analytical methods of validation validation of HPLC.....

1. SEMINAR
ON
Analytical Method Validation
& Validation of HPLC
• GUIDE: • Presented by:
MR. Ishaq Ahmed T.VENKATESH
(Asst. Proff) M. Pharmacy (pharmaceutics)
Sri Kakatiya Institute of
Pharmaceutical Science.

2. CONTENTS
• INTRODUCTION
• PARAMETERS FOR METHOD VALIDATION
-AS PER USP/BP
-AS PER ICH
• VALIDATION OF HPLC
-TYPICAL HPLC DESIGN
-VALIDATION PARAMETERS
• CONCLUSION
• REFERENCES

3. INTRODUCTION
 Validation
Establish a documented evidence which provides a high degree of
assurance that a specific process will consistently produce a product
meeting its predetermined specifications and quality attributes
 Analytical Validation
The principle purpose of analytical validation is to ensure that the
selected analytical procedure will give reproducible and reliable
results that are adequate for the intended purpose.

4. Why validation is necessary?
 It is an important element of quality control.
 Validation helps provide assurance that a measurement will be
reliable.
 In some fields, validation of methods is a regulatory requirement.

5. When is validation needed?
 Before introduction of a new method in to routine use.
 Whenever condition change for which method has been validation e.g.
instrument with different characteristics.
 Whenever the method is changed and the change is outside the scope
of the original method

6. When Revalidation To Be Done?
• Equipment changes
• Formula changed
• Changed suppliers of critical reagents

7. Parameters For Method Validation
Comparison BP/USP/ICH
As per USP and BP As per ICH

8. Accuracy
Accuracy
The accuracy is the closeness of the test results obtained by the
method to he true value. Accuracy should be established across its range.
Accuracy assessed using a minimum of 9 determinations over a
minimum of 3 concentration levels

9. True value
Accurate but
imprecise

10. Precision
 Precision : The precision of an analytical method is the
degree of agreement between a series of measurements
obtained from multiple sampling of the same homogeneous
sample.
Repeatability : Repeatability expresses the precision under
the same operating conditions over a short interval of time.
Repeatability is also termed intra-assay precision .
a minimum of 9 determinations covering the specified range
for the procedure ( e.g., 3 concentrations/3 replicates each);
or
a minimum of 6 determinations at 100% of the test
concentration.

11. Precision
Intermediate Precision:
Intermediate precision expresses within-laboratories variations,
different days, different analysts, different equipment, etc.
Reproducibility:
Reproducibility expresses the precision between laboratories
(collaborative studies, usually applied to standardization of
methodology).

12. Relationship between Accuracy
and Precision
Inaccurate &
imprecise
Inaccurate but Accurate but Accurate AND Precise
precise imprecise

13. Linearity
 The linearity of an analytical procedure is
its ability to obtain test results that are
directly proportional to the concentration of
the analyte in the sample.
 Linearity is usually demonstrated by the
analysis of various concentrations of the
analyte (s) across the indented range and
represented graphically.
A statistical analysis of the data is usually
required, such as the calculation of a
regression line using the method of least
square .
 A minimum of 5 concentration is
recommended.

14. Range
 Range of the analytical procedure is the interval between the upper
and the lower concentration of the analyte for which it has been
demonstrated that the analytical procedure has a suitable precision,
accuracy and linearity.
 For assay the range is usually not less than 80 to 120% of the test
concentration.
 For determination of content uniformity the range is usually not less
than 70 to 130% or the test concentration.
 For determination of impurities the range is usually not less than the
reporting limit of the impurity to 120% or the specification.
 For dissolution testing the range is usually ±20% over the expected
concentration.

15. Specificity/Selectivity
 The ability to assess unequivocally the analyte in
the presence of components that may be
expected to be present.
– Impurities
– degradants
– excipients
 Specificity must be demonstrated for:
– Identification
– Impurities Test
– Assay Test

16. Detection limit (limit of detection)
 Definition :
Limit of Detection is the smallest quantity of an analyte that can be
detected, but not necessarily quantified.
 Approaches to calculation :
• visual evaluation
• signal to noise ratio
• standard deviation of the response and the slope of the calibration
curve

17. Calculation
 1. visual evaluation- DL is determined by the analysis of a series of
samples with known concentrations and establishing the minimum level
at which the analyte can be reliably detected.
 2. signal to noise ratio- For instrumental procedures that exhibit
background noise, it is common to compare measured signals
from samples with known low concentrations of analyte with
those of the blank samples. The minimum concentration at which
the analyte can reliably be detected is established using an
acceptable signal - to - noise ratio of 2 : 1 or 3 : 1.
 3. standard deviation of the response and the slope of the calibration
curve
DL=3σ/S
where σ is the standard deviation of the response and S is the slope
of the calibration curve

18. Limit of Quantitation
• The quantitation limit is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable precision and
accuracy.
• Used particularly for the determination of impurities and/or
degradation products.

19. Limit of Quantitation
 Various approaches of determining the Quantitation
Limits are
- Based on visual evaluation
- Based on signal-to-noise
- Based On Standard Deviation Of Response And Slope
DL= 10 σ/S
σ = the standard deviation of the response
S = the slope of the calibration curve
LOQ vary with detector sensitivity.
Lamp aging, different manufacturer of detector.

20. Ruggedness
 Ruggedness: The ruggedness of an analytical method is the
degree of reproducibility of the test results obtained by the analysis
of the same samples under a variety of conditions, such as
 Day-to-day variations
 Analyst-to-analyst
 Laboratory-to-laboratory
 Instrument-to-instrument
 Chromatographic column-to-column
 Reagent kit-to-kit
 Instability of analytical reagents

21. 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 use.

22. VALIDATION OF HPLC
The goal of equipment validation is to produce constant result with
minimal variation with out compromising the product and performance of
equipment.

23. Qualification
 Qualification is a subset of the validation process that verifies module and system
performance prior to the instrument being placed on-line.

24. Design
Qualification
(DQ)
•For setting the
functional and
performance
specifications
•DQ can be very
simple for similar
equipment e.g. just
another HPLC system

25. Installation
Qualification
(IQ)
•for performing and
documenting the
installation in the selected
user environment
•safety, service
requirements

26. Operational
Qualification
(OQ)
•For testing the
equipment in the
selected user
environment to ensure it
meets our defined
functional and
performance
specifications

27. Performance
Qualification
(PQ)
•For testing that the system
consistently performs as intended
for the selected application
•Periodic calibration /
maintenance
•Must be signed back by user
•May use Method System
Suitability Checks as part of PQ

28. TYPICAL PARAMETERS USED IN
HPLC Method validation
 Accuracy
 Detection limit and quantitation limit
 Linearity
 Precision
Repeatability
Reproducibility
• Recovery
• Robustness
• Sample solution stability
• Specificity

29. System suitability
 The simplest form of an HPLC system suitability test involves a
comparison of chromatogram trace with a standard trace.
 This allows a comparison of the peak shape, peak width, baseline
resolution.
 Parameters to be calculated to provide a system suitability test
report.

30. System Suitability
 Number of theoretical plates (efficiency)
 Capacity factor
 Separation (relative retention)
 Resolution
 Tailing factor
 Relative standard deviation
 These are measured on a peak or peaks of known retention time
and peak width.

31. Retention Factor or capacity factor
•The capacity factor is a measure of how
long each component is retained on the
column.
• k is used in preference to retention time.
•Generally the value ok K’ is > 2.
•Inpractice the k value for the first peak of
interest should be >1 to assure that it is
separated from solvent.
•Hear Tr is retention time of peak of
interest & T0 is unretained peaks
retention time.

32. Relative Retention/ Separation
•This describes the relative position of two adjacent peaks. Ideally it is
calculated using the capacity factor
•Because the peak separation depends on the components interction with
the stationary phase.

33. Number of theoretical plate
•Thisis a measure of sharpness of the peaks and
therefore the efficiency of the column that is ,
how many peaks can be located per unit run
time of the chromatogram.
•This cam be calculated in various ways.
•ex: USP uses the peak width at base and BP at
half the height.
•The theoretical plate number depends on
elution time but in general should be > 2000

34. Tailing factor T
• This is a measure for
asymmetry of peak.
• Peak asymmetry is measured
at 5% of full peak height.

35. Peak Resolution R
• This is not only a measure of separation between two
peaks, but also the efficiency of the column.
• It is expressed as ratio of the distance between the two
peak maxima to mean value of peak width.

36. Precision
• Injection repeatability (i.e. 6)
• If RSD (relative standard
deviation) of ≤ 2% is required
then 5 replicate injections
should be used.
• Data from six injections are used
if the RSD is more than 2.0%

37. CONCLUSION
 This summarizes the validation parameters that are required according
to the requirement of ICH/USP&BP.
 Summarized the extent, need & necessary of validation.
 Validation may cost initially but it avoids the risk of breaking down, for
this reason even small industries are concentrating more on validation
their by fulfilling goal of GMP.
 Validation when done according to standard protocol and used it always
produce a product which meets its predetermined specifications and
quality.

38. References
 Article on Validation of Analytical Procedures: comparison
of ICH vs Pharmacopoeia by katrai Sahil.
 British Pharmacopoeia, 2007 (4) A523 and A159-163
 United states pharmacopoeia 30, 1920-1924 and 2149-2152.
 www.ich.org
 CDER guideline Nov 1994 validation of chromatographic
methods