This presentation was made to solely for students to make them aware/ understand basics of “Analytical Method Validation”. These slides are part of lectures delivered in M. Pharmacy Curriculum & taken up from various books and websites

1. Analytical Method Validation
Dr. ARTI R. THAKKAR
This presentation was made to solely for students to make them aware/ understand basics of
“Validation”. These slides are part of lectures delivered in M. Pharmacy Curriculum & taken up from
various books and websites

2. Analytical Method Development & Validation

3. Analytical Method Development & Validation

4. Analytical Method Development & Validation

5. Analytical Method Development & Validation

6. Method Validation
Thus, Method validation is the process of
demonstrating that analytical procedures are
suitable for their intended use and that they
support the identity, strength, quality, purity and
potency of the drug substances and drug
products.

7. Method Validation
Published Guidances
– ICH-Q2A “Text on Validation of Analytical Procedure:(1994)
– ICH-Q2B “Validation on Analytical Procedures: Methodology:
(1995)
– CDER “Reviewer Guidance: Validation of Chromatographic
Method” (1994)
– CDER “Submitting Samples and Analytical Data for Method
Validations” (1987)
– CDER Draft “Analytical Procedures and Method Validation”
(2000)
– CDER “Bioanalytical Method Validation for Human Studies”
(1999)
– USP<1225> “Validation of Compendial Methods” (current
revision)

8. Considerations Prior to Method Validation
• Suitability of Instrument
– Status of Qualification and Calibration
• Suitability of Materials
– Status of Reference Standards, Reagents, Placebo Lots
• Suitability of Analyst
– Status of Training and Qualification Records
• Suitability of Documentation
– Written analytical procedure and proper approved
protocol with pre-established acceptance criteria

9. Examples of Methods That Require
Validation Documentation
• Chromatographic Methods – HPLC, GC, TLC, GC/MS, etc.
– Pharmaceutical Analysis – In support of CMC.
– Bioanalytical Analysis – In support of PK/PD/Clinical Studies.
• Spectrophotometric Methods – UV-VIS, IR, NIR, AA, NMR, XRD, MS,
etc.
• Capillary Electrophoresis Methods – Zone, Isoelectric Focusing,
Isotachophoresis, etc.
• Particle Sizer Analysis Methods – Laser, Microscopic, Photozone,
Sieving, SEC, etc.
• Dissolution Methods – Method of Analysis – HPLC, UV, Automated,
etc.
• Titration Methods.
• Automated Analytical Methods – Robots, Automated Analysis.

10. Specificity & Selectivity
Specific refers to a method that produces a response for a single
analyte only
selective refers to a method that provides responses for a number of
chemical entities that may or may not be distinguished from each
other. If the response is distinguished from all other responses, the
method is said to be selective.
Since there are very few methods that respond to only one analyte,
the term selectivity is usually more appropriate.

11. Specificity & Selectivity
Identity testing
– To ensure the identity of an analyte
Purity testing
– To ensure the content of impurities of an analyte
Assay
– To allow the content of an analyte in a sample

12. Specificity & Selectivity
• Specificity: Overlay chromatogram of an impurity solution with a
sample solution

13. Analytical Method Development
Specificity andSpecificity and stabilitystability
• Stress stability testing to ensure theStress stability testing to ensure the stability indicating potentialstability indicating potential of anof an
analytical methodanalytical method
– Apply diverse stress factors to the APIApply diverse stress factors to the API
– Apply diverse stress factors to the FPPApply diverse stress factors to the FPP
Assure that the API can be assessed specifically in the presence of knownAssure that the API can be assessed specifically in the presence of known
and unknown (generated by stress) impuritiesand unknown (generated by stress) impurities
Assure that known impurities/degradants can be specifically assessed inAssure that known impurities/degradants can be specifically assessed in
the presence of further degradantsthe presence of further degradants
ByBy peak purity assessmentpeak purity assessment and (overlay of)and (overlay of) chromatogramschromatograms

14. 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
– If there is a linear relationship test results should be
evaluated by appropriate statistical methods
• Correlation coefficient (r)
• Y-intercept
• Slope of regression line
• Residual sum of squares
• Plot of the data

15. Regression equation Y = aX + b
Where “a” is the slope of the line and “b” is the
intercept on the y-axis. When X = 0, “a” & “b” will be
according to equation 1.2.
( )∑ ∑
∑ ∑ ∑∑
−
−
= 22
2
xixiN
xiyixiyixi
b
( )∑ ∑
∑ ∑ ∑−
= 22
xixiN
yixixiyiN
a
( )[ ] ( )[ ]∑ ∑∑ ∑
∑ ∑ ∑
−−−
−
=
yiyiNxixiN
yixixiyi
r
22

16. Analytical Method Development
• Usual acceptance criteria for a linear calibration curve
– r > 0.999; y-intercept a < 0 to 5% of target concentration
RSD (wrt calibration curve) < 1.5-2%
r > 0.997 r < 0.997

18. Range
The range of an analytical procedure is the interval
between the upper and lower concentration
(amounts) of analyte in the sample for which it has
been demonstrated that the analytical procedure
has a suitable level of precision, accuracy and
linearity.

19. Accuracy
• The accuracy of an analytical
procedure expresses the closeness of
true value and the found value.

20. • AccuracyAccuracy
– 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
• Sometimes referred to as TRUENESS
truetruemeanmean

21. To find out whether a method is accurate:
• Drug substance (assay)
– Application of the method to an analyte of known purity (e.g. reference
substance)
– Comparison of the results of one method with those of a second well-
characterised method (accuracy known)
• Drug product (assay)
– Application of the method to synthetic mixtures of the drug product component
to which known quantities of the analyte have been added i.e. spiked
• Drug product may exceptionally be used as matrix
• Drug substance/Drug product (Impurities)
– Application of the method to samples spiked with known amounts of impurities

22. • Accuracy: Application of the method to synthetic mixtures of the drug
product components
to which known quantities
of the analyte
have been added
• Recovery reduced
by ~10 – 15%

23. • When to expect Accuracy problems
– Insufficient selectivity of the method
• Impurity peaks are not resolved and account for assay
value
– Recovery is < 100%
• Irreversible adsorption of analyte to surfaces of the
system
– Incorrect assay value of a reference standard
• Due to decomposition of reference standard
– Incorrect assay value due to change in matrix
• Analytical laboratory still uses the preceding matrix as
standard

24. ( ) ( )( )
( ) ( )
100covRe% 22
×
−
−
=
∑∑
∑∑∑
xxN
yxxyN
ery
100×





=
ionConcentratExpected
ionConcentratmeasuredMean
Accuracy

25. • Precision
– Expresses the closeness of agreement between a
series of measurements obtained from multiple
sampling of the same homogenous sample
– Is usually expressed as the standard deviation (S),
variance (S2
) or coefficient of variation (RSD) of a
series of measurements
– Precision may be considered at three levels
• Repeatability (intra-assay precision)
• Intermediate Precision (variability within a laboratory)
• Reproducibility (precision between laboratories)

26. Precision
• Repeatability expresses the precision under the
same operating conditions over a short interval of
time. Repeatability is also termed intra-assay
precision.
• Intermediate Precision expresses within-
laboratories variations: different days, different
analysts, different equipment, etc.
• Reproducibility expresses the precision between
laboratories (collaborative studies, usually applied
to standardization of methodology).

27. Repeatability
( )∑ −− 1/
2
NXXiS D =
100×
X
S
R S D =

29. Numberoftimeseachvalueoccurs
Valuesσ2σ3σ
σ 2σ 3σ
Normal distribution, probability function [P(x)] and confidence
interval [CI]

30. • Relationship of variability, probability and
reliability of data
– High variability of data (large σ) generate large confidence intervals and thus
lower the reliability of the mean
– Low variability of data (small σ) generate small confidence intervals and thus
increase the reliability of the mean

31. • Reproducibility
– Expresses the precision between laboratories
• Collaborative studies, usually applied to standardisation
of methodology
– Transfer of technology
– Compendial methods

32. Detection Limit
• 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.

33. • Limit of Detection (LOD, DL)Limit of Detection (LOD, DL)
• Determination is usually based on
– Signal to noise ratio (~3:1) (baseline noise)
or
– Standard deviation of response (σ) and Slope (S)
• 3.3 σ/S

34. • Limit of Quantitation (LOQ, QL)Limit of Quantitation (LOQ, QL)
– The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy
• The quantitation limit is used particularly for the
determination of impurities and/or degradation products
• Determination is usually based on
– Signal to noise ratio (~10:1) (baseline noise)
or
– Standard deviation of response (σ) and Slope (S)
• 10 σ/S

35. NoiseNoise
LODLOD
Signal toSignal to NoiseNoise = 3:= 3:11
LOQLOQ
Signal toSignal to NoiseNoise = 10:= 10:11
LOD, LOQ and Signal to Noise Ratio (SNR)LOD, LOQ and Signal to Noise Ratio (SNR)

36. • LOQ
– Quantitation by SNR is accepted
– Quantitation by Standard deviation of response (σ)
and Slope (S) (10 σ/S) is more adequate as it
involves the response of the actual analyte

37. • LOQ and impurities
– In determination of impurities in APIs the LOQ
should be determined in the presence of API
• LOQ should be NMT reporting level
• LOQ should be given relative to the test concentration of
API
– Specificity of impurity determination should always
be demonstrated in the presence of API at API
specification levels
• Spiking of test concentration (API) with impurities at
levels of their specification range

38. • Spiking
– API test concentration (normalised)
• 0.1 mg/ml (100%)
– Impurity spiking concentrations
• 0.001 mg/ml (1%) – specification limit
• 0.0001 mg/ml (0.1%) – limit of quantitation (minimum
requirement)
API at test concentrations
API below test concentrations

39. Impurities (Quantitation)
• Accuracy should be
assessed on samples
(substance / product)
spiked with known
amounts of
impurities.

40. 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.

41. • Robustness
– Robustness of an analytical procedure should show the
reliability of an analysis with respect to deliberate
variations in method parameters
– The evaluation of robustness should be considered during
the development phase
– 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

42. • Influence of buffer pH and buffer concentration in mobile phase on
retention times of API and impurities
• Conclusion: The buffer composition should be maintained in a range of 85
± 0.5%
– Missing: Acceptance criterion for maximal deviation of retention time should be
defined unless justified
API Impurity A Impurity B Impurity C
As is 10.46 3.86 7.43 8.26
buffer pH 5.9 10.45 3.94 7.51 8.38
buffer pH 6.9 10.46 3.94 7.49 8.34
Buffer conc. 83% 7.84 3.43 6.16 6.66
Buffer conc. 87% 15.26 4.77 9.61 11.18

43. System Suitability Testing
• System suitability testing is an integral part of
many 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.

44. • Method stability
– System suitability over time
• Sample solution stability
– A solution of stavudine is stable for ~ 2 h, then it starts to
degrade to thymine
• Impurity-spiked sample solution stability
 A solution containing stavudine spiked with its impurity thymine
does not allow to clearly distinguish between degradation and spike
Should be analysed immediately

45. • When to be „surprised“ about validation data:
– Precision of
impurity determination
– Precision of
API determination
– Method precision of
released API (dissolution)
System precision % RSD 0.33 – 2.25
Method precision % RSD 0.0
Average peak area % RSD 0.08
Acceptance
criterion
% RSD ≤ 2.0
Average peak area % RSD 0.4
Acceptance
criterion
% RSD ≤ 10.0

46. Thank you