2. CONTENTS OF PRESENTATION:
❖ WHAT IS TEST PROCEDURE?
❖ WHAT IS THE ANALYTICAL METHOD VALIDATION?
❖ WHY IT IS IMPORTANT?
❖ TYPICAL CHARACTERISTIC OF ANALYTICAL METHOD VALIDATION
❖ DIFFERENCE BETWEEN VALIDATION AND VERIFICATION
❖ ANALYTICAL METHOD TRANSFER
❖ ANALYTICAL METHOD LIFECYCLE
❖ GUIDANCE AND REFERENCES
3. WHAT IS TEST PROCEDURE/MOA?
❖ Test procedure is a approved documents which describes in detail the steps
necessary to perform each analytical test.
This may include but is not limited to:
➢ Test sample,
➢ Reference standard/Working Standards/CRS
➢ Chemicals and the reagents preparations,
➢ use of the apparatus,
➢ generation of the calibration curve,
➢ use of the formulae for the calculation, etc.
4. WHAT IS ANALYTICAL METHOD VALIDATION
DEFINITION:
❖ Method validation is the process by which it is established, through laboratory
studies, that the performance characteristics of the method meet the
requirements for its intended purpose.
❖ It is a part of the overall validation process that also includes software
validation, instrument qualification, and system suitability.
5. WHY IT IMPORTANT?
GMP Requirement:
ICH Q7 - 12.8 Validation of Analytical Methods
● 12.80 Analytical methods should be validated unless the method employed
is included in the relevant pharmacopoeia or other recognised standard
reference. The suitability of all testing methods used should nonetheless be
verified under actual conditions of use and documented.
21 CFR Part 211: Sec. 211.194 Laboratory records.
The suitability of all testing methods used shall be verified under actual conditions
of use. regulations [21 CFR 211.194(a)(2)], users of analytical methods described
in USP–NF are not required to validate the accuracy and reliability of these
methods, but merely verify their suitability under actual conditions of use
6. WHY IMPORTANT?
EudraLex Volume - 4, Part-1, Chapter 6: Quality Control
● 6.15 Testing methods should be validated. A laboratory that is using a
testing method and which did not perform the original validation, should
verify the appropriateness of the testing method.
EudraLex Volume -4, Annex - 15: Qualification and Validation
● 9.1. All analytical test methods used in qualification, validation or cleaning
exercises should be validated with an appropriate detection and
quantification limit, where necessary, as defined in Chapter 6 of the
EudraLex, Volume 4, Part I.
7. TYPE OF METHODS TO BE VALIDATED
❖ CATEGORY I
➢ Analytical procedures for quantitation of major components of bulk drug substances
or active ingredients (including preservatives) in finished pharmaceutical products.
❖ CATEGORY II
➢ Analytical procedures for determination of impurities in bulk drug substances or
degradation compounds in finished pharmaceutical products. These procedures
include quantitative assays and limit tests.
❖ CATEGORY III
➢ Analytical procedures for determination of performance characteristics (e.g.,
dissolution, drug release, and others).
❖ CATEGORY IV
➢ Identification tests.
9. ● Data Elements Required for Method Validation
*May be required, depending on the nature of specific test
10. Method Validation Parameters:
1.1. 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.
➢ Lack of specificity of an individual analytical procedure may be
compensated by other supporting analytical procedure(s).
11. Method Validation Parameters:
.1. SPECIFICITY (CONT.)
➢ Interference due to diluent/Blank/Placebo
➢ Interference due to Impurities/Degradants
➢ Forced Degradation: this should include samples stored under
relevant stress conditions: light, heat, humidity, acid/base hydrolysis
and oxidation.
➢ Peak purity tests assessed to show that the analyte chromatographic
peak is not attributable to more than one component (e.g., diode
array, mass spectrometry).
12. Method Validation Parameters:
2. 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.
3. RANGE
The range of an analytical procedure is the interval between the upper and
lower concentration (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.
13. Method Validation Parameters:
3. RANGE (CONT.)
The following minimum specified ranges should be considered:
➢ For the assay of a drug substance or a finished (drug) product: normally from
80 to 120 percent of the test concentration;
➢ For content uniformity, covering a minimum of 70 to 130 percent of the test
concentration, unless a wider more appropriate range, based on the nature of
the dosage form (e.g., metered dose inhalers), is justified;
➢ For dissolution testing: +/-20 % over the specified range;
➢ For the determination of an impurity: from the reporting level of an
impurity(LOQ) to 120% of the specification.
16. Method Validation Parameters:
4. ACCURACY (RECOVERY)
➢ 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 also termed as trueness.
➢ Accuracy should be established across the specified range of the
analytical procedure.
17. Method Validation Parameters:
5. 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. Precision may be considered at three levels:
repeatability, intermediate precision and reproducibility.
➢ The precision of an analytical procedure is expressed as the variance,
standard deviation or coefficient of variation of a series of measurements.
18. Method Validation Parameters:
5.1. Repeatability/Method Precision:
➢ Repeatability expresses the precision under the same operating conditions
over a short interval of time. Repeatability is also termed intra-assay precision
➢ Repeatability assessed using:
➢ Minimum of 9 determinations covering the specified range for the procedure
(e.g., 3 concentrations/3 replicates each); or
➢ Minimum of 6 determinations at 100% of the test concentration.
20. Method Validation Parameters:
5.2. Intermediate precision/Ruggedness:
➢ Intermediate precision expresses within-laboratories variations: different
days, different analysts, different equipment, etc.
5.3. Reproducibility:
➢ Reproducibility expresses the precision between laboratories
(collaborative studies, usually applied to standardization of methodology).
22. Method Validation Parameters:
6. LIMIT OF DETECTION & LIMIT OF QUANTIFICATION
LOD
➢ The detection limit of an individual analytical procedure is the lowest amount
of analyte in a sample which can be detected but not necessarily quantitated
as an exact value.
LOQ
➢ The quantitation limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be quantitatively determined with
suitable precision and accuracy.
23. Method Validation Parameters:
❖ Following are the approaches followed for LOD/LOQ
6.1. 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.
24. Method Validation Parameters:
LOD/LOQ (CONTI.)
6.2. Based on Signal-to-Noise
➢ Determination of the signal-to-noise 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 ratio between 3 or 2:1 is generally considered acceptable
for estimating the detection limit.
25. Method Validation Parameters:
LOD/LOQ (CONTI.)
6.3 Based on the Standard Deviation of the Response and the Slope
The detection limit (DL) & quantitation limit (QL) may be expressed as:
may be expressed as:
LOD = 3.3 σ & LOQ = 10 σ
S S
where σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried
out in a variety of ways, for example:
27. Method Validation Parameters:
9. 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 normal usage.
➢ Examples of typical variations are:
➔ stability of analytical solutions;
➔ extraction time.
➔ Filter compatibility Study
28. Method Validation Parameters:
9. ROBUSTNESS (CONTI.)
In the case of liquid chromatography, examples of typical variations are:
➔ Flow rate.
➔ Column Oven Temperature;
➔ Influence of variations of pH in a mobile phase;
➔ Influence of variations in mobile phase composition;
➔ Different columns (different lots and/or suppliers);
29. Method Validation Parameters:
10. SYSTEM SUITABILITY
➢ System suitability testing is an integral part of analytical procedures. The tests
are based on the concept that the equipment, electronics, analytical
operations and 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
depend on the type of procedure being validated.
30. WHAT IS THE METHOD VERIFICATION?
VERIFICATION OF TEST PROCEDURES:
➢ The verification process for compendial test procedures is the assessment of
whether the procedure can be used for its intended purpose, under the actual
conditions of use for a specified drug substance and/or drug product matrix.
➢ All the compendial test procedure needs to be verified.
➢ method validation is usually applied to an “in-house method” developed by a
laboratory; while method verification is applied to a “compendial method or
previously validated method” when it is being use in a particular laboratory for
the first time.
31. ANALYTICAL METHOD TRANSFER
➢ 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), thus ensuring that the receiving unit has the
procedural knowledge and ability to perform the transferred analytical
procedure as intended.
➢ TYPES OF TRANSFERS OF ANALYTICAL PROCEDURE
➢ Comparative Testing
➢ Co-Validation Between Two or More Laboratories
➢ Revalidation/Method Verification
32. Method Transfer Waiver
● The conventional method transfer may be omitted under certain
circumstances. In such instances, the receiving unit is considered to be
qualified to use the analytical test procedures without comparison and
generation of interlaboratory comparative data.
● The following examples give some scenarios that may justify the waiver of
TAP:
• The new product’s composition is comparable to that of an existing product
and/or the concentration of active ingredient is similar to that of an existing
product and is analyzed by procedures with which the receiving unit already
has experience.
33. Method Transfer Waiver
➢ The analytical procedure transferred is the same as or very similar to a
procedure already in use.
➢ The personnel in charge of the development, validation, or routine analysis of
the product at the transferring unit are moved to the receiving unit.
➢ If eligible for transfer waiver, the receiving unit should document it with
appropriate justifications
35. LIFE CYCLE MANAGEMENT OF ANALYTICAL PROCEDURES
➢ Once an analytical procedure (including compendial methods) is successfully
validated and implemented, the procedure should be followed during the life
cycle of the product to continually assure that it remains fit for its intended
purpose.
➢ Over the life cycle of a product, new information and risk assessments (e.g., a
better understanding of product CQAs or awareness of a new impurity) may
warrant the development and validation of a new or alternative analytical
method. New technologies may allow for greater understanding and/or
confidence when ensuring product quality.
36. GUIDELINES REFERENCES:
❏ ICH Q2(R1) - VALIDATION OF ANALYTICAL PROCEDURES
❏ USP <1224> - TRANSFER OF ANALYTICAL PROCEDURES
❏ USP<1225> - VALIDATION OF COMPENDIAL PROCEDURES
❏ USP<1226> - VERIFICATION OF COMPENDIAL PROCEDURES
