by ANALYTICAL METHOD VALIDATION PROTOCOL FOR RELATED SUBSTANCES 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| Superseded Protocol No. | Nil |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 Related Substances of Cetirizine dihydrochloride tablets by considering thefollowing parameters:
| Parameters | Cetirizine Dihydrochloride |
| Specificity | |
| Limit of Detection and Quantification | |
| LOD Precision | |
| LOQ Precision | |
| Precision | |
| System Precision | |
| Method Precision | |
| Intermediate Precision | |
| Linearity | |
| Force Degradation | |
| Range | |
| Stability of Analytical Solution | |
| Filter Effect study | |
| Accuracy | |
| Robustness | |
| System Suitability |
- Scope :
This protocol is applicable for the Validation of Related Substances 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 | Related Substances Impurity A : Not more than 0.20% m/m Impurity G : Not more than 0.20% m/m 4-Chlorobenzophenone : Not more than 0.30% m/m Any other secondary peak : Not more than 0.10% m/m Total Impurities (Sum of all Impurities) : Not more than 0.80% m/m |
- Methodology:
Chemical, reagents and filters:
Table 1.0: Chemical, reagents and filters
| Sr. No | Material /Chemicals/Filters | Grade |
| 1.0 | Ortho phosphoric acid | AR Grade |
| 2.0 | Water | HPLC Grade or Milli Q water |
| 3.0 | Methanol | HPLC Grade |
| 4.0 | Acetonitrile | HPLC Grade |
| 5.0 | 0.45 µm membrane filter | Millipore or Equivalent |
| 6.0 | 0.45 µm Nylon filter | Millipore or Equivalent |
| 7.0 | Hydrochloric Acid | AR Grade |
| 8.0 | Sodium Hydroxide Pellets | AR Grade |
| 9.0 | Hydrogen Peroxide | AR Grade |
Chromatographic conditions (By HPLC):
Instrument : High Performance Liquid Chromatography (UV Visible/DAD Detector)
Column : Inertsil ODS-2 (150 x 4.6 mm), 5 μm
Flow rate : 1.5 ml/min
Detector : 230 nm
Column Temperature : 30ºC
Injection Volume : 10 µl
Sample Temperature : Ambient
Run time : 40.0 minutes
Retention time : Approximately 17.0 min (Cetirizine)
Program : Low pressure gradient
Mobile Phase : Solvent A: Weigh and dissolve 3.45 grams of ortho-Phosphoric acid in
2000 mL of water.
: Solvent B: Acetonitrile filtered through 0.45μ Nylon filter.
: Solvent C: Methanol filtered through 0.45μ Nylon filter.
Diluent : Water: Methanol (20:80) filtered through 0.45μ filter.
Gradient Program :
| S. No | Time | Solvent A | Solvent B | Solvent C |
| 1 | 0.01 | 60 | 5 | 35 |
| 2 | 22.00 | 60 | 5 | 35 |
| 3 | 25.00 | 40 | 60 | 0 |
| 4 | 30.00 | 40 | 60 | 0 |
| 5 | 32.00 | 60 | 5 | 35 |
| 6 | 40.00 | 60 | 5 | 35 |
System Suitability Solutions Preparation:
Impurity-G Stock Solution:
Take 5 mg of impurity-G into 50 mL volumetric flask and add 30 ml of methanol then sonicate for 5 minutes. Dilute to 50 ml with same methanol.
System Suitability Solution:
Take 50 mg of Cetirizine Dihydrochloride working standard into 25 ml volumetric flask and add 20 ml of diluent then sonicate for 5 minutes. Add 1 ml of Impurity-G stock solution and dilute to 25 ml with diluent.
Placebo Preparation:
Weigh approximately 720 mg of Cetirizine dihydrochloride tablets placebo into a 25 ml volumetric flask. Add about 15 ml of diluent and sonicate for 5 minutes and cool to room temperature if required, then make up to volume with diluent, mix the preparation. Then filter the preparation with 0.45μ Nylon filter.
Standard Preparation:
Weigh accurately 40 mg of Cetirizine dihydrochloride working standard and transfer into 100 ml volumetric flask. Add 60 ml of diluent and sonicate till dissolve, cool to room temperature if required. Then make up to the volume with same solvent. Further dilute 1 ml of above solution to 100 ml with diluent. Filter the Standard solution with 0.45 μ Nylon filter.
Sample Preparation: (Related Substances)
Weigh accurately about 5 Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate and approximate 18 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Evaluation of System Suitability:
- Resolution : Resolution between Cetirizine Dihydrochloride and Impurity G peak should not less than 5.0
- % Area RSD : Should not be more than 5.0 % for peak area due to Cetirizine dihydrochloride from six replicate injections of standard solution.
- Theoretical plates : Should not be less than 1000 for peak due to Cetirizine dihydrochloride.
- Tailing factor : Should not be more than 2.0 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 (Related Substances):
Table 2.0: Sequence of Injection for Related Substances
| Preparation | No. of Injections | Action |
| Blank Solution | 1 | Record the blank chromatograms to detect the blank peak. |
| Placebo Solution | 1 | Record the placebo chromatograms to detect the placebo peak. |
| System Suitability solution | 1 | Check for system suitability Criteria (i) |
| Standard solution | 6 | Check for system suitability Criteria (ii), (iii) and (iv). |
| Sample solution 1 | 1 | Inject the solution and calculate impurities. |
| Standard solution (Bracketing standard) | 1 | %, Area RSD of total 7 injections ≤ 5.0% (6 Injection of standard solution + 1 injection of bracketing standard) |
Specified Impurities are eluted in the respective order of, Impurity G, Impurity A, and Cetirizine dihydrochloride principal peak.
Identify the components in sample preparation with following relative retention times (RRT).
| S.No. | Component Name | Relative Retention Time (RRT) | Correction Factor (CF) |
| 1. | Cetirizine dihydrochloride | 1.00 | 1.00 |
| 2. | Impurity – A | 0.71 | 0.71 |
| 3. | Impurity – G | 0.63 | 0.95 |
| 4. | 4-Chlorobenzophenone | 0.89 | 1.4 |
Disregard limit:
Disregard all peaks less than 0.02%. (All peak area lesser than 1/10 times of diluted standard)
Below disregards limit for 4-Chlorobenzophenone is 0.01%m/m
Calculation:
AT WS 1 25 AW P
% Specified Impurity = ——- x ——- x —— x —— x —— x —— x 100 x CF
AS 100 100 WT LC 100
ATU Max WS 1 25 AW P
% Unspecified Impurity = ——- x ——- x —— x —— x —— x —— x 100
AS 100 100 WT LC 100
ATUk Sum WS 1 25 AW P
% Total unspecified Impurity = ——- x ——- x —— x —— x —— x —— x 100
AS 100 100 WT LC 100
% Total Impurities = Sum of Impurity A, Impurity G, 4-Chlorobezophenone and Total Unspecified Impurities
Where,
AS = Average peak area due to Cetirizine dihydrochloride obtained from six replicate
injections of standard solution.
AT = Peak area due to specified impurity from sample solution.
ATU Max = Peak area due to unspecified individual maximum impurity from sample solution.
ATUk Sum = Sum of peak area of all unspecified impurities from sample solution.
WS = Weight of Cetirizine Dihydrochloride RS/WS taken in mg.
WT = Weight of sample taken in mg.
AW = Average weight of tablets in mg.
LC = Label claim of Cetirizine Dihydrochloride in mg/tablet (10).
P = Potency of Cetirizine Dihydrochloride RS/WS in %, on as basis.
CF = Correction Factor
| S. No. | Impurity | Chemical Name |
| 1 | Impurity-A | (RS)-1-[(4-chlorophenyl) phenylmethyl] piperazine. |
| 2 | Impurity-G | 2-[4[(RS)-(4-chlorophenyl) phenylmethyl] piperazine-1yl] ethanol. |
| 3 | 4-Chlorobenzophenone | (4-Chlorophenyl)phenylmethanone |
- Validation parameters:
The following parameters to be perform for the Validation activity.
Specificity
Limit of Detection and Quantization
Precision
Force Degradation
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 (diluent), Placebo, standard solution, sample solution and spiked sample solution. Sample solution shall be prepared as per method of analysis.
Preparation of Identification solutions:
Stock solution of Impurity A:
Weigh accurately about 2.5 mg of Impurity A and transfer into 25 ml of volumetric flask, add 15 ml of diluent and dissolve. Make up the volume to 25 ml with diluent and mix.
Identification solution of Impurity A:
Pipette out 1.0 ml of stock solution of Impurity A and transfer into 25 ml of volumetric flask and make up with diluent, mix.
Stock solution of Impurity G:
Weigh accurately about 2.5 mg of Impurity G and transfer into 25 ml of volumetric flask, add 15 ml of diluent and dissolve. Make up the volume to 25 ml with diluent and mix.
Identification solution of Impurity G:
Pipette out 1.0 ml of stock solution of Impurity G and transfer into 25 ml of volumetric flask and make up with diluent, mix.
Stock solution of 4-chlorobenzophenone :
Weigh accurately about 2.5 mg of 4-chlorobenzophenoneand transfer into 25 ml of volumetric flask, add 15 ml of diluent and dissolve. Make up the volume to 25 ml with diluent and mix.
Identification solution of 4-chlorobenzophenone:
Pipette out 1.5 ml of stock solution of 4-chlorobenzophenone and transfer into 25 ml of volumetric flask and make up with diluent, mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
System Suitability Solutions Preparation:
Impurity-G Stock Solution:
Take 5 mg of impurity-G into 50 ml volumetric flask and add 30 ml of methanol then sonicate for 5 minutes. Dilute to 50 ml with same methanol.
System Suitability Solution:
Take 50 mg of Cetirizine dihydrochloride working standard into 25 ml volumetric flask and add 20 ml of diluent then sonicate for 5 minutes. Add 1 ml of Impurity-G stock solution and dilute to 25 ml with diluent.
(Note: lesser amount of impurities and working standard can be weighed to prepare standard stock solution of same final concentration.)
Placebo Preparation:
Weigh approximately 720 mg of Cetirizine dihydrochloride tablets placebo into a 25 ml volumetric flask. Add about 15 ml of diluent and sonicate for 5 minutes and cool to room temperature if required, then make up to volume with diluent, mix the preparation. Then filter the preparation with 0.45μ Nylon filter.
Standard Preparation:
Weigh accurately 40 mg of Cetirizine dihydrochloride working standard and transfer into 100 ml volumetric flask. Add 60 ml of diluent and sonicate till dissolve, cool to room temperature if required. Then make up to the volume with same solvent. Further dilute 1 ml of above solution to 100 ml with diluent. Filter the Standard solution with 0.45 μ Nylon filter.
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate and approximate 18 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Spiked Test solution:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate, further pipette out 1.0 ml of Impurity A, Impurity G and 1.5 ml of 4-Chlorobenzophenone and transfer, add approximate 12 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Procedure: Inject the preparation of blank solution (diluent), Placebo solution, standard solution, and Identification solution of Impurity A, Impurity G, 4-Chlorobenzophenone, sample solution and spiked sample solution on a HPLC system with a Diode array detector (DAD) as follows in table. Determine the purity of the individual peak of interest. Record the retention times of all peaks obtained from the respective solution and check for system suitability parameters for peak obtained.
Table 3.0: Sequence of Injection
| Solution | No of Injection to be injected in Sequence |
| Blank (diluent) | 1 |
| Placebo Solution | 1 |
| System Suitability solution | 1 |
| Standard solution | 6 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity G | 1 |
| Identification solution of 4-Chlorobenzophenone | 1 |
| 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 | Identification solutions | Impurity A | ||
| Impurity G | ||||
| 4-Chlorobenzophenone | ||||
| 5 | Sample Solution | Cetirizine Dihydrochloride | ||
| 6 | Spiked Solution | Cetirizine Dihydrochloride | ||
| Impurity A | ||||
| Impurity G | ||||
| 4-Chlorobenzophenone | ||||
| Unspecified impurities | NA |
Acceptance Criteria:
- There should be no interference of the diluent, placebo, Known Impurities 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 Known impurities peaks in spiked sample solution should be spectrally pure.
- Limit Of Detection And Quantification
Predictive Linearity
For determining LOQ, prepare a series of seven dilutions of linearity solutions with increasing concentrations. Inject in duplicate progressively lower concentrations and determine the areas. Plot a graph of concentration v/s area and determine slope of the line. The Predictive linearity solutions shall be prepares and inject by assuming the concentration of 1.0% to 200% of limit level for each of Impurities.
Preparation of Impurities Stock
Linearity Stock-I of Impurity A:
Weigh accurately about 2.5 mg of Impurity A and transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
Linearity Stock-I of Impurity G:
Weigh accurately about 2.5 mg of Impurity G and transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
Linearity Stock-II of Impurity A & G:
Pipette out 5.0 ml of stock solution of Impurity A and stock solution of Impurity G and transfer into 100 ml of volumetric flask and make up with diluent, mix.
Linearity Stock-I of 4-chlorobenzophenone :
Weigh accurately about 2.5 mg of 4-chlorobenzophenoneand transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
Linearity Stock-II of 4-chlorobenzophenone:
Pipette out 5.0 ml of stock solution of 4-chlorobenzophenone and transfer into 200 ml of volumetric flask and make up with diluent, mix.
Linearity Stock-I of Cetirizine Dihydrochloride :
Weigh accurately about 50 mg of Cetirizine Dihydrochloride WSand transfer into 100 ml of volumetric flask, add 80 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix. Further Dilute 1.0 of this solution and diluted to 200 ml with diluent and mixed.
Linearity Stock-II of Cetirizine Dihydrochloride:
Weigh accurately about 50 mg of Cetirizine Dihydrochloride WSand transfer into 100 ml of volumetric flask, add 80 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix. Further Dilute 5.0 of this solution and diluted to 50.0 ml with diluent and mixed.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
Table 5.0: Linearity levels preparation
| Level % | Vol. (ml) of Stock | Volume to be diluted with diluent (ml) | ||
| Impurity A & Impurity G | 4-Chlorobenzophenone | Cetirizine Dihydrochloride | ||
| 1 | Stock II = 0.8 ml | Stock II = 1.2 ml | Stock I = 0.5 ml | 25 |
| 2.5 | Stock II = 2.0 ml | Stock II = 3.0 ml | Stock I = 1.0 ml | 25 |
| 5 | Stock II = 4.0 ml | Stock II = 6.0 ml | Stock I = 2.0 ml | 25 |
| 50 | Stock I = 1.0 ml | Stock I = 1.5 ml | Stock II = 0.5 ml | 25 |
| 100 | Stock I = 2.0 ml | Stock I = 3.0 ml | Stock II = 1.0 ml | 25 |
| 150 | Stock I = 3.0 ml | Stock I = 4.5 ml | Stock II = 1.5 ml | 25 |
| 200 | Stock I = 4.0 ml | Stock I = 6.0 ml | Stock II = 2.0 ml | 25 |
Table 6.0: Prediction linearity for Impurity A
| Sr. No. | Level (%) | Theoretical Concentration (ppm) | Actual conc. (ppm) | Peak Area | Mean Peak Area | |
| I | II | |||||
| 1 | 1 | 0.08 | ||||
| 2 | 2.5 | 0.20 | ||||
| 3 | 5 | 0.40 | ||||
| 4 | 50 | 2.00 | ||||
| 5 | 100 | 4.00 | ||||
| 6 | 150 | 6.00 | ||||
| 7 | 200 | 8.00 | ||||
| Correlation Coefficient (r) | ||||||
| Standard Error | ||||||
| Slope | ||||||
| Limit of detection (LOD), ppm | ||||||
| Limit of quantitation (LOQ), ppm |
Table 7.0: Prediction linearity for Impurity G
| Sr. No. | Level (%) | Theoretical Concentration (ppm) | Actual conc. (ppm) | Peak Area | Mean Peak Area | |
| I | II | |||||
| 1 | 1 | 0.08 | ||||
| 2 | 2.5 | 0.20 | ||||
| 3 | 5 | 0.40 | ||||
| 4 | 50 | 2.00 | ||||
| 5 | 100 | 4.00 | ||||
| 6 | 150 | 6.00 | ||||
| 7 | 200 | 8.00 | ||||
| Correlation Coefficient (r) | ||||||
| Standard Error | ||||||
| Slope | ||||||
| Limit of detection (LOD), ppm | ||||||
| Limit of quantitation (LOQ), ppm |
Table 8.0: Prediction linearity for 4-Chlorobenzophenone
| Sr. No. | Level (%) | Theoretical Concentration (ppm) | Actual conc. (ppm) | Peak Area | Mean Peak Area | |
| I | II | |||||
| 1 | 1 | 0.06 | ||||
| 2 | 2.5 | 0.15 | ||||
| 3 | 5 | 0.30 | ||||
| 4 | 50 | 3.0 | ||||
| 5 | 100 | 6.0 | ||||
| 6 | 150 | 9.0 | ||||
| 7 | 200 | 12.0 | ||||
| Correlation Coefficient (r) | ||||||
| Standard Error | ||||||
| Slope | ||||||
| Limit of detection (LOD), ppm | ||||||
| Limit of quantitation (LOQ), ppm |
Table 9.0: Prediction linearity for Cetirizine Dihydrochloride
| Sr. No. | Level (%) | Theoretical Concentration (ppm) | Actual conc. (ppm) | Peak Area | Mean Peak Area | |
| I | II | |||||
| 1 | 1 | 0.05 | ||||
| 2 | 2.5 | 0.1 | ||||
| 3 | 5 | 0.2 | ||||
| 4 | 50 | 1.0 | ||||
| 5 | 100 | 2.0 | ||||
| 6 | 150 | 3.0 | ||||
| 7 | 200 | 4.0 | ||||
| Correlation Coefficient (r) | ||||||
| Standard Error | ||||||
| Slope | ||||||
| Limit of detection (LOD), ppm | ||||||
| Limit of quantitation (LOQ), ppm |
The LOD and LOQ are calculated based on the following formula
LOD = Standard Error (σ) x 3.3
——————————–
Slope (S)
LOQ = Standard Error (σ) x 10
————————————
Slope (S)
LOQ and LOD Precision
To confirm the values of LOQ and LOD obtained from prediction linearity, prepare solution of Cetirizine dihydrochloride, impurity A, Impurity G and 4-chlorobenzophenone at LOQ and LOD concentration by using stock solutions with suitable dilutions and inject six replicate injections to check the %RSD and Signal to noise ratio. Summaries the data obtained in the following tables
Table 10.0: Precision at LOD
| COMPONENT® |
Impurity A, Impurity G, Cetirizine
Dihydrochloride, 4-Chlorobenzophenone | ||
| Peak Area | Signal/Noise Ratio | ||
| Theoretical Conc. (ppm) | |||
| Actual Conc. (ppm) | |||
| 1 | |||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | |||
| 6 | |||
| Mean | |||
| SD | |||
| % RSD | |||
| Acceptance criteria | NMT 30.0% | 3:1 |
Table 11.0: Precision at LOQ
| COMPONENT® | Impurity A, Impurity G, Cetirizine Dihydrochloride, 4-Chlorobenzophenone | |
| Theoretical Conc. (ppm) | ||
| Actual Conc. (ppm) | ||
| Peak Area | Signal/Noise Ratio | |
| 1 | ||
| 2 | ||
| 3 | ||
| 4 | ||
| 5 | ||
| 6 | ||
| Mean | ||
| SD | ||
| % RSD | ||
| Acceptance criteria | NMT 10.0% | 10:1 |
Acceptance criteria:
The correlation coefficient of predictive linearity of Impurity A, Impurity G, Cetirizine Dihydrochloride, 4-Chlorobenzophenone from LOD or LOQ to 200% should not less than 0.99. The % RSD of Peak Area should be NMT 10.0% for Limit of Quantization and % RSD of peak area should be NMT 30.0% for Limit of Detection. Signal to Noise Ratio for LOD should be 3:1 and Signal to Noise Ratio for LOQ should be 10:1.
- 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 12.0: 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 5.0% 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:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the Impurity A, Impurity F and 4-chlorobenzophenone at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Obtained results will be report in tabulated manner as given below.
Table 13.0: Method precision results for Related Substances
| Sr. No. | Spl. Wt. (mg) | Impurity A (%) | Impurity G (%) | 4-Chlorobenzophenone (%) | Any Other Secondary Impurity (%) | %Total Impurities (Sum of All expect 4-chlorobenzophenone) | |
| 1 | 2 | ||||||
| RRT | |||||||
| 1 | |||||||
| 2 | |||||||
| 3 | |||||||
| 4 | |||||||
| 5 | |||||||
| 6 | |||||||
| Mean | |||||||
| SD | |||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- 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 14.0: 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 samples of same batch by another analyst as per standard analytical procedure and analyzed by spiking the Impurity A, Impurity F and 4-chlorobenzophenone at limit level. Un-spiked sample will be analyzed to identify the known impurity in sample. Obtained known impurity in un-spiked sample will be subtracted in spiked sample to calculate the actually known spiked amount. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 15.0: Intermediate precision results for Related Substances
| Sr. No. | Spl. Wt. (mg) | Impurity A (%) | Impurity G (%) | 4-Chlorobenzophenone (%) | Any Other Secondary Impurity (%) | %Total Impurities (Sum of All expect 4-chlorobenzophenone) | |
| 1 | 2 | ||||||
| RRT | |||||||
| 1 | |||||||
| 2 | |||||||
| 3 | |||||||
| 4 | |||||||
| 5 | |||||||
| 6 | |||||||
| Mean | |||||||
| SD | |||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
Table 16.0: Pooled results of Related Substances of analyst – I & analyst – II
| Sr. No. | Spl. Wt. (mg) | Impurity A (%) | Impurity G (%) | 4-Chlorobenzophenone (%) | Any Other Secondary Impurity (%) | %Total Impurities (Sum of All expect 4-chlorobenzophenone) | |
| 1 | 2 | ||||||
| Analyst I | |||||||
| RRT | |||||||
| 1 | |||||||
| 2 | |||||||
| 3 | |||||||
| 4 | |||||||
| 5 | |||||||
| 6 | |||||||
| Analyst II | |||||||
| 1 | |||||||
| 2 | |||||||
| 3 | |||||||
| 4 | |||||||
| 5 | |||||||
| 6 | |||||||
| Mean | |||||||
| SD | |||||||
| % RSD |
Acceptance criteria:
% RSD of known, unknown and total impurity content should be not more than the limits specified below.
Result observed Limit for %RSD
Between 0.11 and 0.99 % 15.0%
Greater than 1.0% 10.0%
Impurity content below 0.10% should not be considered for %RSD.
- Force Degradation Study
For stability indicating method, it is essential to perform forced degradation studies by appling appropriate accelerated stress conditions to the sample and placebo, the proposed stress conditions are:
Procedure:
Prepare blank, placebo, standard solution, untreated test solution and treated test solution. In case of related substances placebo is also treated in same manner as sample treated. Placebo weight shall be taken equivalent to 5 tablets. Inject and calculate the % Impurities. Calculate the % difference between the untreated test solutions and treated test solution results. Check for 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% (Oxidation 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)
- Acid Hydrolysis :
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. Add 3.0 ml of 5M HCl and stand for 60 minutes, further add 3.0 ml of 5M NaOH to neutralize the sample. Add approximate 10 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
- Base Hydrolysis :
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. Add 3.0 ml of 5M NaOH and stand for 60 minutes, further add 3.0 ml of 5M HCl to neutralize the sample. Add approximate 10 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
- Oxidative Degradation:
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. 5.0 ml of 30% H2O2 and stand for 60 minutes add approximate 10 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
- Thermal Degradation (at 80°C/ 24 hours)
Tablets store at control temperature condition of 80°C for thermal degradation for 24 hour.
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. Add approximate 18 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
- Humidity Degradation (at 25°C/ 90% RH)
Tablets store at control temperature condition of 25°C and 90% RH for Humidity degradation.
Preparation of Humidity Chamber:
Prepare super saturated solution of Potassium Nitrate and keep this solution in a well closed desiccator. Keep the sample and place in the chamber.
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. Add approximate 18 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
- Water Hydrolysis :
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 10 ml of water, sonicate until tablets disintegrate. Heat the sample at 105°C for 60 minutes. Add more water when the sample gets dry. Cool the sample and add approximate 18 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
- Photolytic Degradation (Ultraviolet light at 365 nm for 2-4 hours):
Tablets store under ultraviolet light at 365 for 2-4 hour for Photolytic degradation.
Sample Solution:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 mL volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate. Add approximate 18 ml of methanol and sonicate for 5 minutes, cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter by discarding first two ml and inject.
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.
Mass Balance Determination has to be verified by demonstrating that the decrease the concentration of substance exposed to stress conditions.
- 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 (spiked) at room temperature (ambient temperature) during analytical measurement stages of the testing.
Test Procedure:
Prepared the blank solution, standard solution and test solution by spiking known impurities (Impurity A, Impurity G and 4-Chlorobenzophenone) at specification limit level 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 % Related Substances 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 area % RSD of standard solution should be not more than 5.0 %. Cumulative % RSD of peak area for known impurities and main peak shall not be more than10%.
- 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 Nylon syringe filter, further analyse as per the method and inject. From the data calculate the % Related Substances.
Table 17.0: Effect of Filtration (Impurity A, Impurity G and 4-Chlorobenzophenone)
| Sample | Filter Used | Mean Peak area | % Impurities | Mean % Impurities | Difference (%) |
| Impurity A, Impurity G and 4-Chlorobenzophenone | |||||
| 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 % Impurities values should be NMT 10.0%.
- 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 by spiking Impurity A, Impurity G and 4-chlorobenzophenone with test solution to the place at about LOQ, 50%, 100% and 200%, of specification limit 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.
Preparation of Identification solutions:
- Accuracy stock-I of Impurity A:
Weigh accurately about 2.5 mg of Impurity A and transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
- Accuracy stock-I of Impurity G:
Weigh accurately about 2.5 mg of Impurity G and transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
- Accuracy stock-I of 4-chlorobenzophenone :
Weigh accurately about 2.5 mg of 4-chlorobenzophenoneand transfer into 50 ml of volumetric flask, add 30 ml of diluent and dissolve. Make up the volume to 50 ml with diluent and mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
Placebo Preparation:
Weigh approximately 720 mg of Cetirizine dihydrochloride tablets placebo into a 25 ml volumetric flask. Add about 15 ml of diluent and sonicate for 5 minutes and cool to room temperature if required, then make up to volume with diluent, mix the preparation. Then filter the preparation with 0.45μ Nylon filter.
Standard Preparation:
Weigh accurately 40 mg of Cetirizine dihydrochloride working standard and transfer into 100 ml volumetric flask. Add 60 ml of diluent and sonicate till dissolve, cool to room temperature if required. Then make up to the volume with same solvent. Further dilute 1 ml of above solution to 100 ml with diluent. Filter the Standard solution with 0.45 μ Nylon filter.
Sample Preparation:
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate and approximate 18 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Spiked Test solution: (limit Level)
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate, further pipette out 1.0 ml of Impurity A, Impurity G and 1.5 ml of 4-Chlorobenzophenone and transfer, add approximate 12 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Level I: LOQ
LOQ sample solution would be spiked at LOQ level after deciding the LOQ from limit of detection and quantification test.
Level I: 50%
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml of volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate, further pipette out 1.0 ml of each of Impurity A, Impurity G and 1.5 ml of 4-Chlorobenzophenone and transfer into same volumetric flask of 25 ml, add approximate 10 ml of methanol and sonicate for 5 minutes and cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two ml and inject.
Level II: 100%
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 5 ml of water, shake and sonicate until tablets disintegrate, further pipette out 2.0 ml of each of Impurity A, Impurity G and 3.0 ml of 4-Chlorobenzophenone and transfer, add approximate 5 ml of methanol and sonicate for 5 minutes cool to room temperature if required, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Level II: 200%
Weigh accurately 5 intact Tablets (Equivalent to 50mg of Cetirizine dihydrochloride) and transfer into a 25 ml volumetric flask. Add about 2 ml of water, shake and sonicate until tablets disintegrate, further pipette out 4.0 ml of each of Impurity A, Impurity G and 6.0 ml of 4-Chlorobenzophenone and transfer, add approximate 5 ml of methanol and sonicate for 5 minutes cool to room temperature, then make up to volume with methanol, mix the solution and filter the preparation through 0.45μ Nylon filter discarding first two and inject.
Calculation:
Amount Recovered in ppm
% Recovery = ————————————— x 100
Amount added in ppm
Table 18.0: Accuracy for Impurity A, Impurity G and 4-Chlorobenzophenone
| Levels (%) | Amount added in Sample (ppm) | Amount recovered in Sample (ppm) | Recovery % | Mean Recovery % | SD | RSD% |
Acceptance criteria
The recovery of known impurities of the drug at each spiking level shall be between 85% and 115% and RSD from replicate analysis shall be not more than 10%.The overall average recovery shall be between 85% to 115% with RSD of not more than 10%.
- 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 Sonication time of test solution by time Interval of 5 minutes.
System suitability parameters are performed as per testing procedure for each deliberate variation.
Test Procedure:
The standard solution and test solution are prepared by spiking with known impurities at specification limit level in sample as per the method by deliberate variations made in the method for each condition as mentioned in protocol and analyze. Calculated the % Impurity at each deliberate variation with respect to the replicate injection of standard solution is tabulate below.
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
System suitability was evaluated by injecting replicate injection of standard solution and single injection of system suitability during various days of validation. The area % relative standard deviation, theoretical plates and tailing factor for the peak of Cetirizine Dihydrochloride in standard solution and the resolution of peak due to impurity G and Cetirizine dihydrochloride in system suitability solution were verified at each stage. Evaluation of system suitability is tabulated in below Table 21.0.
Table 19.0: System suitability parameters
| Sr. No. | Parameter | Set limits |
| 1. | Resolution between Cetirizine Dihydrochloride and Impurity G peak. | NLT- 5.0 |
| 2. | RSD for the peak area of the six replicate injections of Cetirizine dihydrochloride. | NMT – 5.0% |
| 3. | Theoretical Plates for Cetirizine dihydrochloride peak | NLT – 1000 |
| 4. | Tailing factor for Cetirizine dihydrochloride peak | NMT – 2.0 |
Summary of system suitability of Cetirizine Dihydrochloride is tabulated below in Table 45.0.
Table 45.0
| Sr. No. | Name of experiment | Date | Tailing Factor | Theoretical plates | % RSD | Resolution |
| 1 | Specificity | |||||
| 2 | Predictive Linearity | |||||
| 3 | LOD and LOQ Precision | |||||
| 4 | Methanol Precision | |||||
| 5 | Intermediate Precision | |||||
| 6 | Solution stability | |||||
| 7 | Force Degradation | |||||
| 8 | Accuracy | |||||
| 9 | Filter effect study | |||||
| 10 | Robustness | |||||
| Flow rate 1.35 ml /min | ||||||
| Flow rate 1.65 ml /min | ||||||
| Oven Temp 25°C | ||||||
| Oven Temp 35°C | ||||||
| Change in Sonication time |
- 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 Related Substances test of Cetirizine Dihydrochloride and Related Substances of Cetirizine Dihydrochloride tablets by HPLC.
Summary of validation report shall be prepare and accordingly standard testing procedure to be updated.
Abbreviation
REL : Related Substances
VAL : Validation
P : Protocol
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 |
