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Product / Material
Active Ingredient
Types of Analytical Procedures
Batch No.
Approvals
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TABLE OF CONTENTS
Sr. # Content Page No.
1. Objective 03
2. Scope 03
Material Used (General):
3. 1.1 Glassware Used: 03
1.2 Traceable Material Used:
Procedure:
03
4.1 Specificity
4.2 Accuracy 05
4.2.1 Accuracy-Spiked Placebo 05
4.2.2 Accuracy-Standard Addition Method 06
4.3 Linearity 07
4.4 Precision 08
4.
4.4.1 Repeatability 08
4.4.2 Intermediate Precision 08
4.5 Detection Limit 13
4.6 Quantitation Limit 14
4.7 Range 14
4.8 Robustness 15
4.9 System Suitability 16
5 Conclusion 16
6 Signature Verification Sheet 17
7 List of Appendices 17
8 Equipment used in Validation activity 17
9 Post Approval 17
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1.0 PRE-VALIDATION/ VERIFICATION REQUIREMENTS.
Chemicals, such as reagents and standards, should be available in sufficient quantities, accurately
identified, sufficiently stable and checked for purity. Other materials and consumables, for example,
chromatographic columns, should be qualified to meet the column’s performance criteria. Validation on the
analytical procedure should be performed with homogeneous samples, and validation data should be
obtained by repeatedly analyzing aliquots of a homogeneous sample, each of which has been
independently prepared according to the analytical method procedure.
1.1 ANALYTICAL EQUIPMENT QUALIFICATION.
Before undertaking the validation study, it is necessary to verify that the analytical system is adequately
designed, maintained, qualified.
1.1.1 INSTALLATION QUALIFICATION (IQ).
Documented Validation that all key aspects of an installed High performance Liquid chromatography system
(Shimadzu) adhere to the approved design specification and that the recommendations of the Shimadzu
have been suitably considered.
Protocol Number; _______________
1.1.2 OPERATIONAL QUALIFICATION (OQ).
Operational Validation carried out after installation that shows High Performance Liquid chromatography
system (Shimadzu performs in accordance with (Shimadzu) specifications and process requirements and
that the appropriate GMP systems (e.g. training, calibration, and maintenance, etc.) are in place.
Protocol Number; _______________
1.1.3 PERFORMANCE QUALIFICATION (PQ).
Performance Qualification carried out after Operational (vendor) that shows the tests carried out for the
Performance Qualification of the HPLC are in complete agreement with the required limits and criteria.
Method being used for the determination of test Methods produces consistent, reproducible and reliable
results therefore it is suitable for its intended purpose. I-e (Quantification and Qualification).
Protocol Number; _______________
1.1.4 CALIBRATION STATUS OF EQUIPMENT.
Equipment was calibrated and bears calibration sticker of the external calibrator.
Calibration Date: _____________________________
Next Calibration Date: ________________________________
Calibration Certificate No. _____________________
Calibrated by: ______________________________________
1.2 STABILITY OF THE ANALYTE(S) IN THE SOLUTIONS.
Method development process shows that it generates reproducible, consistent, the stability of the analyte(s)
in the solutions, and that of mobile phase give reliable results.
1.3 FACILITIES:
The validation of the method of Determination will be carried out in the Chemistry Lab “Name of
organization”
1.4 IDENTIFICATION OF MACHINE/ EQUIPMENT USED:
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High Performance Liquid Chromatography / Analytical Balance.
Lab Identification
Sr. # Instrument Name Make & Model Serial No.
No.
01 PUMP
02 Auto Sampler
03 Column Oven
04 CBM
05 PDA Detector
06 Analytical balance
07 HPLC Column
1.5 TRACEABILITY OF MATERIAL OR PRODUCT USED FOR STUDY:
Sr. Manufacturer of material / Lot No. /
Type of material Name Expiry
# Product Batch No.
Reference
01
Standard.
02 Raw Material
03 Raw Material
04 Tablets
05 Tablets
06 Tablets
07 Tablets
1.6 CHEMICALS / GLASSWARE/ APPARATUS USED:
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Sr. Type / Lot No. / Batch No.
Name Manufacturer Expiry
# uncertainty / Serial No.
01 Methanol
02 Water
03 Nylon Filters (M.P) -
04 Syringe Filters -
05 50ml volumetric flasks. -
06 1.0 ml Graduated pipet. -
07 3.0 ml Gradated pipet. -
08 5.0 ml Gradated pipet. -
09 Glass Beakers 50,100,500ml -
10 Magnetic Stirrer - -
11 Spatula(s) - -
Weighing Boats / Butter
12
papers
12 Motor & pastel - -
13 Sonicate bath
14 Filtration Syringe
15 Auto sampler HPLC Vials
2.0 SCOPE
3.0 OBJECTIVE
4.0 METHOD TO BE VARIFIED/ VALIDATE.
Use HPLC Work instruction for HPLC Operations. Work Instruction # ____________
Use Analytical Balance Work instruction for weighing. Work Instruction # ____________
Test Specification / Method Reference
CHROMATOGRAPHIC CONDITIONS
Column
Mode
flow rate
Column temperature.
Quantification / Wavelength
Identification Injection Volume
Run Time
Injection Procedure
System Suitability
Mobile Phase
Diluent
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STANDARD SOLUTION PREPRATION:
SAMPLE SOLUTION PREPRATION:
INJECTION PROCEDURE:
CALCULATIONS:
Where ;
90%-110% of the labelled amount of
Precautions:
1. Use Pyrex type glassware and broken glassware should not be used.
2. During analysis temperature should be in between 20-25°c.
3. Use dried glassware and rinse with solvent to be used before use.
4. Use HPLC grade solvents only.
5. All samples must be filtered with 0.45µ Filter.
6. Always degas the mobile phase, as air bubbles may tend to form during
solvent mixing or during temperature or pressure changes. Air bubbles may
cause pump malfunctions and detector signal noise.
7. Never run the column dry. Make sure there is enough solvent in the
reservoir.
8. Use a 0.2µ sized syringe filter for sample. Always filter your sample before
injection.
Method Reference: In-House
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5.0 SPECIFICITY:
Specificity is the ability to assess unequivocally the analyte in the presence of components which may
be expected to be present. Typically these might include impurities, degradants, matrix, etc.
5.1 Identification Tests.
Specificity ensure the identity of an analyte.
Methodology: specificity is demonstrated by the ability to discriminate between compounds of closely
related structures, or by comparison to known reference materials.
5.2 Purity Test
Specificity ensure that all the analytical procedures (Method) performed allow an accurate statement of
the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents
content, etc.
Methodology: Impurities available: specificity is demonstrated by using spiking the drug substance or
product with appropriate levels of impurities.
5.3 Assay (content or potency):
To provide an exact result which allows an accurate statement on the content or potency of the analyte
in a sample.
Methodology: specificity is demonstrated by using spiked samples to show that method results are
unaffected by the presence of impurities or excipients etc.
Placebo Interference
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Sr. # Excipients Name Peak Time Peak Area
The inactive material did not show significant absorbance /
peak at λ max 228 nm.
Acceptance Criteria Known degradation products, synthetic impurities and sample
matrix present in the commercial product do not interfere with
the determination of the active constituent.
6.0 LINEARITY & RANGE.
Prepare a range of standards containing at least five different concentrations of analyte in expected
working range. Verify that the method provides acceptable precision, accuracy, and linearity when
applied to samples at the extreme as well as within range. For assay 80-120%, determination of
impurity 50-120%, content uniformity 70-130%, Dissolution ±20% of over specified range of the test
concentration.
Concentration Sample
Avg. Peak area
Concentration % mg/ml Sample 1 2 Sample 3
Linearity
1240000
1205000
1170000
1135000 Prepared by Reviewed by Approved by
1100000
Sign &1065000
Date
Area
Name/ 1030000
995000
Designation
960000
925000
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Slope = 9400.39
Conclusion
Parameters Criteria Result Qualified√ - Disqualified×
Correlation coefficient NLT 0.9990 Qualified Not Qualified
y−intercept Criteria NMT 2.0% Qualified Not Qualified
7.0 ACCURACY AND PRECISION
7.1 ACCURACY AND RECOVERY
In the study of accuracy / recovery, Known amount of sample corresponding to three concentration
levels i.e. 50%, 100 % and 150 % were taken. Results were obtained using nine determinations over
three concentration levels (50%, 100%, and 150 %) and three replicate of each concentration. 50
percent is (lowest concentration) and 150 percent (highest concentration) of the expected working
range.
The average of individual recovery was found with in ±2 % of the theoretical amount.
Acceptance criteria: (theoretical amount ± 4%)
Level Peak Area of Replicate Peak Area of Replicate %age Average
% (Samples) (Standard ) Recovery Recovery
50 %
100%
150%
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Conclusion
Criteria Results
Qualified√ - Disqualified×
For 50 % Level 48 – 52 % 49.64% Qualified Not Qualified
Limits
For 100 % Level 96 – 104 % 100.12 % Qualified Not Qualified
For 150 % Level 144 – 156 % 153.82 % Qualified Not Qualified
7.2 REPEATABILITY PRECISION (System Precision).
Six replicates of a 100% test solution and measure the responses peak of each sample. The relative standard deviation
of the six readings should not be greater than 1%.
Observations
S. No.
Retention Time (min) Peak Area
1 2.847 9188940
2 2.841 9149437
3 2.834 9140982
4 2.837 9121143
5 2.840 9104771
6 2.836 9045541
Mean 2.8392 9125135.66667
Standard Dev. 0.004622409 48329.34105
Relative Standard dev (%RSD) 0.162808659 0.52963
Acceptance Criteria Conclusion
Criteria Result Qualified√ - Disqualified×
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RSD < 1.0 % Qualified Not Qualified
7.3 INTERMEDIATE PRECISION (Method Precision).
The Precision of a method is determined by assaying aliquots of a homogeneous sample to be able to
calculate statistically significant estimates of standard deviation or relative standard deviation. Assay should
be of samples that have all gone through the entire analytical procedure from sample preparation through
final analysis.
Typical variations to be studied include days, analysts, equipment, etc.
7.3.1 WITHIN DAYS AND BETWEEN DAYS VARIATION (Reproducibility)
Peak Area of Peak Area of Average Assay
Day(s) Assay% STDV %RSD
Sample Standard %
01
02
03
Conclusion
Criteria Day (s) Result Qualified√ - Disqualified×
Qualified Not Qualified
Acceptance
Criteria Qualified Not Qualified
RSD < % Qualified Not Qualified
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7.3.2 ANALYST TO ANALYST VARIATION (RUGGEDNESS).
Peak Area of Peak Area of Average Assay
Analyst(s) Assay% STDV %RSD
Sample Standard %
JB
AJ
7.3.3 EQUIPMENT TO EQUIPMENT VARIATION (RUGGEDNESS).
Peak Area of Peak Area of Average Assay
Analyst(s) Assay% STDV %RSD
Sample Standard %
JB
AJ
Conclusion
Criteria Analyst(s) Result Qualified√ - Disqualified×
Qualified Not Qualified
Acceptance RSD < % Qualified Not Qualified
Criteria Conclusion
Criteria Equipment Result Qualified√ - Disqualified×
Qualified Not Qualified
Qualified Not Qualified
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8.0 ROBUSTNESS:
The robustness of an analytical method is a measure of its capacity to remain unaffected by small but
deliberate variations in method parameters to ensure reliability during normal usage (e.g., a small change
in the composition of an HPLC mobile phase + 2 %, column temperature + 5 ºC, reagents, pH of buffer of
an HPLC mobile phase 0.1 pH units and Injection volume by + 2 µL.
Flow rate Peak area Avg. Peak Area SD %RSD
1.1
1.3
1.5
Mobile Phase
Peak area Avg. Peak Area SD %RSD
composition
Column Oven Peak area Avg. Peak Area SD %RSD
pH Buffer Peak area Avg. Peak Area SD %RSD
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Injection
Peak area Avg. Peak Area SD %RSD
volume
Conclusion
Qualified√ -
Criteria Parameters Result Disqualified×
Acceptance Flow Rate Qualified Not Qualified
Criteria for MP Composition Qualified Not Qualified
Robustness
Column Oven
RSD < % pH buffer Qualified Not Qualified
Injection Volume Qualified Not Qualified
9.0 LIMIT OF DETECTION (LOD).
The detection limit of an analytical procedure is the lowest analytical concentration at which an analyte(s)
could be detected qualitatively. LOD can be calculated at levels approximating the LOD according to the
formula LOD =3.3(SD/S). Where SD: Standard deviation and S: Slope
Conc. (ug/ml)
Conc. Avg. Peak Standard at LOD
Sample Conc. % (mg/ml) Area Deviation Slope 3.3 x [Link] /slop Area at LOD
1 80
2 90
3 100
4 110
5 120
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10.0 LIMIT OF QUANTITATION (LOQ).
LOQ is the lowest concentration of an analyte in a sample that can be determined (quantitated) with
acceptable precision and accuracy under stated operational conditions of the method. LOQ can be
calculated at levels approximating the LOQ according to the formula LOQ = 10(SD/S). Where SD:
Standard deviation and S: Slope
Conc. (ug/ml)
Conc. Avg. Peak Standard at LOQ
Sample Conc. % (mg/ml) Area Deviation Slope 10 x [Link] /slop Area at LOD
1 80
2 90
3 100
4 110
5 120
11.0 SYSTEM SUITABILITY
System Suitability is the checking of a system to ensure system performance before or during the analysis
of Unknowns. SST accomplished by summarizing data on reproducibility, efficiency, tailing and resolution
for replicate injections.
Conclusion
Day(s) Qualified√ -
Creteria Parameters SST Avg. SST Disqualified×
RSD < 1% Inj. Precision for
Qualified Not Qualified
Peak Area (n=6)
Acceptanc
e Criteria
for SST Rs= > 2.0 Resolution (R1) Qualified Not Qualified
T= < 2.0 Tailing Factor (T) Qualified Not Qualified
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Capacity Factor
N= > 2.0 Qualified Not Qualified
(k)
Theoretical Plates
N= > 2000 Qualified Not Qualified
(N)
12.0 OBSERVATIONS / DEVAITIONS:
Protocol Section No: Page No: Deviation #:
Protocol Requirements
State the protocol requirements:
Deviation Description
12.1 JUSTIFICATION FOR ACCEPTANCE:
Deviation Cause/ Investigation
12.2 IMPACT ON OPERATIONS:
Does this Deviation Potentially Impact If so Reference Investigation
Distributed Product? (Yes/No) and/or CAPA No.
Recommended Corrective Action
Results of any corrective action taken
12.3 DEVIATION REPORT WRITTEN BY:
Originator/Function Name Signature Date
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Submitted By
13.0 CONCLUSIONS
14.0 POST APPROVAL:
Responsibility Designation Name Signature Date
Written By
Reviewed By
Approved By
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Sign & Date
Name/
Designation
