloride:
X P
Amount added (ppm) = ——— x ————- x 1000
900 100
AT WS 10 5 P
Amount Recovered (ppm) = ——— x ———- x ————- x ———— x ———— x 1000
AS 100 50 50 100
Amount Recovered in ppm
% Recovery = ————————————— x 100
Amount added in ppm
Where,
AT : Area of Cetirizine dihydrochloride in spiked test solution
AS : Area of Cetirizine dihydrochloride in the standard solution
WS : Weight of Cetirizine dihydrochloride taken in mg for standard solution.
X : Weight of Cetirizine dihydrochloride taken in mg at different Level (8, 10 & 12)
P : Potency of Cetirizine dihydrochloride
Table 13.0: Accuracy for Cetirizine Dihydrochloride
| Levels (%) | Amount added in placebo (ppm) | Amount recovered in placebo (ppm) | Recovery % | Mean Recovery % | SD | RSD% |
| 80% | ||||||
| 100% | ||||||
| 120% | ||||||
Acceptance Criteria:
The mean recovery at each level should be between 95.0% and 105% of theoretical value and the % RSD not more than 5.0%, the overall average should be between 95.0% and 105.0% with % RSD not more than 5.0%
- Robustness:
The method should show reliability of an analysis with respect to deliberate variation in method parameters of cleaning.
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%.
- By Changing RPM ±5 RPM.
- By Change in pH.
System suitability parameters shall be performed as per 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. Calculate the % release at each deliberate variation with respect to the initial replicate injection of standard solution.
Acceptance criteria
System suitability parameter should meet as per acceptance limit of system suitability criteria. Overall % RSD shall be not more than 10 with of the method precision data of individual experiments.
- System Suitability
To ensure that during each analysis, the analytical procedure is giving accurate and precise results, system suitability parameters have been set. The set limits are given below.
The data obtained will be summarized in table.
Table 14.0: System suitability parameters
| Parameter | Set limits |
| Theoretical plate for Cetirizine dihydrochloride peak | NLT – 2000 |
| Tailing factor for Cetirizine dihydrochloride peak | NMT – 2.0 |
| RSD for the peak area of the five replicate injections of Cetirizine dihydrochloride | NMT – 2.0% |
- Incident /Deviation:
Any incident or deviation observed during analytical method validation should be recorded and reported in validation report.
- Summary/ Conclusion / Recommendation:
Final conclusion should be drawn from analytical method validation for its use to analyze the Dissolution test of Cetirizine Dihydrochloride and content uniformity of Cetirizine Dihydrochloride tablets by HPLC.
Summary of validation report shall be prepare and accordingly standard testing procedure to be updated.
- Abbreviation
DIS : Dissolution
VAL : Validation
R : Report
SD : Standard deviation
HPLC : High performance liquid chromatography
DAD : diode-array detector
RT : Retention Time
mL : Milliliter
mg : Milligram
min. : Minutes
QA : Quality Assurance
QC : Quality Control
% : Percentage
ºC : Degree centigrade
Corr. coeff. (r) : Correlation coefficient
hrs : Hours
µm : Micrometer
µl : Microlitre
BP : British Pharmacopoeia
RSD : Relative standard deviation
NLT : Not less than
NMT : Not more than
WS : Working standard
Vol : Volume
AS : Standard Area
AT : Test Area
Revision History:
| Revision No. | Details of changes | Reason |
| 00 | Nil | New Document |
