4. Method validation means establishing through
documented evidence, a high degree of assurance that an
analytical method will consistently yield results that
accurately reflect the quality characteristics of the
products tested.
4
5. The currently available guidelines for analytical method
validation were
ICH (Q2R1)
USP (Chapter <1225>)
ANVISA (RE 899)
AOAC
IUPAC
FDA (CDER, CBER - Draft guideline Feb 2014 )
5
6. Four most common types of analytical procedures to be
validated:
• Identification tests
• Quantitative tests for impurities content
• Limit tests for the control of impurities
• Quantitative tests of the active moiety
Compendial Vs Non-compendial methods
6
7. For identification of source of
potential errors.
Determination if Method is
Acceptable for Intended Use.
Establish that test data obtained
was consistent, reliable &
accurate.
Validation of analytical methods
is also required by most
regulations.l
Establish proof that method can
be used for decision making.
7
9. Characteristic Identific
ation
Impurities Testing Assay
Quantitative Limit
Accuracy _ + _ +
Precision
a. Repeatability _ + _ +
b. Intermediate precision _ + _ +
Specificity + + + +
LOD _ _ + _
LOQ _ + _ _
Linearity _ + _ +
Range _ + _ +
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated
9
10. What analyte should be detected?
Should information be qualitative or quantitative?
What are expected concentration levels?
What are sample matrices?
What precision and accuracy is required?
Are there any specific requirements?
What are required detection and quantitation limits?
10
11. Suitability of Instrument - Status of Qualification and
Calibration.
Suitability of Materials - Status of Reference Standards,
Reagents, Sample, Placebo Lots.
Suitability of Analyst - Status of Training and Qualification
Records.
Suitability of Documentation - Written analytical procedure
and proper approved study plan with pre-established
acceptance criteria.
Stability of samples & standard.
11
12. System suitability testing is an integral part of many
analytical procedures.
To verify proper functioning of the operating system
Parameters Recommendation
Resolution R ≥ 1.5, between the peak of interest and
the closest potential interferent
(degradant, Impurity, excipient, etc.)
Tailing T ≤ 2
Theoretical plates (N) In general N > 2000
Repeatability % CV ≤ 2.0% (n ≥ 5)
12
“No sample analysis is acceptable unless the suitability of the
system have been demonstrated.” (USP Chapter 621)
13. The ability to assess unequivocally the analyte in the
presence of components which may be expected to be
present.
Interfering substances
- Active ingredients
- Excipients
- Impurities
- Degradation products
13
14. Forced degradation study (for stability indicating methods)Forced degradation study (for stability indicating methods)
Should include samples stored under relevant stress conditions: light, heat,
humidity, acid/base hydrolysis and oxidation.(Target : 5-20% Degradation)
Purpose:
Stability indicating power of analytical method
Address the stability of compound
Establish the degradation pathway
Identify the degradation products (if required)
Specificity Evaluation:
Identification test
Assay
PDA – peak purity
14
15. Ability to obtain test results that are directly (or by a well-
defined mathematical transformation) proportional to the
concentration of analyte in samples within a given range.
The range of an analytical procedure is the interval
between the upper and lower levels of analyte (including
these levels) that have been demonstrated to be determined
with a suitable level of precision, accuracy, and linearity.
15
16. For establishment of linearity, minimum 5 concentrations
are recommended.
Linearity results should be established by appropriate
statistical methods.
Assay:- 80% to 120% of test concentration is recommended
16
17. The following parameters should be determined:
correlation coefficient (> 0.99)
y-intercept
slope of the regression line
Residual sum of squares
The range of the procedure is validated by verifying that the
analytical procedure provides acceptable precision, accuracy,
and linearity when applied to samples containing analyte at
the extremes of the range as well as within the range.
17
18. 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.
18
21. Repeatability
• Repeatability expresses the precision under the same
operating conditions (with-in a laboratory over a short
period of time).
• Repeatability should be assessed
using a minimum of 6 determinations at 100% level
(or)
9 preparations covering the specified range (3 preparations
each at 3 levels).
Evaluation:
Standard deviation
Relative standard deviation (coefficient of variation)
21
22. • Intermediate precision expresses variations within
laboratories, such as
• different days,
• different analysts,
• different equipment,
• different columns etc.
Evaluation:
Standard deviation
Relative standard deviation (coefficient of variation)
22
23. • Reproducibility expresses the precision between
laboratories. It is assessed by means of an inter-laboratory
trial. (Defined as ruggedness in USP)
23
24. Closeness of agreement between the conventional true
value or an accepted reference value and the value found.
Accuracy = trueness + precision
24
Low Accuracy
High Precision
25. 1. Assay
a)Known amount of drug substance spiked with synthetic
mixtures of drug product components (excipients)
b)Accuracy should be assessed using a minimum of 9
determinations over a minimum of 3 concentration levels
covering the specified range (e.g., 3 concentrations/3
replicates each of the total analytical procedure).
Evaluation
% recovery from the amount added
to amount found
% CV at each level (for 3 replicates)
DRUG PRODUCT
25
26. Limit of Detection:
It is the lowest amount of analyte in a sample which can
be detected but not necessarily quantitated.
Limit of Quantitation:
It is the lowest amount of analyte in a sample which can
be quantitatively determined with suitable precision and
accuracy.
26
27. Method
Based on visual evaluation
Based on standard deviation of response and slope
Signal to noise ratio 2:1 or 3:1
Limit of Detection Limit of Quantitation
LOD = 3.3 σ / Slope LOQ = 10 σ / Slope
27
28. 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.
If measurements are susceptible to variations
in analytical conditions, the analytical
conditions should be suitably controlled or a
precautionary statement should be included in
the procedure.
Typical variations:
Stability of analytical solution
Extraction time
28
29. In the case of liquid chromatography, examples of typical
variations are:
pH of a mobile phase (± 0.2 units)
mobile phase composition (Organic ± 2-5%)
buffer concentration (± 10%)
Temperature (± 5°C)
flow rate (± 10%)
Wavelength (± 2 nm)
Evaluation:
System suitability criteria should meet at each variable
condition
Comparison of results under varying conditions with precision
under normal conditions.
Expressed as % CV (or) as ratio of results.
29
30. Standard and sample solutions were analyzed at initial and
at regular intervals by storing at Room temperature/
refrigerator conditions (2-8°C).
Determine the period of time, a solution can be held before
analysis without compromising accuracy.
Evaluation
The difference b/w initial response to specified interval
(expressed as % difference or as ratio).
30
31. VALIDATION REPORT
Generally method validation report shall have (but not limited to)
Objective and scope of the method (applicability, type).
Standard, Sample and Placebo details.
Detailed list of chemicals, reagents and equipment used
Summary of methodology.
Validation data (parameter wise – procedure, results, conclusion etc.,)
Summary of data (results in brief – parameter wise)
Summary of report (overall view of validation exercise, any critical
issues, recommendations etc., for the application of method)
Representative plots, e.g., chromatograms, spectra, peak purity data and
calibration curves.
31
32. There are no official guidelines on the sequence of validation
experiments and the optimal sequence can be depending on the
method itself. Based on experience, for HPLC method the following
sequence has been proven to be useful for time management.
If the method is proved as
stable and robust under
pre-validation program
1.Specifity
2.Linearity
3.LOD,LOQ*(if required)
4.Precision
5.Accuracy
6.Range
7.Solution stability
8.Robustness
In case if stability and
robustness data is not
available
1. Solution stability
2. Robustness
3.Specifity
4.Linearity
5.LOD,LOQ*(if required)
6.Precision
7.Accuracy
8.Range
32
33. Change in the analytical procedure, drug substance, drug
product may necessitate revalidation of the analytical
procedures.
“The degree of revalidation depends on the nature of the
change.”
Revalidation should accompany
formulation changes (eg. new matrices)
manufacturing changes (eg. Synthetic route)
modification of analytical parameters.
REVALIDATION
33
34. When the method is properly validated – consistent,
reliable and accurate results are obtained.
METHOD VALIDATION =ERROR ASSESSMENT
34
35. 1) ICH harmonised tripartite guideline - Validation of Analytical
Procedures : Text and methodology - ICH Q2(R1)
2) United States Pharmacopoeia, Chapter <1225> Validation of
compendial methods
3) http://www.anvisa.gov.br/hotsite/genericos/legis/resolucoes/20
03/899_03re_e.pdf
4) http://www.aoac.org/imis15_prod/AOAC_Docs/StandardsDev
elopment/SLV_Guidelines_Dietary_Supplements.pdf
5) http://www.iupac.org/publications/pac/
2002/pdf/7405x0835.pdf
1) http://www.labcompliance.com/tutorial/
methods/default.aspx? sm=d_d
35
Validation is defined as the process of demonstrating that the analytical procedure is suitable for its intended purpose
The FDA, under the authority of existing cGMP regulations guidelines and directives, considers validation is necessary and because it makes good sense of science / engineering.
Compendial – suitability of all testing procedures shall be verified under actual conditions of its use
Validated analytical methods play a major role in achieving this goal - consistent, reliable & accurate
No exact methodology given for each parameter
Only ICH – Q2B and CDER guidelines are provided but not to the extent of 100%
Good understanding of each performance characteristics most important. This understanding must be beyond the basic definition of each parameter
Understanding must be anchored by sufficient years of practical experience and knowledge.
It will permit sound and logical decisions, even under the most intense situation
LOD and LOQ
6 blank injections – find the average noise (µV)
LOD = 3 (S/N) (2-3.4)
LOQ = 10 (S/N) (9-11.4)
%RSD should be within 10% if impurity observed more than 0.1% and below 0.5%.if experiment performed by spiking the impurity at 0.5℅ level then %RSD should be NMT 5% in case of UHPLC or UPLC
Identification Tests: Positive response for compound of interest only
Assay : No peak should be found at the retention time of analyte peak in balnk and placebo and Peak purity of analyte peak should pass.
Impurities : Should pass Peak purity of -main analyte and Impurity peaks. (synthetic precursors, enantiomers)
No peak should be found at the retention time of analyte/Impurity
Dissolution: No peak should be found at the retention time of analyte.
Degradation : 5-20% in atleast one condition and 1-20% where ever possible
Degradation also need to be done for placebo
Forced degradation or stress testing is undertaken to demonstrate specificity when developing stability-indicating methods
A stability-indicating method is one that accurately quantitates the active ingredients without interference from degradation products, process impurities, excipients or other potential products.
EVALUATION
% Difference of assay for Control (Un-stressed) and each Stressed samples
Peak purity of analyte peak for Control and stressed sample
Points to remember
If the degradation media degrades the drug substance/drug product to too great extent or do not degrade the drug substance/drug product at all, then alternative action should be taken (e.g., change the strength of the degradation medium or exposure time or apply heat over a period of time to achieve minimum level of degradation.
Some compound may not necessarily degrade under a given stress condition. No further stressing is advised in these cases.
Over stressing may lead to the formation of secondary degradants, causes disturbance to Over stressing may lead to the formation of secondary degradants, causes disturbance to the selectivity of the method, requires further development
The forced degradation studies should mimic the conditions to which the drug substance / drug product is actually exposed during its shelf life
A simple logic behind these studies is, ability to separate the analyte of interest from its degradation impurities formed, if any, caused by quality of chemicals used for its manufacturing, processing, packaging, storage, shipment, and/or any unexpected exposure during shelf life of the drug substance/drug product during shelf life of the drug substance/drug product
In order to determine the quantity of any analyte present in unknown sample, some kind of relation ship (mathematical/empirical) between concentration and response is essential
ANALYTE ------ PROCEDURE ------ RESPONSE EXPLANATORY VARIABLE VS RESPONSE VARIABLE
ICH and USP encouraging Linear and non-Linear relation ships
IUPAC – more than single injections of each of the standards
RRF in RS = slope of impurity / slope of main analyte
Method: dilution of stock solution/separate weightings
Assay:- 80% to 120% of test concentration
Content Uniformity :- 70% to 130% test concentration
Dissolution:- ± 20% of expected release (Q) for immediate release
0 to 120% (for extended release)
Impurities:-Impurities:- LOQ to 200% of specification
EVALUATION
Slope- indicates sensitivity of the method
Intercept- indicates response for no analyte (interference)
Residual sum of squares- indicates uncertainty of intercept(in blank response)
Correlation Coefficient (R)- indicates the relation ship chosen is correct (how strong the relationship is)
% Y INTERCEPT = 10% OF 100% PEAK AREA (RS)
Bias = nmt 2% (y-intercept/100%peak area x 100)
Getting same result over and over again
RS = SPIKED SAMPLE (Will check recovery)
Accuracy = unknown impurity (main Analyte – at 100%, loq) (Spike main analyte to placebo)
Recovery = spike impurities stock solution to sample (subtract spiked sample from control sample)
If 100% analyte conc. Is 1000ppm and if impurity is 0.5% then spike at 5 ppm
A simple logic behind this performance characteristic is whether the procedure is capable of estimating a true value or not
Spiking should be done in a similar manner as that of the weight of the std. (in case of 10mg, 20mg dose formulations then 300% accuracy need to be done to cover 20mg dose)
Sample spiking
Placebo spiking
Dissolution:- 95% to 105%
Impurities:- if, Specification is ≤ 0.2% : 85% to 115%
if, Specification is &gt; 0.2% : 90% to 110%
At LOQ level : 80% to 120%
Resiudal std dev of regression line or standard deviation of y-intercepts of regression lines
RSD of six replicate injections is ≤ 10.0% for LOQ and between &gt; 10.0% and10.0% and ≤ 33.0%
According to ICH guidelines, robustness should be considered early in the development stage of a method, but it is not required to be included as part of a registration application
If measurements are susceptible to variations in analytical conditions, these should be suitably controlled, or a precautionary statement should be included in the procedure.
Series of system suitability parameters is established to ensure that the validity of the analytical procedure is maintained whenever used.
MOBILE PHASE STABILITY – Atleast for 3-5 days (assess system suitability)
Robustness – evaluate system suitability (in RS – check resolution and report RT and RRT)
Assay: Analysis of Resolution (if applicable), Standard & Test samples (2replicates) by proposed analytical methodology and the method operated at variable conditions.
Impurities:Analysis of Resolution & Test sample by proposed analytical methodology and the method operated at variable
EVALUATION
Assay:System Suitability parameters at all variable conditions
% Assay of samples at all variable conditions
Impurities: System Suitability parameters at all variable conditions
RRTs at all variable conditions
( monitor the separation at each variable condition)
A simple logic behind this study is to determine the period of time, a solution can be held before analysis without compromising accuracy.
It is often essential that solutions (standards, test samples) be stable enough to allow for delays covering instrument break downs / overnight analyses. Solutes may readily decompose prior to chromatographicinvestigations e.g. during sample preparation, extraction, cleanup, phase transfer or storage of prepared vials
Methods for which vialed solutions may remain on an auto-samplers at ambient temperatures due to various delays
ACCEPTANCE CRITERIA
% Difference is not more than:
Assay, CU : 2.0
Dissolution : 3.0 (also stability at 37°C in disso bowl need to be performed – Accuracy including recovery of API to placebo in bowl for low dose)
Impurities : 10.0
The validity of an analytical procedure can be verified only by laboratory studies. Therefore, documentation of the successful completion of such studies is a basic requirement for determining whether a procedure is suitable for its intended application
Every DMF / ANDA / COS data package submitting for US FDA and European community etc., should consist method validation data. For easy to review, method validation report is usually attached to package
new instruments with different characteristics,
new location with different environmental conditions,
new chemicals and/or reference standards and