Analytical Method Validation (AMV)

&
Analytical Method Transfer (AMT)

M Jagadeesh
AD
Systematic Steps for Validation

Analytical Method
Plant Specifications & STPS
Development
(Q.C)

Test Method

Analytical Method Validation Analytical Method

Protocol (AMVP) Validation Report (AMVR)

Analytical Method Transfer

(AMT)
 Validation
 Definition:

Establishing documented evidence that provides a high degree of

assurance that a specific process will consistently produce a product
meeting its pre determined specifications and Quality Attributes.

 Guidelines for Validation:

ICH (Q2) R2: Analytical Method Validation

USP <1225>: Validation of Compendial procedures

WHO: Good Manufacturing Practices: Guidelines on Validations

Purpose of Method Validation

 Identification of sources and Quantification of potential errors

 Establish proof that a method can be used for regular purpose

 Satisfy Regulatory requirements

Examples of Methods that requires Validation
 Spectrophotometric Methods:
UV/Vis , IR ,NMR ,XRD ,MS etc.
 Chromatographic Methods:
HPLC,TLC,GC,GC/MS etc.
 Capillary Electrophoresis Methods
 Particle size analysis Methods:
Laser, Microscopic , Sieving etc.
 Bio Analytical Methods:
PK/PD/Clinical studies
Requirements of Validation
 Impurities Quantities along with COA’s

 API,WRS or RS Quantities along with COA’s

 Sample Quantities along with COA’s

 Placebo Quantities along with COA’s

 Batch Analysis COA’s

 Specifications

 Chemicals and Columns atleast 2 No’s

 Test Methods or STP

 MSDS
 Validation Parameters
 System Suitability and System Precision
 Specificity/Selectivity
 Method Precision
 Intermediate Precision
 Accuracy/Recovery
 LOQ & LOD Establishment
 Linearity
 Range
 Solution Stability
 Mobile Phase Stability
 Robustness
System Suitability and System Precision
 System Suitability is to prove that system is working perfectly before
analysis.

 System precision related to reproducibility and repeatability, under same

conditions show the same results.

Acceptance Criteria:
Dissolution by HPLC:%RSD-NMT 2.0%
Dissolution by UV: %RSD-NMT 3.0%
Assay: %RSD-NMT 2.0%
RC:%RSD-NMT10.0%
 Specificity/Selectivity
 Specificity is the ability of the analytical method to distinguish between the
analyte (s) and the other components in the sample matrix.

 Blank, Placebo, Standard , impurities, Samples free from interference.

 Specificity plays a major role in

1. Forced Degradation Studies (FDS)

2. Interference Study

3. Filter study
 Forced Degradation Studies
 Types of Degradation:
1. Physical conditions: Thermal , Photolytic , Humidity.
2. Chemical conditions: Acid, base, Peroxide, Hydrolysis

 Degradation:5%-20%

 Peak Purity Should be passed for standard ,sample and impurities

 Always PDA HPLC Systems use.

 Normalized Mass Balance: 95% - 105%

 Mass Balance: (%Assay of Stressed sample+% Total imp.)/% Assay of Unstressed

SampleX100

Acceptance Criteria:

Assay and RC: Peak Purity should be passed.

Mass Balance: should be achieved.

 Interference Study
 Interference occurs when a substance or process falsely alters an test result.

 Blank , Placebo, Standard,Samples,Impurities plays a major role.

Acceptance Criteria:

Dissolution and Assay: No interference at RT of Active peak

RC: Disregard below LOQ Level

 Filter Study
 Also known as Filter Saturation study.
 Blank, Placebo , Standard and Sample solution to be established.
 (Centrifuged or Unfiltered)
 Discarding and collect and inject 1st mL,2nd mL,3rd mL solution.
 Atleast 2 different types of filters required (PVDF, Nylon)
Filter solution-Unfiltered solution
Filter Study = Unfiltered solution X100
Acceptance Criteria:
Dissolution: NMT 3.0%
Specification %Difference
Assay: NMT 2.0% Level Acceptance Criteria
RC: Individual impurities
≤0.5% ±0.05 absolute difference
>0.5% ± 10%of Relative from Initial value
Total impurities
≤1.0% ±0.1% absolute difference
>1.0% ± 10%of Relative from Initial value
 Method Precision (Repeatability)
 Definition:
Closeness of Agreement between a series of measurements obtained from
multiple sampling of the same homogenous sample.
 Control and Spiked Samples(Spec. Level) injected (n=6)
Acceptance Criteria:
 Product should meet specification limit (Control Sample)
 Dissolution : %RSD NMT 2.0%
 Assay :%RSD NMT 2.0%
 RC:
%w/w %RSD of
(Mean Value) %w/w
impurities
≤0.05 or LOQ NA
>0.05 To 0.1 30.0%
0.1 To 0.2 15.0%
>0.2 10.0%
 Intermediate Precision
 Expresses within laboratories variations: different days,analysts,systems,columns.
 Another set of six samples (Control, Spiked samples) will be performed.
Acceptance Criteria:
 Product should meet specification limit (Control Sample)
 Dissolution : %RSD NMT 2.0%
 Cumulative %RSD(n=12)-NMT 5.0%
 Assay :%RSD NMT 2.0%
 Cumulative %RSD(n=12)-NMT 2.0%
 RC:
 Accuracy/Recovery
 Definition:
Closeness of agreement between the value which is accepted as either as
conventional true value or an accepted reference value and the value found.
 Prepare 3 Samples for each level-Total of 9 samples.
 Dissolution: 10%-150% test concentration(NLT 3 Levels)
 Assay:25%-150% test concentration(NLT 3 Levels)
 RC-LOQ to 300% of specification level
 Weighing powder method or by spiking method on placebo or on samples.
Acceptance Criteria:
Dissolution: %Recovery -95-105% and %RSD NMT 5.0%
Assay: %Recovery -98-102% and %RSD NMT 2.0%
RC: Specification Acceptance
Level(%) criteria %RSD
%Recovery
≤0.05 or LOQ 75.0-125% 15.0
>0.05 To 1.0 85.0-115% 10.0
0.1 To 0.2 90.0-110% 5.0
>0.2 90.0-110% 5.0
 LOQ and LOD Establishment
 Limit of Quantification:
Lowest amount of analyst in a sample, which can be quantitatively
determined with suitable precision and accuracy.
LOQ=10σ/s Where σ = Standard deviation of Response
S = Slope of Calibration curve

Limit of Detection:
Lowest amount of analyst in a sample, which can be detected but not
necessarily quantitated as an exact value.
LOD=3.3σ/s Where σ = Standard deviation of Response
S = Slope of Calibration curve
Acceptance Criteria:
LOQ: s/n should be 10
LOD: s/n should be 3
 Linearity
Definition:
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.
Minimum of 6 levels required
Dissolution: 0% to 150% of Target Standard concentration
Assay: 0% to 150% of Target standard concentration
RC: LOQ-300% of shelf life specification
Acceptance criteria:
Dissolution: r2 NLT 0.999,Y-intercept-NMT 2.0%
Assay: r2 NLT 0.99,Y-intercept-NMT 2.0%
RC:r2 NLT 0.98,Y-intercept-NMT 2.0%
Report RRF value
 Range
 Range of an analytical method define as interval from the upper to the

lower concentration of the analyte/impurities in the sample with

Accuracy, Linearity and precision experiments.

 Solution Stability
 Solution stability plays a major role in sample and standard solution
establishments.
 Intial,24hrs,48hrs-on day basis will be established. If fails-hourly basis.
 Establishes in both RT and Rf conditions.

Acceptance Criteria:
For Standard : Similarity Factor should pass 0.98 to 1.02
Dissolution: Sample from initial to Next time point NMT3.0%
Assay: Sample from initial to Next time point NMT2.0%
RC: Specification Level %Difference
Acceptance Criteria
Individual impurities
≤0.5% ±0.05 absolute difference
>0.5% ± 10%of Relative from Initial value
Total impurities
≤1.0% ±0.1% absolute difference
>1.0% ± 10% of Relative from Initial value
 Mobile Phase Stability

 Stability of Mobile phase plays a major role in Validation.

 Mobile phase stability was established for 2 days on BT
 Every day fresh SST will injecting and calculating against aged standard
and sample solutions
 Any Colour change or particles observed .Mobile phase stability will be
aborted.
Acceptance criteria:
Dissolution: %Difference for Sample NMT 2.0%
Assay: %Difference for Sample NMT 2.0%
Specification Level (%w/w) %Relative Variation
RC:
Individual impurities
<0.10 ±30
0.10 To 0.50 ± 20
>0.50 ± 10
Total impurities
0.10 To 0.50 ±25
>0.50 ±15
 Robustness
 Definition:
It is a measure of its capacity to remain unaffected by small, but deliberated
variations in method parameters and provides an indication of its reliability
during normal usage.
Dissolution:
Chromatographic Conditions:
Flow Rate, Column Temperature, Buffer pH, Organic Variations, Wave length
Sampling Conditions:
Auto Vs Manual, RPM Variation, Media Volume Variation, Media PH Variation
Media Temperature.
Assay and RC:
Chromatographic Conditions:
Flow Rate, Column Temperature, Buffer pH, Organic Variations, Wave length
Sampling Conditions:
Sonication Time, Shaking Time, Centrifuge Time, Diluent organic composition
 Analytical Method Transfer (AMT)
 Analytical Method transfer is the documented process that qualifies a laboratory
(the receiving unit) to use an analytical test procedure that originated in another
laboratory (the transferring unit).
 Guidelines for Analytical Method Transfer :
USP <1224>: Transfer of Analytical Procedures
 Types of Analytical Method Transfer:
 Direct Transfer
 Physical demonstration and joint analysis of sample by Transferring unit, Scientist
(R&D – Analytical) and the Receiving unit Chemist/analyst, (QC/other laboratory).
 Data will be comparable.
 Indirect Transfer
 This type of method transfer shall include only Protocol data will be shared to
Transferring unit
 Data will be comparable.
Flow Chat for AMT
 Conclusion
 Summarized the extent, need and necessary of validation.

 Validation may cost initially but it avoids the rise of break down ,
for this reason small companies were concentrating more on
validation their by fulfilling the goals of regulatory markets.
Thank Q