Analytical Method Validation Procedure

1.0 OBJECTIVE 
1.1 To lay down the detailed procedure for the Validation of Analytical Methods / Procedures.

2.0 SCOPE
2.1 This Procedure is applicable for the Validation of Analytical Methods being executed using Chromatographic Systems (For Assay, Related Substances, Residual Solvents, Chromatographic Purity, and Content Uniformity etc.), Rate, Particle Size, Viscosity etc. in the Quality Control Department

3.0 RESPONSIBILITY
3.1 QC Officer or QC Executive

4.0 ACCOUNTABILITY
4.1 QC Manager

5.0 PROCEDURE



5.1 Definitions
5.1.1 Validation of an Analytical Method (Procedure) is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications.
5.1.2 Validation is performed in order to demonstrate that the result(s) generated by a particular analytical procedure are accurate and reliable.
5.1.3 Revalidation of an approved analytical procedure may be necessary in the following circumstances:-
a) Changes in the Synthesis of the Drug Substance.
b) Changes in the Composition of the Finished Product / Drug Product.
c) Changes in the Analytical Procedure.
NOTE: The degree of Revalidation required, depends upon the nature of the changes.

5.1.4 If the method is Compendial (Official in IP / BP / EP / USP, etc), then only Partial Validation/Verification of the said method shall be carried out.
5.1.5 Typical Analytical Performance Characteristics (Parameters) that should be considered in the Validation activity are listed below:
a) Specificity / Selectivity.
b) Precision
c) Repeatability.
d) Reproducibility.
e) Ruggedness (Intermediate Precision).
f) Accuracy.
g) Linearity and Range.
h) Limit of Detection (LOD).
i) Limit of Quantitation (LOQ).
j) Robustness.
k) Filter Equivalency.
l) Solution Stability.

5.1.6 SPECIFICITY / SELECTIVITY
This is the ability of the Method to assess unequivocally the analyte in the presence of components which may be expected to be present in the test mixture.
Typically these might include Impurities, Degradants, and Matrix etc.
Specificity is usually demonstrated by measuring the response of the sample matrix and any expected or known species (For example: Excipients, Impurities or degradation products).
It would normally be expected that no significant response would be obtained that interferes with the measurement of the analyte(s).
However it is not always possible that an analytical procedure is specific for a particular analyte.
In this instance a combination of two or more analytical procedures may/shall be necessary to achieve the required results.



5.1.7 PRECISION
The Precision of an analytical procedure expresses the closeness of agreement (Degree of Scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.
The Precision of an analytical procedure is usually expressed as the Variance, Standard Deviation or Coefficient of Variation of a series of measurements.

Precision may be considered at Three Levels: Repeatability, Ruggedness (Intermediate Precision) and Reproducibility

a) Repeatability
Repeatability is also termed as Intra-Assay Precision.
Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time using the same analyst and the same equipment.

Repeatability should be assessed using:
1) A minimum of Nine (9) determinations covering the specified range for the procedure (e.g. 3 Concentrations / 3 replicates each);
OR
2) A minimum of Six (6) determinations at 100% of the test concentration.

b) Ruggedness (Intermediate Precision):
The extent to which intermediate Precision should be established depends on the circumstances under which the procedure is intended to be used.
Ruggedness (Intermediate Precision) expresses within-laboratory variations.

Typical variations to be studied include:
Days, Analyst(s), Equipment(s) etc.
It is not necessary to study these effects (Variations) individually, and all can be studied by using an experimental design (Matrix).
c) Reproducibility:
Reproducibility expresses the Precision Between Different Laboratories.
(Collaborative Studies, usually applied to Standardization of Methodology).
Reproducibility is assessed by means of an Inter-Laboratory exercise.

5.1.8 ACCURACY
The Accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed as Trueness.
a) Accuracy should be established across the specified range of the analytical procedure.
b) In the case of the Assay of a Drug Substance, Accuracy may be determined by application of the analytical procedure to an analyte of known purity, e.g. a Reference Standard.
c) In the case of the Assay of a Drug in a formulated product, Accuracy shall be determined by application of the Analytical Procedure to the synthetic mixtures of the Drug Product components to which known amount of analyte have been added within the range of the procedure.
d) For estimation of Assay the mean recovery should be 98% to 102%.
e) In case of Impurities Accuracy should be assessed on samples (Drug Substance / Drug Product) spiked with known amounts of impurities. (Accuracy by Recovery).
f) For estimation of Related Substances / Impurities the mean recovery should be 90% to 110%.
g) In cases where it is impossible to obtain samples of certain impurities and / or degradation products, it is acceptable to compare results obtained by an independent analytical procedure.
h) The Response Factor of the Drug Substance can be used.
i) Accuracy should assessed using a minimum of Nine (9) determinations over a minimum of Three (3) concentration levels covering the specified range (e.g. three concentrations and three replicates of each concentration).
j) Accuracy should be reported as Percent Recovery of the known added amount of the analyte in the sample.

5.1.9 LINEARITY
The Linearity of an analytical procedure is its ability (Within a given Range) to obtain test results which are directly proportional to the concentration (Amount) of analyte in the sample.
A Linear relationship should be evaluated across the Range of the analytical procedure.
Linearity should be evaluated by inspection of a plot of signals as a function of analyte concentration.
Linearity should be established across the Range of the analytical procedure.
For the establishment of Linearity, a minimum of Five (5) Concentrations should be used.



5.1.10 RANGE
The Range of an analytical procedure is the interval between the Upper and the Lower concentrations (Amounts) of analyte in the sample (Including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of Precision, Accuracy and Linearity.
The Range is usually expressed in the same units as test results, for example, Percent, Parts Per Million (PPM) obtained by the analytical procedure.
The specified Range is normally derived from Linearity studies, and depends on the intended application of the analytical procedure.
The following minimum specified Ranges should be considered:
a) For the Assay of a Drug Substance or a Drug Product: From 80% to 120 % of the Test Concentration.
b) For Content Uniformity (CU): Covering a minimum of 70% to 130 % of the Test Concentration.
c) For testing: ± 20% over the Specified Range.
i. For Example:
If the Specification for a Controlled Released Product cover a region from 20% after One (1) Hour, up to 90% after Twenty Four (24) Hours, the validated range would be 0 to 110% of the label claim.
d) For the determination of an Impurity, from 50% to 120% of the Acceptance Criteria.
e) If Assay and Purity are performed together as one test, and only a 100% Standard is used, Linearity should cover the Range from the reporting level of the Impurities to 120% of the Assay Specification.

5.1.11 LIMIT OF DETECTION (LOD)
The Limit of Detection of an analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
The Detection Limit (DL) is usually expressed as the concentration of analyte, e.g. Percentage, Parts Per Million (PPM), in the sample.
Detection Limit can be determined by using various approaches, depending on whether the procedure is Instrumental or Non-Instrumental:-

a) Based on Visual Evaluation:
Visual evaluation may be used for Non-Instrumental methods, but may also be used with Instrumental methods.
The Detection Limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.

b) Based on Signal-To-Noise (S/N) Ratio:
This approach can only be applied to analytical procedures (Instruments) which exhibit Baseline Noise.
Determination of the Signal-To-Noise (S/N) Ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of Blank samples and establishing the minimum concentration at which the analyte can be reliably detected.
A Signal-To-Noise (S/N) Ratio between 3 or 2:1 is considered acceptable for estimating the Detection Limit.

 

c) Based on the Standard Deviation of the Response and the Slope:

The Detection Limit (DL) is expressed as:

DL= 3.3 σ/s

 

Where,

σ          = STEYX – Standard Deviation of the Response.

S          = Slope of the Calibration Curve.

The Slope “S” shall be estimated from the Calibration Curve of the analyte.

The Standard Deviation of Response (σ) can be estimated using the following ways:
1) Based on the Standard Deviation of the Blank:
Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the Standard Deviation of these responses.
2) Based on Calibration Curve:
A specific Calibration curve shall be studied using samples containing an analyte in the Range of Detection Limit (DL).
The Residual Standard Deviation of a regression line or the standard deviation of y-intercepts of regression lines can be used as the Standard Deviation.

5.1.12 LIMIT OF QUANTITATION (LOQ)
The Quantitation limit of an analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.
The Quantitation Limit (QL) is usually expressed as the concentration of analyte, e.g. Percentage, Parts Per Million (PPM), in the sample.
Quantitation Limit (QL) can be determined by using various approaches, depending on whether the procedure is Instrumental or Non-Instrumental:-

a) Based on Visual Evaluation:
Visual evaluation may be used for Non-Instrumental methods, but may also be used with Instrumental methods.
The Quantitation Limit (QL) is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can reliably be quantified with acceptable accuracy and precision.

b) Based on Signal-To-Noise (S/N) Ratio:
This approach can only be applied to analytical procedures (Instruments) which exhibit Baseline Noise.
Determination of the Signal-To-Noise (S/N) Ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of Blank samples and establishing the minimum concentration at which the analyte can reliably be quantified.
A Signal-To-Noise (S/N) Ratio 10:1 is considered acceptable for estimating the Quantitation Limit (QL).

c) Based on the Standard Deviation of the Response and the Slope:

The Quantitation Limit (QL) is expressed as:

QL= 10 σ /S

 

Where,

σ          = STEYX  Standard Deviation of the Response.

S          = Slope of the Calibration Curve.

The Slope “S” shall be estimated from the Calibration Curve of the analyte.



The Standard Deviation of Response (σ) can be estimated using the following ways:

1) Based on the Standard Deviation of the Blank:
Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the Standard Deviation of these responses.
2) Based on Calibration Curve:
A specific Calibration curve shall be studied using samples containing an analyte in the Range of Quantitation Limit (QL).
The Residual Standard Deviation of a regression line or the standard deviation of y-intercepts of regression lines can be used as the Standard Deviation.

5.1.13 ROBUSTNESS
The Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during routine usage.
One consequence of the evaluation of Robustness should be that a series of System Suitability parameters (For example: Resolution) is established to ensure that the Validity of the analytical procedure is maintained.

Examples of Typical Variations are:

a) Variations of pH Value in a Mobile Phase (In HPLC).
b) Variations in Mobile Phase Composition (In HPLC & TLC).
c) Usage of Columns, of different Lots / Different Suppliers / Manufacturers (In GC & HPLC).
d) Variation in Column Oven Temperature (In GC & HPLC).
e) Variation in Flow Rate (In GC & HPLC).
f) Stability of Analytical Solutions.
g) Extraction Time.
h) Variation in Thermostat Time and Temperature (In GCHS).

5.1.14 Filter Equivalency
The different types / from different manufacturers filters shall be studied and then suitable filters shall be chosen for respective procedure.

5.1.15 Solution Stability
This parameter gives an idea of stability of analytical solutions. After the confirmation of the period of the solution stability, the same solutions can be used for analysis for the stability period established. If the solution is unstable at room temperature, then the stability of analytical solutions shall be studied at low temperatures.
5.1.16 SYSTEM SUITABILITY (SS)
System Suitability (SS) testing is an integral part of almost all the analytical procedures. The tests are based on the concept that the Equipment, Electronics, Analytical Operations and the samples to be analyzed constitute an integral system that can be evaluated as such.
System Suitability test parameters to be established for a particular procedure depends on the type of procedure being validated.
Usually following System Suitability parameters shall be studied and established (But the list is not limited to these parameters):-
a) Resolution.
b) Tailing Factor.
c) Theoretical Plates.
d) Relative Retention Time (RRT).
e) Retardation Factor (RF).
f) Relative Retardation Factor (RF).
g) % Relative Standard Deviation (RSD).

 

5.2 VALIDATION PROTOCOL
Prior to executing the validation study a Validation Protocol shall be prepared by Quality Control personnel for the Analytical Method which is / are to be validated.
The preparation of Protocol shall depend on the method of the analysis to be employed / validated
The Validation Protocol shall provide all the details on how the validation is to be carried out and should include the Acceptance Criteria for each parameter.
Validation Protocol should contain the following (But not limited to):
a) Cover Page.
b) Index.
c) Protocol Approval.
d) Introduction.
e) Objective.
f) Scope.
g) Reference [If Any].
h) Parameters to be validated.
i) Instruments, Chemicals / Reagents, Standards Required.
j) Analytical Method / Procedure (Methodology).
k) Acceptance Criteria.
l) Abbreviations.
m) Annexure(s) [If Any].
n) Table(s) [If Any].
o) Conclusion.
Validation activity shall be executed by addressing the following parameters (But not limited to):
a) Specificity / Selectivity.
b) Precision.
c) Repeatability.
d) Limit of Detection (LOD).
e) Limit of Quantitation (LOQ).
f) Linearity and Range.
g) Ruggedness (Intermediate Precision).
h) Accuracy (By Recovery).
i) Filter Equivalency.
j) Solution Stability.

5.2.1 Linearity and Range
a) Determine from 50% to 150%.
b) Determine for 80%, 90%, 100%, 110% and 120%.
c) Each level to be injected in Duplicate.
d) Plot a graph of Concentration V/s Area.
e) Correlation coefficient, y-intercept, slope of the regression line to be reported.
f) Range to be reported.
i) If the Assay and CU method is the same then Linearity to be done from 70% to 130%.
j) Calculate Coefficient of Correlation (R¬2).

5.2.2 Accuracy

a) Determine at 80%, 100% and 120% in Triplicate.
b) Each sample to be Injected in Duplicate.
c) % RSD to be determined.
d) % Recovery to be calculated at each level and mean % recovery also to be calculated.
e) Accuracy should be reported as % Recovery.

5.2.3 Precision:
A. System Precision:
1) System Suitability to be established.
2) % RSD of Five (5) Replicate Injections/Absorbance values of standard to be calculated.
B. Method Precision
1) Six determinations of the Assay of the Single Batch of sample, with independent weighing.
2) % RSD of six assay values to be calculated.

5.2.4 Ruggedness (Intermediate precision)
a) Six determinations of Assay of the same sample to be analyzed on another day, by a different analyst, using different instrument and different column.
b) % RSD of twelve assay values (Method Precision and Ruggedness) to be calculated.
5.2.5 Robustness
Following Studies shall be Performed:
a) Influence of Variations of pH in a Mobile Phase (if pH adjustment is present in Mobile phase).
b) Influence of Variations in Mobile Phase Composition.
c) Influence of Variation of Wavelength.
d) Temperature (If Temperature condition is present in method).
e) Influence of Flow rate of Mobile phase.
f) % Assay and % RSD to be calculated for samples at each variable condition

5.2.6 Filter Equivalency
a) Check with at least two different filters (Nylon, PTFE [Poly Tetra Fluoro Ethylene], GFC, etc.)
b) Check with Membrane filters (0.45µ)
c) Compare with centrifugation sample.

5.2.7 Solution Stability
a) Check the solution at room temperature for at least 24Hrs.
b) Check for Standard as well as Sample.
c) Difference between the initial assay value for standard and sample to be calculated against a fresh against a fresh standard.
d) The analyte should be considered stable as long as there is no significant rise in the impurity levels.
5.2.8 System Suitability
System suitability shall be established on each day of validation exercise, as per the method of analysis.
Therefore Data on different days, for ruggedness, for robustness to be compiled for the following:
a) Retention Time.
b) Relative Retention Time (RRT).
c) %RSD.
d) Tailing Factor.
e) Theoretical Plates.
f) Resolution (if applicable), etc.
After studying these parameters, particular system suitability parameters are to be set for the method for routine usage.
Acceptance Criteria for various validation parameters, For Assay, are mentioned in the following Table-:

Sr. No. VALIDATION PARAMETER ACCEPTANCE CRITERIA
1.0

 

SPECIFICITY:

Identification.

 

Placebo Interference.

 

Known Impurity Interference *.

 

Forced degradation studies *.

 

Results should be comparable with respect to Retention Time.

Diluent and Placebo should not show any peak at the Retention Time of Analyte Peak.

Difference in mean of Spiked and Unspiked (control) Sample Assay should Not be more than 1.0%.

Peak Purity should Pass in Control Sample and Spiked Sample.

Analyte Peak should be Homogeneous and there should     be  no co- eluting Peaks.

Peak Purity for analyte peak should pass.

 

2.0 LINEARITY & RANGE. ·      Correlation Coefficient should not be less than 0.99.
3.0 ACCURACY (By Recovery).   Mean Recovery should be in the range of 98.0% to 102.0%.

The RSD should not be more than 2.0%.

4.0

 

PRECISION:

·     System Precision.

·     Method Precision.

 

·      RSD should not be more than 1.0%.

·      RSD should not be more than 2.0%.

 

5.0 RUGGEDNESS                     (INTERMEDIATE PRECISION) ·     Overall RSD for Twelve Results should not be more than 2.0%.

 

6.0 STABILITY OF ANALYTICAL SOLUTION. ·      Standard Solution: Assay of old Standard against freshly prepared Standard should be between 98.0% to102.0%.

·    Sample Solution: Correlation of old sample solution against initial Assay should be between 98.0-102.0%.

7.0 ROBUSTNESS.

 

  System suitability should meet as per the test method for each variable condition.

Overall RSD should not be more than 2.0% for the results obtained for both, control and variable conditions.

 

8.0 FILTER EQUIVALENCY.   Filters shall be considered suitable, if the correlation lies between 98.0% and 102.0%.
9.0 SYSTEM SUITABILITY Should comply as per the specified criteria.

 

5.2.9 Ruggedness (Intermediate precision)
a. One set of shall be repeated on another day, by a different analyst, using
Another instrument.
b. % RSD of Twelve values (Method precision and Ruggedness) shall be calculated.
c. Standard deviation, relative standard deviation (coefficient of variation) shall be reported for each type of Precision investigated.

5.2.10 Filter Equivalency
a) Check with at least two different filters (Nylon, PTFE [Poly Tetra Fluoro Ethylene],GFC, etc.)
b) Check with Membrane filters (0.45µ)
c) Compare with centrifugation.
5.2.11 Solution Stability
a) Check the Solution at Room Temperature for at least 24Hrs.
b) Check for Solution Stability of Standard as well as Sample.
c) Difference between the initial assay value for standard and sample shall be calculated against a fresh standard.

5.2.12 System Suitability shall be Established on Each Day, As per the Method of Analysis
Therefore Data on different days, shall be compiled for the following:
a) Retention time (For HPLC).
b) %RSD.
c) Tailing Factor (For HPLC).
d) Theoretical Plates (For HPLC).
e) Resolution (For HPLC if applicable), etc.
After studying these parameters, particular System Suitability parameters shall be established for method for routine usage.
Acceptance Criteria for various parameters during Validation are given in the following Table:-
Acceptance Criteria for Test

Sr. No. VALIDATION PARAMETER ACCEPTANCE CRITERIA
1.0 SPECIFICITY:   There should be No interference from Blank and Placebo at the Retention Time / Wavelength of analyte.

Peak purity should pass.

2.0 LINEARITY AND RANGE. ·       Correlation coefficient should not be less than 0.99.
3.0 ACCURACY (By Recovery).  Mean Recovery should be between 95% to 105%.
 

4.0

 

 

PRECISION:

§  System Precision.

§  Method Precision.

 

·         RSD of Five (5) Replicate injections of Standard should not be more than 2.0%.

·         RSD of Six samples should not be more than 5.0%.

5.0 RUGGEDNESS

(INTERMEDIATE PRECISION)

§  Overall RSD of Twelve samples should not be more than 5.0%.
6.0



   
STABILITY OF ANALYTICAL SOLUTIONS.

 

·        Standard solution: Assay of old Standard against freshly prepared should be between 98.0-102.0%.

·        Sample solution:  Correlation of old Sample solution against initial value should be between 98.0-102.0%.

7.0 FILTER EQUIVALENCY.      Filters shall be considered suitable, if the Correlation lies between 98.0% and 102.0%.
8.0 SYSTEM SUITABILITY.   Should comply as per the specified criteria.

Acceptance Criteria for Related Substances / Residual Solvents / Chromatographic Purity

Sr. No. VALIDATION PARAMETER ACCEPTANCE CRITERIA
1.0

 

SPECIFICITY:

Identification.

 

Placebo Interference.

 

Forced Degradation Studies.

 

 

Results should be comparable with respect to Retention time.

Peak purity should pass for analyte and known impurities in control sample and spiked sample.

Diluent and Placebo should not show any peak at the retention time of analyte Peak.

Analyte peak should be homogeneous and there should be no co-eluting peaks.

Peak purity for analyte peak should pass.

2.0 LOD/LOQ: 

Analyte and Known impurities

   RSD for LOD : NMT 33%

RSD for LOQ : NMT 10%

3.0 LINEARITY & RANGE   Correlation Co-efficient should not be less than 0.99.

Method Precision:
 One set of viscosity (6 times) shall be carried out.
 % RSD of Six (6) values shall be calculated.

5.4.1.2 Ruggedness (Intermediate precision)
• One set of viscosity shall be repeated on another day, by different analyst.
• % RSD of Twelve values (Method Precision and Ruggedness) shall be calculated.
5.4.1.3 Robustness
Following studies shall be done (But not limited to):
• Temperature (Temperature Condition if present in method).
• Speed (RPM) shall be varied, etc.
• Viscosity shall be calculated for samples at each variable condition.

Acceptance Criteria For Viscosity

Sr. No. VALIDATION PARAMETER ACCEPTANCE CRITERIA
1.0

 

METHOD PRECISION.

 

RSD should not be more than 10.0%.
2.0 RUGGEDNESS.

 (INTERMEDIATE PRECISION).

Overall RSD for Twelve results should not be more than 10.0%.
3.0 ROBUSTNESS. Overall RSD should not be more than 10.0% for the results obtained at control and variable conditions.

5.4 Revalidation
Revalidation is necessary in following circumstances (But not limited to):
a) Changes in the impurity profiling of Drug substance used in the Drug Products.
b) Changes in the composition of the Drug Products.
c) Changes in the analytical Procedure.
d) Source variation of drug substance.
e) Revalidation may require Partial Validation of previously validated method or Complete validation depending on the changes done. This decision shall be taken by Head of the Department with proper justification and shall be documented in the Report.

5.6 Documentation of Analytical Data
5.6.1 All the experiments done shall be documented properly.
5.6.2 All the raw data and results shall be compiled.
5.6.3 All the raw data (chromatograms and calculation sheets) shall be signed by the Analyst and
the checker.
5.6.4 All the calculation sheets shall be checked and signed by the Section In-charge or the Designee.
5.6.5 After Validation is complete a final validation Report shall be prepared.
5.6.6 At the end of the Validation report a Summary and Conclusion of Validation shall be given.
5.6.7 Annexure, if any shall be attached at the end of Validation Report.
5.6.8 If all the parameters are within limits specified in Validation Protocol the method shall be considered Valid. If any parameter fails as per Validation Protocol, the same shall be investigated and corrective action shall be taken accordingly.
5.6.9 The Head of the Department shall review the Validation protocol, raw data and Validation report and then the same shall be approved by Head – Quality.
5.6.10 This Validated Method shall be incorporated in the respective MOA / STP of that Material / Product.

6.0 ABBREVIATION

Sr. No. Abbreviation used Extended Form
2. QC. Quality Control.
3. SOP. Standard Operating Procedure.
4. MOA. Method of Analysis.
5. COA Certificate of Analysis.
6. LOD Loss on Drying.

7.0 REFERENCE

Sr. No. Reference Title
01. Indian Pharmacopeia

 

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