The document discusses the development, optimization, and validation of HPLC methods. It begins by outlining reasons why new HPLC methods may need to be developed, such as when existing methods are not suitable for a new drug or formulation. The document then describes the general steps in HPLC method development, including defining separation goals based on the sample properties, choosing sample pretreatment and detection methods, optimizing the separation conditions, and checking for any problems. Key parameters that require optimization are also outlined, such as the stationary and mobile phases, column, and detector. The document concludes by discussing the process of validating the method, including evaluation of accuracy, precision, linearity, range, specificity, limits of detection and quantification, robustness

1. ANALYTICAL METHOD DEVELOPMENT
OPTIMISATION AND VALIDATION OF
HPLC
Sudheer kumar kamarapu
Assistant professor
Sri Shivani college of pharmacy

2. INTRODUCTION
2

3. • The drug or drug combination may not be official in any pharmacopoeias.
• A proper analytical procedure for the drug may not be available in the
literature due to the patent regulations.
• Analytical procedures may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients.
• Analytical methods for the quantisation of the drug in the biological fluids
may not be available.
• Analytical methods for a drug in combination with other drugs may not be
available.
• The existing analytical procedures may require expensive reagents and
solvents.
• The existing analytical procedures involve cumbersome extraction and
separation procedures and these may not be reliable.
3

4. • As simple as possible.
• Most specific.
• Most productive economical and convenient.
• As accurate precise as required.
• Multiple source of key components(reagents,columns,TLC
plates) should be avoided.
• To be fully optimized before transfer for validation of
characteristics such as accuracy,precision,sensitivity,
ruggedness etc.
4

5. STEPS IN HPLC METHOD
DEVELOPMENT
5

6. Information on sample,
define separation goals.
Need for special HPLC procedure, sample
pretreatment..etc..
Choose detector
Choose LC method ; preliminary run;
estimate best separation conditions
Optimize separation
conditions.
6

7. Check for problems or
requirement for special
procedure
Validate method for
release to routine
laboratory.
7

8. Important information concerning
sample composition and properties:
 Number of compounds present.
 Chemical structures (functionality) of compounds.
 Molecular weights of compounds.
 Pka values of compounds.
 UV spectra of compounds.
 Concentration range of compounds in samples of interest.
 Sample solubility.
8

9. SEPARATION GOALS
 Is the primary goal quantitative analysis ,the detection of an substance, the
characterization of unknown sample components or the isolation of purified
material ?
 Is it necessary to resolve all sample components?
 If quantitative analysis is requested , what levels of accuracy and precision
are required
 For how many different sample matrices should the method be designed
 How many samples will be analyzed at one time
 What HPLC equipment and operator skills are present in the laboratory that
will use the final method ?
9

10. SAMPLE PRE TREATMENT
AND DETECTION
Samples come in various forms:
• Solutions ready for injection.
• Solutions that require dilution, buffering, addition of an
internal standard, or other volumetric manipulation.
• Solids that must first be dissolved or extracted.
• Samples that require sample pretreatment to remove
interferences and/or protect the column or equipment
from damage.
10

11. SAMPLE PRETREATMENT
• Removal of insoluble material
Filteration
Centrifuge
• Control of concentration
Dilution
• Extraction
Liquid phase extraction
Solid phase extraction
• Derivatization for detection
11

12. Selection of detectors
• UV-VIS Ultraviolet/ visible detector
• PDA Photodiode Array Detector
• RF Fluorescence detector
• CDD Conductivity detector
• RID Refractive index detector
• ECD Electrochemical detector
• ELSD Evaporative light scattering detector
• MS Mass spectrometer detector
12

13. DEVELOPING THE SEPARATION
Ref:Practical HPLC method development 13
2nd edition by Lioyd R.Snyder Chapter 1

14. Improving The Separation
Ref:Practical HPLC method development 14
2nd edition by Lioyd R.Snyder Chapter 1

15. Checking for problems
Problem Comment
Low plate numbers Poor choice of column
Column variability Poor choice of column
Short column life Poor choice of column, need for
sample pretreatment
Retention shift Insufficient column equilibrium, need
for sample pretreatment, loss of
bonded phase
Poor quantitative precision Need for better calibration,
identification of sources of error
New interference peaks discovered Initial inadequate or initial samples
not representative.
Ref:Practical HPLC method development 15
2nd edition by Lioyd R.Snyder Chapter 1

16. METHOD OPTIMIZATION
Optimization of a separation is principally directed by the following
goals:
• To separate better (higher resolution),
• To separate faster (shorter retention time),
• To see more (lower detection limit),
• To separate at lower cost (economic effort),
• To separate more (higher throughput).
Optimization of a method can follow either of two general
approaches:
• Manual .
• Computer driven.
16

17. The various parameters that include to be optimized
during method development are :
• Mode of separation .
• Selection of stationary phase .
• Selection of mobile phase.
• Selection of detector .
Mode of separation:
For the separation of polar or moderately polar
compounds, the most preferred mode is reverse
phase
17

18. Selection of stationary phase/column:
The appropriate choice of separation column includes different
approaches:
• The particle size and the nature of the column packing.
• The physical parameters of the column i.e. the length and
the diameter.
Selection of mobile phase :
The following are the parameters, which shall be taken into
consideration while selecting and optimizing the mobile
phase:
• Buffer and its strength: The retention times are depend on
the molar strengths of the buffer – Molar strength is
increasingly proportional to retention times.
18

19. • pH : It is important to maintain the pH of the mobile
phase in the range of 2.0 to 8.0 as most columns
does not withstand to the pH which are outside this
range.
• Mobile phase composition:
• Experiments were conducted with mobile phases
having buffers with different pH and different
organic phases to check for the best separations
between the impurities.
• A mobile phase which gives separation of all the
impurities and degradants from each other and from
analyte peak should be preferred
19

20. Selection of detector:
The characteristics that are to be fulfilled by a detector to
be used in HPLC determination are:
• High sensitivity, facilitating trace analysis
• Negligible baseline noise. To facilitate lower detection
• Large linear dynamic range
• Non destructive to sample
• Inexpensive to purchase and operate
20

21. VALIDATION OF HPLC METHOD
21

22. VALIDATION, USP:
“Validation of an analytical procedure is the process by which
it is established, by laboratory studies, that the performance
characteristics of the procedure meet the requirements for
the intended analytical applications.”
22

23. Basic Parameters for the Validation
of Method:
Specificity
Linearity
Accuracy
Method
validation Limit of detection
Limit of quantification
Precision
Range
Robustness
23
System suitability

24. Validation Characteristics
Identificatio Impurities Assay
n quantita
tive limit
Accuracy - + - +
Precision - + - +
Specificity + + + +
Detection Limit - - + -
Quantitation - -
Limit + -
Linearity - + - +
Range - + - +
Robustness + + + +
Ref:Quality assurance of pharmaceuticals(A compendium of guidelines 24
and related materials),volume 1 ,Chapter 4
World Health Organization Geneva

25. Accuracy:
• Definition: The accuracy of an analytical procedure expresses the
closeness of agreement between the value that is accepted either
as a conventional true value or as an accepted reference value and
the value found.
According to the ICH, accuracy should be determined using a minimum of
nine determinations over a minimum of three concentration levels
covering the range .
Obtained value - Expected value
%Error = ----------------------------------------- * 100
Expected value
%Error
<1% Highly accurate
1 to 5% Moderately accurate
>5% Low accurate
25

26. Precision :
Definition :
The Precision is a measure of the ability of the method to
generate reproducible results. The precision of a method is
evaluated for repeatability, intermediate precision and
reproducibility.
26

27. Limit of Detection (LOD, DL):
The LOD of an analytical procedure is the lowest amount of
analyte in sample which can be detected but not necessarily
quantitated as an exact value. Determination is usually based on
– Signal to noise ratio (~3:1) (baseline noise)
or
– Standard deviation of response (s) and Slope (S)
3.3 s/S
SNR = H/h
Where,
H = height of the peak corresponding to the component.
h = absolute value of the largest noise fluctuation from
the baseline of the chromatogram of a blank solution .
27

28. 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 (s) and Slope (S)
10 s/S
- The Quantitation limit of a method is affected by both the detector
sensitivity and the accuracy of sample preparation.
28

29. LOD, LOQ and Signal to Noise Ratio (SNR)
LOQ
Signal to Noise = 10:1
Signal to Noise = 3:1
LOD
Noise
29

30. Range:
ICH Definition:
The range of an analytical procedure is the interval
between the upper and lower concentrations of
analytes in the for which it has been demonstrated
that the analytical procedure has a suitable level of
precision, accuracy, and linearity.
30

31. Range For Different Tests:
• Assay
80 to 120% of test concentration.
• Content uniformity
70 to 130% of test concentration
• Dissolution
Q-20% to 120%
• Impurities
Reporting level – 120% of specification limit (with respect
to test concentration of API)
• Assay & Impurities
Reporting level to 120% of assay specification
31

32. Linearity:
Definition : Linearity of an analytical procedure is its ability
(within a given range) to obtain test results which are
directly proportional to the concentration of analytes in the
sample.
•If there is a linear relationship test results should be
evaluated by appropriate statistical methods like,
•Correlation coefficient
•Y-intercept
•Slope of regression line
•Plot of the Data
32

33. Linearity Ranges and Acceptance Criteria for
Various Pharmaceutical Methods
33
Ref:Modern HPLC by Michel Dong.

34. SYSTEM SUITABILITY TESTING (SST)
System suitability testing (SST) is used to verify resolution,
column efficiency, and repeatability of the analysis system
to ensure its adequacy for performing the intended
application on a daily basis.
Which Parameters??
•Number of theoretical plates (efficiency)
•Capacity factor,
•Separation (relative retention)
•Resolution,
•Tailing factor
•Relative Standard Deviation (Precision) 34 34

35. •Plate number or number of theoretical plates (n)
n=L/H, where L is Length of Column
H is HETP or height of one theoretical plate
•Capacity factor (capacity ratio) k
k= (tr-tm) /tm where tr is retention time
tm is dead time
•Separation Factor (relative retention)
α=k1/k2
where k1 is capacity factor of compound a
and k2 is capacity factor of compound b
•Tailing factor ,T
T=W/2f
where W is width at 5% at peak height,
f is distance between max and leading edge of
the peak
35

36. Ref:Modern HPLC by Michel Dong, 36

37. Robustness :
Definition :
Robustness is reliability of an analytical procedure with
respect to deliberate variations in method parameters.
•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.
37

38. PARAMETERS TO BE EVALUATED FOR ROBUSTNESS
•Mobile Phase
• pH (±0.1–0.2 units)
• Buffer concentration (±5–10mM)
• Percentage organic modifier (±1–2% MP)
•Sample
•Injection volume
• sample concentration
•Column temperature (±5°C)
•Detector wavelength (±3nm)
38

39. Selectivity and Specificity :
•Selectivity is the ability to measure accurately and specifically the
analyte in the presence of components that may be expected to
be present in the sample matrix.
•Specificity for an assay ensures that the signal measured comes
from the substance of interest, and that there is no interference
from excipients and/or degradation products and/or impurities.
39

40. REFERENCES
1. Modern HPLC by Michel Dong, Chapter 9.
2. Analytical Method Validation and Instrument Performance
Verification by Herman Lam and Y.C. Lec, Chapter 3 and
Chapter 11.
3. Practical HPLC method development 2nd edition by Lioyd
R.Snyder Chapter 1
4. Quality assurance of pharmaceuticals(A compendium of
guidelines and related materials),volume 1 ,Chapter 4
World Health Organization Geneva
40