by RESUDIAL SOLVENT METHOD VERIFICATION PROTOCOL OF CETIRIZINE DIHYDROCHLORIDE Ph. Eur.
| Superseded Protocol No. | Nil |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 verification is to provide documentary evidence that analytical methodology used for residual solvents of Cetirizine Dihydrochloride by vendor method is consistent and reliable results within the predetermined acceptance criteria.
Analytical method verification will be performed by considering thefollowing parameters:
| Parameters | Cetirizine Dihydrochloride |
| Specificity | yes |
| Precision | |
| System Precision | yes |
| Method Precision | yes |
| Intermediate Precision (Ruggedness) | yes |
| Linearity | yes |
| Stability of Analytical Solution | yes |
| System Suitability | yes |
- Scope :
The scope of this protocol is applicable for the verification of method of residual solvents of Cetirizine Dihydrochloride.
- 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 | Active Product Ingredient |
| Reason for Verification | In House Validated Method |
| Active Ingredient | Cetirizine Dihydrochloride |
| Method Reference | API Manufacturer ( In-House) |
| Specification Limits | Methanol : Not More than 3000 ppm Acetone: Not More than 5000 ppm Methylene Chloride : Not More than 600 ppm Chloroform : Not More than 60 ppm Ethyl Acetate : Not More than 5000 ppm Toluene : Not More than 890 ppm Dimethyl Formamide : Not More than 880 ppm Benzene : Not More than 2 ppm |
- Methodology:
Solvents:
Table 1.0
| Solvents | Grade |
| 1-Methyl-2-Pyrrolidinone(NMP) | LR/ AR Grade |
| Methanol | LR/ AR Grade |
| Acetone | LR/ AR Grade |
| Methylene Choride | LR/ AR Grade |
| Chloroform | LR/ AR Grade |
| Ethyl Acetate | LR/ AR Grade |
| Toluene | LR/ AR Grade |
| Dimethyl Formamide | LR/ AR Grade |
| Benzene | LR/ AR Grade |
Residual Solvents (By GC)
Instrument : Gas Chromatography with Head Space
Make of the Instrument : Agilent Technologies
Detector : FID
Column : Capillary Column DB0624, Length 30 M,ID 0.53 Film 3.0 µm
Oven temperature (1) : 40˚C
Injector temperature : 220˚C
Detector temperature : 260˚C
Oven temperature (I) : Initial Temperature 4°C for 10 min
Ramp Rate-I : 8°C/min
Oven temperature (II) : 175°C
Ramp Rate- : 35°C/min
Split ratio : 1: 5
Carrier gas : Nitrogen
Column flow : 3.0 ml/min
Head space parameters:
Vial temperature : 90˚C
Transfer line temperature : 115˚C
Vial Equilibrium Time : 50 min
Loop Fill Time : 0.2 min
Injection time : 1.0 min
Vial Shake Time : 5.0 min
Loop temperature : 110˚C
GC cycle time : 50 min
Pressurization time : 2.0 min
Loop Equilibrium Time : 0.1 min
Injection Volume : 1.0 ml of Vapor
Blank preparation (Diluent):
Transfer 2.0 ml of 1-methyl-2-pyrrolodine (NMP) into the head space vial and seal the cap tightly.
Standard Solution preparation:
Solution-I: Take accurately 11.4 µl from Benzene standard into a 100 ml volumetric flask Containing about 50 ml NMP and dilute to volume with NMP.
Solution-II: Table-I
| Sr.No. | Name of the Solvent | Volume Taken | Diluted With | Diluted to |
| 01 | Methanol | 189.9 µl | NMP | 100 ml |
| 02 | Acetone | 316.5 µl | ||
| 03 | Methylene Choride | 22.7 µl | ||
| 04 | Chloroform | 2.0 µl | ||
| 05 | Ethyl Acetate | 277.8 µl | ||
| 06 | Toluene | 51.4 µl | ||
| 07 | Dimethyl Formamide | 46.4 µl | ||
| 08 | Benzene (Solution-I) | 1.0 ml |
Solution-II: Contain the PPM value of solvent as follows:
Table-II
| Sr.No. | Name of the Solvent | Concentration (PPM) |
| 01 | Methanol | 1500 |
| 02 | Acetone | 2500 |
| 03 | Methylene Choride | 300 |
| 04 | Chloroform | 30 |
| 05 | Ethyl Acetate | 2500 |
| 06 | Toluene | 445 |
| 07 | Dimethyl Formamide | 440 |
| 08 | Benzene (Solution-I) | 1 |
Take 2.0 ml form solution-II into Headspace vial (Six Vials).
Test Solution Preparation:
Take 1.0 gm of Cetirizine Dihydrochloride sample in a 20 ml headspace vial, add 2.0 ml of
1-Methyl-2-Pyrrolidinone (NMP). Immediately crimp the cap.
System Suitability:
Calculateaverage peak areas and % RSD for 6 Chromatogram NMT 10.0 %.
Procedure: Insert the following vials in following order
| Solutions | No of Injection to be injected in Sequence |
| Blank (NMP 2.0 ml) | 1 |
| Standard Solution-II Six vials(each 2.0 ml) | 6 |
| Blank (NMP 2.0 ml) | 1 |
| Test Solution | 2 |
| Standard Solution-II (Bracketing) | 1 |
AT DS Purity
Calculation: =—————–X—————–X——————-X Density X 106
AS DT 100
Where,
AT : Area of the solvent peak in test solution
As : Average area of the solvent peak in standard solution
DS : Concentration of standard solution
DT : Weight taken of test solution (g)
- Validation parameters:
The following parameters to be perform for the verification activity.
- Specificity
- Precision
- Stability of solution
- System Suitability
- Linearity
- Specificity:
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.
The diluent (1-methyl-2-pyrrolidone) and solution of each solvent were prepared separately and analyzed. Also of mixture these solvents was prepared as their specification limit concentration and analyzed (Refer to Table-II).
Specificity of test method should be established by separately injecting blank solution (1-methyl-2-pyrrolidone) and Identification solution as per Table-II. Spiked test solution and test solution. Identification solution and spiked test solution will be prepared at specification level. Test solution to be prepared as per method of analysis.
Preparation of Identification solutions:
Standard solution of Methanol (1500 ppm):
Weigh accurately about 189.9 µl of Methanol and transferred to 100 ml of volumetric flask, add 60 ml diluent and dissolve. Make up the volume to 100 ml with diluent.
Standard solution of Acetone (2500 ppm):
Weigh accurately about 316.5 µl of Acetone and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
Standard solution of Methylene Chloride (300 ppm):
Weigh accurately about 22.7 µl of Methylene Chloride and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
Standard solution of Chloroform (30 ppm):
Weigh accurately about 2.0 µl of Chloroform and transferred to 100 ml volumetric flask, add 50ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
- Standard solution of Ethyl Acetate (2500 ppm):
Weigh accurately about 277.8 µl of Ethyl Acetateand transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
- Standard solution of Toluene (445 ppm):
Weigh accurately about 51.4 µl of Tolueneand transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
- Standard solution of Dimethyl Formamide (440 ppm):
Weigh accurately about 46.4 µl of Tolueneand transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
- Standard solution of Benzene (1 ppm):
Weigh accurately about 11.4µl of Benzene and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Further dilute 1.0ml this solution to 100.0 ml with diluent.
Spiked test solution:
Transfer Take 1.0 gm of Cetirizine Dihydrochloride sample in a 20 ml headspace vial, add 2.0 ml of flask; add 30 ml of diluent to dissolve further add 5.0 ml stock solution of impurity A and 3.0 ml of stock solution of impurity F and mix well, make up the volume to 50 ml with diluent.
Procedure: Inject the preparation of blank solution, reference solution (a), and identification solution of impurity A, identification solution of impurity F, test solution and spiked test solution on a HPLC system with a Diode array detector (DAD) as follows in Table 3.0. Determine the purity of the individual peaks of interest. Record the retention times and system suitability parameters for all peaks. System suitability criteria should be checked.
Tablet 3.0
| Solution | No of Injection to be injected in Sequence |
| Blank (Mobile Phase) | 1 |
| Reference solution (a) | 6 |
| Identification solution of Impurity A | 1 |
| Identification solution of Impurity F | 1 |
| System suitability solution | 1 |
| Test Solution | 1 |
| Spiked Test Solution | 1 |
| Reference solution (a) + Bracketing | 1 |
Acceptance Criteria:
- There should be no interference of the diluent, impurities at the retention time of analyte peak,
- Impurity peaks should be well resolved from active peak and each other,
- Analyte peak in standard solution and known Impurity peaks in spiked sample solution should be spectrally pure.
Table 4.0 Specificity data
| Sr. No | Sample | RT (min.) | RRT | Peak purity | ||
| 1 | Blank | NA | ||||
| 2 | Reference solution (a) | Diclofenac Sodium | ||||
| 3 | System Suitability solution | Impurity A | ||||
| Impurity F | ||||||
| 4 | Identification solution of Impurity A | |||||
| 5 | Identification solution of Impurity F | |||||
| 6 | Test Solution | Diclofenac Sodium | ||||
| 7 | Unknown Impurity | NA | ||||
Table 5.0 Spiked test solution
| Sr. No | Sample | RT (min.) | RRT | Peak purity |
| 1 | Blank | NA | ||
| 2 | Diclofenac Sodium | |||
| 3 | Impurity A | |||
| 4 | Impurity F | |||
| 5 | Unknown Impurity | NA |
- 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 of main analyte peak. And calculate the % area RSD and % RT RSD of main analyte peak.
Table 6.0 System Precision- Repeatability of Standard Injections
| Sr. No. | Diclofenac Sodium | |
| 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.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity 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 of impurity. Record the area on data sheet and calculate the % impurity, mean, standard deviation and % relative standard deviation.
Table 8.0 Method precision results spiked Sample at limit level
| Sr. No. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 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. Reporting threshold value is 0.05%.
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.
Test Procedure:
Prepare the six samples of same batch as per standard analytical procedure and analyzed by spiking the known impurity 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 10.0 Method precision results spiked sample at limit level
| Sr. No. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 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. Reporting threshold value is 0.05%.
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 11.0 Pooled results of analyst – I & analyst – II
| Sr. No. | Spl. Wt. (mg) | Diclofenac Impurity A (%) | Diclofenac Impurity F (%) | Unknown Impurities (%) | %Total imp. (≥0.05%) | |
| 1 | 2 | |||||
| RRT | ||||||
| ANALYST I | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| RRT | ||||||
| ANALYST II | ||||||
| 1 | ||||||
| 2 | ||||||
| 3 | ||||||
| 4 | ||||||
| 5 | ||||||
| 6 | ||||||
| Mean | ||||||
| SD | ||||||
| % RSD |
Acceptance criteria:
Pooled % 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.
- 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 at room temperature (ambient Temperature) during analytical measurement stages of the testing.
Test Procedure:
Prepared the Blank solution, standard solution and test solution as per method of analysis and spiked the test solution with Impurity A & Impurity F at limit level 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 cumulative RSD of peak area for known Impurity and main peak.
The obtained area of known impurities in test solution after different time interval will be calculated. The obtained area of Reference solution (a) after different time interval will be considered to calculate the area % RSD of initial replicate standard.
Acceptance criteria
Cumulative % RSD of peak area for known Impurities and main peak shall not be more than 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.
Determine the linearity by preparing and inject the Reference solution (a) and Impurity A and Impurity F in the range of reporting threshold value of 0.05 % of test concentration to 200% of limit level and calculate the correlation coefficient “r”.
Prepare the solution at below threshold value, threshold value, 50%, 100%, 150% and 200%.
Preparation of Linearity solutions:
i) Linearity Stock solution of Impurity A (20.0 ppm):
Weigh accurately about 2.0 mg of Impurity A and transferred to 100 ml of volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
ii) Linearity Stock solution of Impurity F (25.0 ppm):
Weigh accurately about 2.5 mg of Impurity F and transferred to 100 ml volumetric flask, add 70 ml of diluent and dissolve. Make up the volume to 100 ml with diluent and mix.
(Note: Depending on Purity of Impurity, adjust the weight of Impurity to achieve final concentration of impurity.)
iii) Stock solution of Diclofenac Sodium:
Transfer 50.0 mg of Diclofenac sodium CRS/WS in 50.0 ml of volumetric flask, add 30.0 ml of diluent and dissolve, make up the volume to 50.0 ml with mobile phase.
Further dilute 2.5 ml of this solution to 100.0 ml with mobile phase.
Table 12: Linearity levels preparation
| Level % | Vol. (mL) of Stock | Volume to be diluted with diluent (ml) | ||
| Impurity A | Impurity F | Diclofenac Sodium | ||
| Below Threshold | 2.0 | 1.8 | 1.6 | 100 |
| Threshold value | 2.5 | 2.0 | 2.0 | 100 |
| 50% | 2.5 | 1.5 | 1.0 | 50 |
| 100% | 5.0 | 3.0 | 2.0 | 50 |
| 150% | 3.0 | 1.8 | 1.2 | 20 |
| 200% | 5.0 | 3.0 | 2.0 | 25 |
Table 13: Linearity of Impurity A
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.04 | 0.4 | |||
| Threshold | 0.05 | 0.5 | |||
| 50 | 0.10 | 1.0 | |||
| 100 | 0.20 | 2.0 | |||
| 150 | 0.30 | 3.0 | |||
| 200 | 0.40 | 4.0 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Table 14: Linearity of Impurity F
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.045 | 0.45 | |||
| Threshold | 0.05 | 0.50 | |||
| 50 | 0.75 | 0.75 | |||
| 100 | 0.15 | 1.50 | |||
| 150 | 0.225 | 2.25 | |||
| 200 | 0.30 | 3.00 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Table 15: Linearity of Diclofenac Sodium (Unspecified Impurity)
| %Level | Conc. (%) | Theoretical Conc. (ppm) | Actual Conc. (ppm) | Peak Area | Mean peak area |
| Below Threshold | 0.04 | 0.4 | |||
| Threshold | 0.05 | 0.5 | |||
| 50 | 0.05 | 0.5 | |||
| 100 | 0.10 | 1.0 | |||
| 150 | 0.15 | 1.5 | |||
| 200 | 0.20 | 2.0 | |||
| Slope | |||||
| Intercept | |||||
| Corr. coeff.(r) |
Acceptance criteria
Plot the calibration curve of peak area v/s concentration for the studied peak. The correlation coefficient (r), of known impurities and Diclofenac sodium should not be less than 0.99.
- System Suitability:
System suitability tests are based on concept that the equipment, electronics, analytical operations and sample to be analyzed, system suitability test provide the added assurance that on specific occasion the method is given accurate and precise results.
The system suitability should be as per below mention criteria in Table 16.0.
Table 16.0
| Parameters | System Suitability Criteria | Limit |
| Resolution | The resolution between the peaks due to impurity F and Diclofenac | Minimum 4.0 |
| Area % RSD | The area %RSD of six replicate injection of reference solution (a) | NMT – 5.0% |
- Incident/Deviation:
Any Incident or Deviation observed during Analytical Method validation should be recoded and reported in Validation Report.
- Summary/Conclusion/recommendation:
Final Conclusion should be drawn from analytical method verification for its use to analyze the related substances of Diclofenac sodium by pharmacopeia method and if any differences shall be observed between methodology and experiments than accordingly will be revised and update our methodology. Abbreviations:
REL : Related substance
VAL : Validation
R : Report
RSD : Related standard deviation
HPLC : High performance liquid chromatography
mL : Milliliter
mg : Milligram
min. : Minutes
QA : Quality Assurance
QC : Quality Control
% : Percentage
ºC : Degree centigrade
hrs : Hours
µm : Micrometer
µl : Microlitre
BP : British Pharmacopoeia
RSD : Relative standard deviation
NLT : Not less than
NMT : Not more than
Imp : Impurity
Ws : Working standard
Vol : Volume Revision History :
| Revision No. | Details of changes | Reason for change |
| 00 | Nil | New Document |
