RELATED SUBSTANCES METHOD VALIDATION PROTOCOL FOR CITALOPRAM TABLET

RELATED SUBSTANCES METHOD VALIDATION PROTOCOL FOR CITALOPRAM TABLET

Superseded Protocol No.	Nil
Effective Date

Table of contents :

Sr. No.	Subject	Page No.
	Protocol Approval
	Objective
	Scope
	Responsibility
	Product profile
	Methodology
	Specificity
	Response factor/ correction Factor
	Precision
	Force Degradation
	Linearity
	Range
	Accuracy
	Limit of Detection (LOD)
	Limit of Quantification (LOQ)
	Stability of Analytical solution
	Filter paper Selection Study
	Robustness
	Incident/Deviation
	Validation Summary
	Final Summary/Conclusion
	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 validation is to provide documentary evidence that analytical methodology used in the determination of Related Substance in Citalopram Tablet (10mg, 20mg and 40mg) will yield consistent and reliable results within the predetermined acceptance criteria.

Analytical method validation will be performed by considering thefollowing parameters:

Parameters	10mg	20mg	40mg
Specificity	yes	yes	yes
Precision
System Precision	yes	yes	yes
Method Precision	yes	yes	yes
Intermediate Precision(Ruggedness)	yes	yes	yes
Force degradation			yes
Linearity	yes	yes	yes
Accuracy			yes
Range	yes	yes	yes
Stability in Analytical Solutions	–	–	yes
Robustness	–	–	yes
Filter Paper Selection Study	–	–	yes

• Scope :

This protocol is based on SOP and applicable for the validation of Related Substance of Citalopram in Citalopram Hydrobromide tablet.

• Responsibility of Validation Team:

Departments	Responsibilities
QC	Preparation and Review of Validation protocol.
Analysis of samples and recording of data.
Compilation and checking of data
Preparation and review of Validation report.
To impart training of protocol to concerned department/persons.
QA	Review and approval of Validation protocol.
Co-ordination with QC to carryout Validation.
Review and approval of Validation report.
Head Quality	Authorization of protocol.

• Product Profile:

Category	Antidepressant
Reason for Validation	First validation
Active Ingredient	Citalopram
Strength	Each Film Coated tablet contains: Citalopram Ph. Eur(10mg, 20 mg & 40 mg)
Methodology	In House
Method Reference	In House
Specification Limits
Impurities at 230 nm
Impurity A (amide)	≤0.2%
Impurity B (acid) (IH)	≤0.2%
Impurity C (cyano phthalane)	≤0.1%
Impurity D (demthyl Citalopram)	≤0.2%
Impurity B (acid) (Ph. Eur.)	≤0.3%
Other individual impurity	≤0.1%
Total impurities	≤0.5%
Impurities at 254 nm
Impurity G	≤0.3%
Other individual impurity	≤0.1%

• Methodology:

Related Substance

Materials and Methods

Reagents:

Ammonium format (AR grade)

Methanol (HPLC grade)

Acetonitrile (HPLC grade)

Milli-Q water

Preparation of mobile phase A:

Prepare a suitable quantity of a mixture of acetonitrile, methanol and water in the ratio of 4: 32: 64. Dissolve about 1.58 g of ammonium format in 500 ml of above prepared mixture. Mix well and degas.

Preparation of mobile phase B:

Prepare a suitable quantity of a mixture of water and acetonitrile in the ratio of 32: 68. Dissolve about 1.58 g of ammonium format in 500 ml of above prepared mixture. Mix well and degas.

Preparation of diluent:

Use mobile phase A as diluent.

Preparation of system suitability solution:

Dissolve the contents of a vial Citalopram for system suitability CRS (containing impurities B, D, F and G) in 1.0 ml of diluent.

Alternatively:

Accurately weigh and transfer about 1 mg each of impurity a standard and impurity G standard to a 25 ml volumetric flask. Add about 15 ml of diluent and sonicate to dissolve. Make up the volume with diluent and mix (Impurity mixture)

Accurately weigh and transfer about 1.5 mg of impurity D standard and Citalopram Hydrobromide working standard / Citalopram Hydrobromide CRS equivalent to about 0.25 mg of Citalopram to a 50 ml volumetric flask. Add about 30 ml of diluent and 1 ml of impurity mixture. Sonicate to dissolve and make up the volume with diluents and mix.

Preparation of standard solution:

Accurately weigh and transfer Citalopram Hydrobromide working standard / Citalopram Hydrobromide CRS equivalent to about 50 mg of Citalopram to a 100 ml volumetric flask. Add about 70 ml of diluent and sonicate to dissolve. Make up the volume with diluent and mix (stock solution).

Dilute 1 ml of stock solution to 100 ml with diluent and mix. Further dilute 1 ml to 10 ml with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Preparation of sample solution:

Determine the average weight of not less than 10 tablets and crush them to a fine powder. Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask. Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Note: Sample solution is stable for at least 27 h at 5°C and 25 h at 25°C.

Preparation of Placebo solution:

Weigh and transfer placebo powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask. Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45µm nylon membrane filter.

Chromatographic parameters:

Column                               : Phenomenex synergi hydro-RP80A, 4µm (250mm x 4.6 mm)

Column oven temperature : 40°C

Injection volume                 : 40µl

Wavelength                                    : UV at 230 and 254 nm

Flow rate                             : 1.0 ml/min

Run time                             : 40 min

Gradient program

Time (min)	Mobile Phase A	Mobile Phase B
0	100	0
2	100	0
25	40	60
32	40	60
33	100	0
40	100	0

Evaluation of system suitability:

1. Inject the system suitability solution into the chromatograph and record the chromatogram at 230 nm.

The system is suitable for analysis if and only if:

The resolution between impurity D and Citalopram peaks is not less than 1.5.

• Inject the standard solution into the chromatograph and record the chromatograms.

The column efficiency determined from Citalopram peak is not less than 40000 theoretical plates at 230 nm.

The relative standard deviation for six replicate injections is not more than 5.0 % at 230 nm and 254 nm.

Make the adjustments if necessary to meet the system suitability requirements.

Procedure:

Inject the placebo and sample solution into the chromatograph and record the chromatograms. Disregard the peaks observed in the sample chromatogram which are observed at the same retention time in the placebo chromatogram. Also disregard the peak due to impurity G at RRT 0.63 in the sample solution at 230 nm. The retention time of Citalopram peak is about 19 min.

Note:

Extract the chromatograms of placebo and sample at 254 nm for the calculations of Impurity G.

The relative retention times (RRT’s) of the eluting peaks are:

Sr. No.	Name of the impurities	RRT	RF	LOD (% m/m)	LOQ (% m/m)	Origin
1.	Impurity B (IH)	~ 0.40	1.51	0.020	0.040	P/D
2.	Impurity A	~ 0.57	1.23	0.020	0.039	P/D
3.	Impurity G	~ 0.63	0.46	0.038	0.095	P
4.	Impurity B (Ph. Eur.)	~ 0.73	1.0	_	_	P
5.	Impurity D	~ 0.97	1.13	0.020	0.039	P/D
6.	Impurity C (IH)	~ 1.65	0.67	0.020	0.040	P

RRT’s are calculated with respect to Citalopram peak (with delay volume of the instrument about 1 ml)

P = Process, D = Degradation, LOD = Limit of detection and LOQ = Limit of Quantitation

Any unknown impurity observed at level below 0.05 % m/m, shall not be considered.

Note: Peak due to impurity A and impurity G may get swapped. Retention time of Impurity G can be confirmed from system suitability solution 254 nm.

Calculations:

                                           AT            DS            P                       A             324.44

1. Impurity G                   = ————- x ——— x ———- x 100 x ———– x —————– x RF

(% m/m) (At 254 nm)           AS           DT           100                      C              405.35

                                                        AT₁            DS            P                       A             324.44

• Any individual known Impurity= ————- x ——— x ———- x 100 x ———– x —————– x RF

(% m/m) (At 230 nm)                       AS₁            DT        100                      C             405.35

                                              AT₂           DS            P                       A             324.44

• Other individual Impurity= ————- x ——— x ———- x 100 x ———– x —————– x RF

(% m/m) (At 230 nm)            AS₁            DT          100                     C             405.35

                                              AT₃           DS            P                        A             324.44

• Other individual Impurity= ————- x ——— x ———- x 100 x ———– x —————– x RF

(% m/m) (At 254 nm)             AS            DT          100                      C             405.35

• Total (% m/m)        =2 + 3

Where,

AT = Area counts of impurity G peak in the chromatogram of the sample solution at 254 nm.

AT₁ = Area counts of any individual known impurity peak in the chromatogram of the sample solution at 230 nm

AT₂ = Area counts of other individual impurity peak in the chromatogram of the sample solution at 230 nm

AT₃ = Area counts of other individual impurity peak in the chromatogram of the sample solution at 254 nm

AS = Average area counts of the Citalopram peak in the chromatogram of the standard solution at 254 nm

AS₁ = Average area counts of the Citalopram peak in the chromatogram of the standard solution as obtained under system suitability at 230 nm

DS = Dilution factor for standard solution

DT = Dilution factor of sample solution

A = Average weight

P = Percent potency of Citalopram Hydrobromide working standard / Citalopram Hydrobromide CRS, on as is basis.

C = Label claim of Citalopram per tablet in mg

324.44 = Molecular weight of Citalopram

405.35 = Molecular weight of Citalopram Hydrobromide

RF = Response factor.

Note:

Impurity B (Ph. Eur.): 1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-3-hydroxy-1,3-dihydroisobenzofuran-5-carbonitrile.

Impurity D: (1RS)-1-(4-fluorophenyl)-1-[3-methylamino)propyl]-1’3-dihydroisobenzofuran-5-carbonitrile.

Impurity F: 3-[(1RS)-5-bromo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl]-N,N-dimethylpropan-1-amine.

Impurity G: 4-(Dimethylamino)-1-[(1RS)-1-[3-dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl]butan-1-one.

Impurity A: (1RS)-1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide

Impurity B (IH): 1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-2-benzofuran-5-carboxylic acid.

Impurity C (IH): 1-(4’-fluorophenyl)-5-phthalane carbonitrile

Requirement: As per current version of approved specification.

• 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 placebo.

Prepare and inject blank, Placebo, reference solution, known impurity individually 0.1mg/ml of target concentration of test and sample solution spiked with known impurities at specification level. Check the peak purity of main peak and known impurities.

Analysis sequence

Sample	Type	Injections
System Suitability	Standard	1
Blank Solution	Unknown	1
Placebo Solution	Unknown	1
System Suitability	Standard	1
Reference Solution	Standard	6
Known impurity solution	Standard	1
Sample Solution	Unknown	1
System Suitability (Bracketing)	Validate	1
Reference Solution (Bracketing)	Validate	1

Each chromatograph shall be monitored individually to examine the interference.

Acceptance criteria

No peak shall be eluted at the retention time of Citalopram. Peak purity of the Citalopram and impurity should be passed.

• Response / Correction factor

To evaluate the correction factor for Impurity-A, Impurity-B (IH), Impurity-B (Ph.Eur), Impurity-C,  Impurity-D & Impurity-G with respect to Citalopram, the standards having concentration 1.0% with respect to the target concentration was prepared and injected. Response and correction factor of -A, Impurity-B (IH), Impurity-B (Ph.Eur), Impurity-C, Impurity-D & Impurity-G with respect to Citalopram should calculated and the results are recorded.

Analysis sequence

Sample	Type	Injections
System Suitability	Standard	1
Blank Solution	Unknown	1
Placebo Solution	Unknown	1
System Suitability	Standard	1
Reference Solution	Standard	6
Standard Solution	Standard	2
Known impurity solution (A, B, C, D & G)	Standard	2
System Suitability (Bracketing)	Validate	1
Reference Solution (Bracketing)	Validate	1

Where

AS :           Areas of Citalopram standard

AI   :           Area of Impurity

PI   :           Purity of Impurity

PS :           Purity of Citalopram standard

CI  :           Concentration of Impurity

CS :           Concentration of Citalopram standard

• Precision:

• System Precision:

The system precision is the closeness of agreement between the responses of detector. It is usually expressed as the standard deviation (SD) or the relative standard deviation (RSD).

Prepare and inject separately System Suitability diluents (Blank), Placebo and Reference solution as per methodology procedure and record the area response of main peak in the Reference solution. Calculate the % RSD of response for six replicate.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
System suitability solution (Bracketing)	Validate	1

Acceptance criteria

The relative standard deviation for peak area and retention time should be not more than 2.0% & 1.0%.

• Method Precision: (Repeatability)  

The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatability to multiple samplings of homogenous sample. It is usually expressed as the standard deviation and the relative standard deviation.

Prepare six individual sample of same batch and analyze as per Methodology.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
Placebo solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
Sample 1	Unknown	1
Sample 2	Unknown	1
Sample 3	Unknown	1
Sample 4	Unknown	1
Sample 5	Unknown	1
Sample 6	Unknown	1
Blank solution	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria

For known and unknown impurities in related substances method, relative standard deviation of six analyses shall be not more than 10.0%.

• Ruggedness (Intermediate Precision):

Intermediate precision expresses within laboratory variation with different analysts or equipment or different column on different days using same batch of drug product as per Methodology

Test Procedure:

The analysis of the same batch will be done in six replicate analyses by using different columns by different analyst, by different system on different day. The mean, standard deviation and relative standard deviation will be calculated.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
Placebo solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
Sample 1	Unknown	1
Sample 2	Unknown	1
Sample 3	Unknown	1
Sample 4	Unknown	1
Sample 5	Unknown	1
Sample 6	Unknown	1
Blank solution	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria

For known and unknown impurities in related substances method, relative standard deviation of six analyses shall be not more than 10.0%.

Forced Degradation:

Prepare System Suitability solution, Blank, Placebo, Reference solution, untreated sample solution and treated sample solution (By acid, base, oxidation, thermal and humidity Oxidative degradationUV-Light).

Inject and calculate the % impurities. Calculate the % difference between the untreated sample and treated sample result. Check the peak purity of main peak.

Stress the sample at the following conditions and evaluate the peak purity and all degradation study procedure shall be recorded in data sheet and mentioned in method validation report.

• Degradation by Hydrochloric Acid
• Degradation by Sodium Hydroxide
• Degradation by hydrogen peroxide 30% (Oxidative degradation)
• Degradation by thermal
• Degradation by humidity
• Degradation by UV-Light
• Degradation by water Hydrolysis
  • Degradation by Hydrochloric Acid (Acid treated sample):

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask add 10ml of 1MHCl Stand for 60minutes at room temperature Neutralize with 10ml of 1MNaOH add 170ml of diluent and shake on a rotator shaker at 200rpm for about 30minutes make up volume up to mark with diluent and mix.  Filter the solution through 0.45µm membrane filter.

Degradation by Sodium Hydroxide (Base treated sample):

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask add 10ml of 1M NaOH Stand for 60minutes at room temperature Neutralize with 10ml of 1MHCl add 170ml of diluent and shake on a rotator shaker at 200rpm for about 30minutes make up volume up to mark with diluent and mix.  Filter the solution through 0.45µm membrane filter.

Degradation by UV-Light (2-4Hrs):

Place the tablets on UV Chamber for 4hrs and take out the sample.

Test solution:

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask. Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Degradation by Humidity (25ºC/90%RH-24Hrs treated sample):

Place the tablets on Humidity Chamber for 24hrs and take out the sample.

Test solution:

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask. Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Degradation by Thermal (105ºC/48Hrs treated sample):

Place the tablets on hot air oven for 24 to 48hrs and take out the sample.

Test solution:

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask. Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Degradation by Hydrogen Peroxide (Peroxide treated sample)

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask add 10ml of 30%H2O2 Stand for 60minutes at room temperature Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Degradation by water Hydrolysis:

Transfer tablet powder equivalent to about 125 mg of Citalopram to a 250 ml volumetric flask add 10ml of water Stand for 60minutes at 105ºC on water bath sonicate for 10minutes Add about 170 ml of diluent and shake on a rotary shaker at 200 rpm for about 30 min. Make up the volume with diluent and mix. Filter through 0.45 µm nylon membrane filter.

Acceptance Criteria:

The difference between treated and untreated sample shall be not more than 15 %.

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 (amount) of analyte in the sample.

Determine the linearity by preparing the solution (standard and impurity standard solution) at different levels (six labels) over a specified range from LOQ to 200 % of the specification limit. Each level inject in duplicate. Calculate correlation co-efficient.

Test Procedure:          

Prepare and inject the known impurities solution and standard solution in the range of LOQ to 200 % concentration level calculate the correlation coefficient.

Preparation of linearity solution

Level	Level % to the limit concentration	Linearity standard  solution	Make up to diluent (ml)
1	LOQ
2	50%
3	100 %
4	150 %
5	200 %

Analysis sequence

Sample	Type	Injections
System Suitability	Standard	1
Blank	Unknown	1
System Suitability	Standard	1
Reference solution	Standard	6
Linearity solution-LOQ% level	Standard	2
Linearity solution-50% level	Standard	2
Linearity solution-100 % level	Standard	2
Linearity solution-150% level	Standard	2
Linearity solution-200 % level	Standard	2
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria

Plot the calibration curve of peak area v/s concentration for the studied peak. The   correlation coefficient (r), after the regression analysis of the results should not be less than 0.99.

Range:

The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The range is normally expressed in the same units as test results obtained by the analytical method. (e.g. percent, parts per million)

Accuracy:

The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy shall be established across the specified range of the analytical procedure.

Prepare System Suitability solution, Blank, Placebo, Reference solution, known impurity solution) and inject the sample solution. inject, prepare the sample solution in triplicate by spiking with known impurities at about LOQ %,50%, 100 % and 150 % of specification limit and calculate the % overall average recovery for known impurities.

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%.

Limit of Detection (LOD):

Limit of detection is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified under the stated experimental condition. To determine the lowest amount of analyte in a sample which the analytical method can:

Detect but not necessarily quantitate as an exact value (LOD)

System suitability was performed.

The LOD was determined for Citalopram, Impurity-A, Impurity-B, Impurity-C, Impurity-D & Impurity-G. Calculated the concentration at which the signal to noise ratio is about 3:1.Prepare the LOD Solution using blank, placebo spiked with known impurities at determined LOD level and inject in six replicates. Calculate % RSD for six replicates of known impurities.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Standard solution (0.1%)	Unknown	1
Blank solution	Unknown	1
System Suitability	Standard	1
Reference solution	Standard	6
LOD-Initial	Unknown	1
LOD-I/1	Unknown	1
LOD-I/2	Unknown	1
LOD-I/3	Unknown	1
LOD-I/4	Unknown	1
LOD-I/5	Unknown	1
LOD-I/6	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria:

%RSD for six replicates response of known impurities shall be not more than 30. The signal to noise ratio should be more than 3:1.

Limit of Quantitation (LOQ):

Limit of Quantitation is the lowest concentration of analyte in a sample that can be quantitate with acceptable precision under the stated experimental condition.

To determine the lowest amount of analyte in a sample which the analytical method can:

Quantitate with suitable precision and accuracy (LOQ)

The LOQ determined Citalopram, Impurity-A, Impurity-B, Impurity-C, Impurity-D & Impurity-G. Calculated the concentration at which the signal to noise ratio is about 10:1. Prepare the LOQ Solution using blank, placebo spiked with known impurities at determined LOQ level and inject in six replicates. Calculate % RSD for six replicates of known impurities.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
System Suitability	Standard	1
Reference solution	Standard	6
LOQ-Initial	Unknown	1
LOQ-I/1	Unknown	1
LOQ-I/2	Unknown	1
LOQ-I/3	Unknown	1
LOQ-I/4	Unknown	1
LOQ-I/5	Unknown	1
LOQ-I/6	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria:

%RSD for six replicates response of known impurities shall be not more than 10.

The signal to noise ratio should be more than 10:1.

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 during analytical measurement stages of the testing.

Test Procedure:

Prepare the standard solution and sample solution spiked with known impurities at the specification level as per finish product testing procedure. Analyze the standard solution and sample solution at the different time intervals and calculate the % cumulative RSD of peak area for known impurities and main peak.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
Placebo solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
Test Solution	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria:

Cumulative % RSD of peak area for known impurities and main peak shall not be more than10.

Filter Paper Selection Study:

Test Procedure:

Prepare sample solution (Spiked sample solution).A portion of sample solution shall be filtered with 0.45µm Nylon filter) inject all samples and calculate the % impurity for each sample and calculate the % impurity difference between Method precision vs. filtered samples.

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
Placebo solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
Test Solution-discard Volume 0.0ml	Unknown	1
Test Solution-discard Volume 2.0ml	Unknown	1
Test Solution-discard Volume 4.0ml	Unknown	1
Test Solution-discard Volume 6.0ml	Unknown	1
System suitability solution (Bracketing)	Validate	1
Reference solution (Bracketing)	Validate	1

Acceptance criteria

If the impurity is less than 0.1 %, no comparison shall be made. For known impurities and Total impurities % difference shall be not less than +10 %.

Robustness:

To establish the robustness of test method and to demonstrate its reliability for minor changes in chromatographic conditions.

Test Procedure:

Robustness of the method shall be investigated by checking the system suitability parameters by deliberately varying the instrumental condition such as   

• By changing the flow rate by ±10%
• By changing the column oven temperature by ±5°C
• By changing the solvent Concentration by ±5%

Prepare the standard solution and sample solution spiked with known impurities at the specification level as per test method by deliberate variations made in the method for each condition as mentioned in protocol and analyze.

Calculate the RRT for known impurities and compare with RRT from method precision experiment. 

Analysis sequence

Sample	Type	Injections
System suitability solution	Standard	1
Blank solution	Unknown	1
Placebo solution	Unknown	1
System suitability solution	Standard	1
Reference solution	Standard	6
Test Solution-1	Unknown	1

Acceptance criteria

System suitability criteria should be met under all conditions and result should be complies all condition.Deviation /Incident

Any deviation during validation exercise shall be reported and justified. Raw data and chromatograms of all non-satisfactory results shall be kept along with validation report.

• Validation summary

Data obtained through validation exercise shall be analyzed with regard to various parameters of validation and their acceptance criteria. A validation report shall be prepared summarizing all data. All prints, chromatograms and calculations shall be captured in this report.

• Final Conclusion/Recommendation

Based on finding of data analysis conclusion shall be drawn.           

• Revision History:

Revision No.	Details of changes	Reason for changes
00	Nil	New Document