ANALYTICAL METHOD VALIDATION PROTOCOL OF ASSAY AND CONTENT UNIFORMITY OF CETIRIZINE DIHYDROCHLORIDE TABLETS

ANALYTICAL METHOD VALIDATION PROTOCOL OF ASSAY AND CONTENT UNIFORMITY 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Superseded Protocol No.	Nil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

1. 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 assay and content uniformity of Cetirizine dihydrochloride tablets by considering the following parameters:

Parameters	Cetirizine Dihydrochloride
Specificity
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 assay and content uniformity 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	Assay: Cetirizine Dihydrochloride : Between 95.0 % and 105.0 % of stated amount Content Uniformity: Cetirizine Dihydrochloride : Acceptance value L1 ≤ 15.0 (For 10 Dosage Units) Acceptance value L2 ≤ 25.0 (For 30 Dosage Units)

• 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

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: Acetonitrile (800:100:100) mix well and add 1.0 ml of

ortho phosphoric acid and filter 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 50 mg of Cetirizine dihydrochloride working standard, transfer into a 200 mL volumetric flask. Add 160ml of diluent and sonicate till dissolve and dilute to volume with diluent, filter through 0.45μ nylon filter.

Further dilute 2 ml of above solution to 20 ml with diluent, mix. (Concentration 25 ppm)

Sample Preparation: (Assay)

(Note: Prepare two samples inject in duplicate.)

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 400ml of diluent and mix sonicate for 5.0 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

Sample Preparation: (Content Uniformity)

Transfer one tablet to a 100 ml volumetric flask, Add 70 ml of diluent and sonicate for 5 minutes with occasional shaking. Make volume up to the mark with diluent and mix. Filter the solution through 0.45 μm Nylon filter; collect the filtrate by discarding first two ml of the filtrate.

5 ml of above filtrate diluted to 20 ml with diluent mix well and inject

Perform content uniformity on 10 tablets.

 Evaluation of System Suitability:

1. Area % RSD             :   Should not be more than 2.0 % for peak area due to Cetirizine dihydrochloride from 5 replicate injections of standard solution.
2. Tailing factor           :   Should not be more than 2.0 for peak due to Cetirizine dihydrochloride.
3. 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 (Assay):

Table 2.0: Sequence of Injection for assay

Preparation	No. of Injections	Action
Blank Solution	1	Record the blank Chromatograms
Standard solution	5	Check for system suitability Criteria (i), (ii) and (iii).
Sample solution 1	2	Inject the solution and calculate for mean injection and report
Sample solution 2	2	Inject the solution and calculate for mean injection and report
Standard solution (Bracketing standard)	1	%, Area RSD of total 6 injections ≤ 2.0% (5 Injection of standard solution + 1 injection of bracketing standard)

Sequence of Injections (Content Uniformity):

Table 3.0: Sequence of Injection of content uniformity

Preparation	No. of Injections	Action
Blank Solution	1	Record the blank Chromatograms
Standard solution 1	5	Check for system suitability Criteria (i), (ii) and (iii)
Sample solution 1 to 10	1	Inject the individual solution, Maximum 10 in a set.
Standard solution 1 (Bracketing standard)	1	%, Area RSD of total 6 injections ≤ 2.0% (5 Injection of standard solution  + 1 injection of bracketing standard)

Calculation for Assay (% labeled amount):

Calculate the percentage of Cetirizine hydrochloride, based on the label claim, in the portion of Tablets taken by the formula:          

                                                                                   AT         WS           2      500     20        AW      P                        

% Cetirizine Dihydrochloride =    ——- x ——- x —— x —— x —— x —— x —— x 100

                                                              AS       200       20       W_T       5         LC      100

Where,

AS    =       Average peak area due to Cetirizine Dihydrochloride obtained from five replicate

                  injections of standard solution.

AT    =       Average Peak area due to Cetirizine Dihydrochloride obtained from sample Solution

WS   =       Weight of Cetirizine Dihydrochloride RS/WS taken in mg.

W_T    =       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.

Calculation for Content Uniformity (% labeled amount):

                                                                     AT          WS          2         100      20       P          

%Drug Content           =        ——- x ——- x —— x —— x —— x —— x 100

                                                        AS       200      20        LC      5      100

Where,

AS    =       Average peak area due to Cetirizine Dihydrochloride obtained from five 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

       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 to be prepared as per method of analysis.

Blank: Diluent

Standard Preparation:

Weigh accurately about 50 mg of Cetirizine dihydrochloride working standard, transfer into a 200 mL volumetric flask. Add 160ml of diluent and sonicate till dissolve and dilute to volume with diluent, filter through 0.45μ nylon filter.

Further dilute 2 ml of above solution to 20 ml with diluent, mix. (Concentration 25 ppm)

Placebo Solution:

Weigh and transfer about 718 mg of placebo powder into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking. Add 400ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Sample Preparation: (Assay)

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 400ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

Spiked Test solution:

Weigh accurately 718 mg of placebo powder and transfer into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking. Add 400ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Pipette out 5.0 ml of this solution, 2.0 ml Cetirizine Dihydrochloride standard stock solution and transfer into 20 ml volumetric flask. Make up the volume with diluent and mix.

Procedure: Inject the preparation of blank solution (diluent), Placebo solution, standard solution, sample solution and spiked sample solution on a HPLC system with a Diode array detector (DAD) as follows in Table 4.0. 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 4.0: Sequence of Injection

Solution	No of Injection to be injected in Sequence
Blank (diluent)	1
Placebo Solution	1
Standard solution	5
Test Solution	1
Spiked Test Solution	1
Standard Solution + Bracketing	1

Table 5.0: Specificity data

Sr. No	Sample	RT (min.)	Peak purity
1	Blank		NA
2	Placebo		NA
3	Standard Solution	Cetirizine Dihydrochloride
6	Sample Solution	Cetirizine Dihydrochloride
7	Spiked  Solution	Cetirizine Dihydrochloride

Acceptance Criteria:

1. There should be no interference of the diluent, placebo at the retention time of analyte peak,
2. Blank peak and Placebo peak should be well resolved from active peak and each other,
3. 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 6.0: 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 Assay:

Prepare the six sample of same batch and analyze as per method of analysis, record the area on testing data sheet and calculate the % assay, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

Table 7.0: Method precision results for Assay

Sample No.	Sample wt. (mg)	Peak area	Mean % Assay	% Mean	SD	% RSD
1
2
3
4
5
6

Acceptance Criteria:

Calculate the assay for each analysis and calculate the mean, SD and % RSD.

% RSD for assay values should be NMT 2.0%.

Test Procedure for Content Uniformity:

Prepare the 10 samples of Cetirizine dihydrochloride tablets of same batch for the content uniformity and analyze as per method of analysis, record the area on testing data sheet and calculate the minimum, maximum, average and acceptance value. Obtained results will be report in tabulated manner as given below.

Table 8.0: Method Precision for Content Uniformity of Cetirizine Dihydrochloride

Tablet No.	Cetirizine Dihydrochloride
Peak area	% release
1
2
3
4
5
6
7
8
9
10
Mean
Minimum
Maximum
SD
% RSD
≤ L1% , L1 = 15

Acceptance value

Acceptance value L1 ≤ 15.0 (For 10 Dosage Units)

Acceptance value L2 ≤ 25.0 (For 30 Dosage Units)

• 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 9.0: 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 % assay, mean, standard deviation and % relative standard deviation. Obtained results will be report in tabulated manner as given below.

          Table 10.0: Intermediate precision results for Assay

Sample No.	Sample wt. (mg)	Peak area	Mean % Assay	% Mean	SD	% RSD
1
2
3
4
5
6

Acceptance Criteria:

Calculate the assay for each analysis and calculate the mean, SD and % RSD.

% RSD for assay values should be NMT 2.0%.

Table 11.0: Intermediate Precision for Content Uniformity of Cetirizine Dihydrochloride

Tablet No.	Cetirizine Dihydrochloride
Peak area	% release
1
2
3
4
5
6
7
8
9
10
Mean
Minimum
Maximum
STDV
% RSD
≤ L1% , L1 = 15

Acceptance value

Acceptance value L1 ≤ 15.0 (For 10 Dosage Units)

Acceptance value L2 ≤ 25.0 (For 30 Dosage Units)

Table 12.0: Pooled results of Assay of analyst – I & analyst – II

Sample No.	Sample wt. (mg)	% Assay
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 assay values of Analyst I and Analyst II should be NMT 2.0%.

• 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 50 mg of Cetirizine dihydrochloride working standard, transfer into a 200 mL volumetric flask. Add 160ml of diluent and sonicate till dissolve and dilute to volume with diluent, filter through 0.45μ filter.

Table 13.0: Linearity levels preparation

Level %	Vol. (mL) of Stock	Volume to be diluted with diluent (ml)
Cetirizine Dihydrochloride
80	1.6	20
90	1.8	20
100	2.0	20
110	2.2	20
120	2.4	20

Table 14.0: Linearity of Cetirizine Dihydrochloride

Level (%)	Theoretical Conc. (ppm)	Actual Conc. (ppm)	Peak Area	Mean peak area
80	20.0
90	22.5
100	25.0
110	27.5
120	30.0
Slope
Intercept
Corr. coeff.(r)

Acceptance criteria

The correlation coefficient (r) should be not less than 0.999.

• Force Degradation Study

For stability indicating method, it is essential to perform forced degradation studies by appling appropriate accelerated stress conditions to the sample, the proposed stress conditions are:

Procedure:

Prepare blank, place, standard solution, untreated test solution and treated test solution. Inject and calculate the % Assay. 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 Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 10.0 ml of 1 M Hydrochloric acid and stand for 60 minutes at room temperature, further neutralize this solution by adding 1 M sodium hydroxide. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• Base Hydrolysis :          

Sample Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 10.0 ml of 1 M sodium hydroxide and stand for 60 minutes at room temperature. Neutralize this solution by adding 1 M Hydrochloric acid. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• Oxidative Degradation:

Sample Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 10.0 ml of 30% H₂O₂ and stand for 60 minutes at room temperature. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• Thermal Degradation (at 80°C/ 24 hours or more)

Tablets store at control temperature condition of 80°C for thermal degradation.

Sample Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• 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 Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• Water Hydrolysis :

Sample Solution:

Weigh and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Heat the sample at 105°C for 60 minutes. Add more water when the sample gets dry. Cool the sample and add 400 ml of diluent and mix sonicate for 5 minutes, make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

• 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 and transfer 5 tablets into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking to disperse the tablets. Add 400 ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 5 ml of above filtered solution to 20 ml with diluent and mix. (Concentration 25 ppm)

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.

• 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:

Prepared 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 % Assay 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% from initial results. The area % RSD of standard solution should be note more than 2 %.

• 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 % assay.

Table 15.0:  Effect of Filtration (Cetirizine Dihydrochloride)

Sample	Filter Used	Mean Peak area	% Assay	Mean % Assay	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 % assay values should be NMT 2.0%.

• Accuracy

The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy should be established across the specified range of the analytical procedure. 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 70%, 100% and 130% 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 50 mg of Cetirizine dihydrochloride working standard, transfer into a 200 mL volumetric flask. Add 160ml of diluent and sonicate till dissolve and dilute to volume with diluent, filter through 0.45μ nylon filter.

Further dilute 2 ml of above solution to 20 ml with diluent, mix. (Concentration 25 ppm)

(Note: lesser or more amount of Cetirizine Dihydrochloride RS/WS can be weighed to prepare standard stock solution of same final concentration.)

Placebo Solution:

Weigh and transfer about 718 mg of placebo powder into a 500 ml clean, dry volumetric flask, add about 50 ml of water sonicate with intermediate shaking. Add 400ml of diluent and mix sonicate for 5 minutes, cool to room temperature and make up to volume with diluent. Filter a portion of solution with 0.45 μ Nylon filter discarding first two ml.

Further dilute 2 ml of above solution to 20 ml with diluent, mix

.Accuracy Stock Preparation:

Weigh accurately about 50 mg of Cetirizine dihydrochloride working standard, transfer into a 200 mL volumetric flask. Add 160ml of diluent and sonicate till dissolve and dilute to volume with diluent, filter through 0.45μ nylon filter.

(Note: lesser or more amount of Cetirizine dihydrochloride RS/WS can be weighed to prepare standard stock solution of same final concentration.)

Level I: 70%

Pipette out 5.0 ml of placebo solution, 1.4 ml Cetirizine dihydrochloride accuracy stock solution and transfer into 100 ml volumetric flask. Make up the volume with diluent and mix.

Level II: 100%

Pipette out 5.0 ml of placebo solution, 2.0 ml Cetirizine dihydrochloride accuracy stock solution and transfer into 100 ml volumetric flask. Make up the volume with diluent and mix.

Level II: 130%

Pipette out 5.0 ml of placebo solution, 2.6 ml Cetirizine dihydrochloride accuracy stock solution and transfer into 100 ml volumetric flask. Make up the volume with diluent and mix.

Calculation:

Cetirizine Dihydrochloride:

                           WS            X           P          

Amount added                  =          ——— x ——— x ——— x 1000

                                                         200           20             100               

                                                          AT          WS        2                      P           

Amount Recovered          =          ——— x ———x ——— x ———    x 1000

                                                          AS          200        20               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 mix standard solution

WS        :           Weight of Cetirizine dihydrochloride taken in standard solution

X           :           Volume of Cetirizine dihydrochloride stock withdrawn in ml (i.e.1.4, 2.0 & 2.6)

P           :           Potency of Cetirizine Dihydrochloride

Table 17.0: Accuracy for Cetirizine Dihydrochloride

Levels (%)	Amount added  in placebo (ppm)	Amount recovered  in placebo (ppm)	Recovery %	Mean Recovery %	SD	RSD%

Acceptance Criteria:                     

The mean recovery at each level should be between 98.0% and 102% of theoretical value and the % RSD not more than 2.0%, the overall average should be between 98.0% and 102.0% with % RSD not more than 2.0%

• 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 the Sonication time of test solution by time Interval of 5 minutes.

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 % assay 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 2 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 17.0:  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 assay 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.

1. Abbreviation

ASS                 :           Assay

            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