presentation related to method development, trouble shooting and validation according to ICH guidelines

1. R DEEPTHI
Assistant Professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. HPLC/HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
HPLC Normal phase chromatography
Reverse phase chromatography
Normal chromatography Reverse phase chromatography
Stationary phase: polar (ex silica) Non-polar (ex: C8, C18, C4)
Mobile phase : non-polar (ex n-hexane) Polar (ex: water, methanol)

3. Normal phase: non-polar component comes 1st
Reverse phase: polar components comes 1st
Column
 Brand name
 Polarity
 Column length × diameter
 Stationary phase particle size
Brand names: Thermo hypersil, Iorbax, Ace, xterra,
inertsil, phenomenex, agilent
Polarity: silica cyano, phenyl, C4, C8, C18
polar Mid polar Non-polar

4. Column length:
50
100
150
200
250**
300
Column diameter: 4.6mm (Fixed)
Stationary phase particle size:
5µ( generally)
3.5µ
2.9µ
Ex: Agilent, c8, 150×4.6mm, 3.5µ

5. System suitability: The purpose of the system suitability is
to ensure that the Instrument, Method and Analyst is
suitable for the intended purpose.
Retention time
Resolution
Tailing
Theoretical plates
Relative standard deviation
Similarity factor
Bracketing standard

6. Retention time : The time taken by the component to
pass through the column is called Rt.
Resolution The distance between two peaks
Tailing The gap between the midpoint of the
peak to tail of the peak is called tailing
Theoretical plates Efficiency of method
Relative standard
deviation(%RSD)
It is used to determine “Repeatability
of the result”
Similarity Factor To check analyst repeatability
Bracketing standard To check whether system is suitable
NLT2
NMT2
NLT
2500
NMT
2%
NMT2%

7. Analytical Research & Development
Method Development Validation Transfer
Method development
1. Mobile phase
2. Column
3. Injection volume
4. Column temperature
5. Flow rate
6. Standard preparation
7. Sample preparation
8. Diluent
9. Wavelength
10. Run time

8.  Mobile phase: Buffer + solvent(Methanol/Acetonitrile)
If component is acidic Basic buffer(pH 7-14)
If component is Basic Acidic buffer(pH 0-7)
Acidic buffers
 Orthophosphoric acid
 Potassium dihydrogen phosphate
 Sodium dihydrogen phosphate
 Ammonium acetate
Basic Buffers
Dipotassium hydrogen phosphate
Disodium hydrogen phosphate

9. 2. Column: Choosing of the column depends upon the
polarity of the components.
 Component (strong Polar)- choose strong non-polar
column (C8, C18)
 Component (weak polar)-choose weak non-polar
column (CN,C4)
3. Injection Volume: 10µL
4. Column Temperature: 250C
5. Flow rate: 1mL/min
6. Diluent: Solubility of the Components
7. Run time: Depends on the Retention time
8. Standard preparation: 1000µg/mL

10. 9. Sample Preparation:
Wt to be taken = Std wt. × Avg wt
Label claim
10. Wavelength:
Detector: UV -1st scan in UV and Find λmax
Detector : PDA –No need to scan in UV

11. Trouble shooting
1. There is no peak?
Check the solubility in Diluent
Check the Mobile phase
Check the Detector wavelength
Check the column Polarity
2. High Retention Time?
Increase the solvent ratio, reduce buffer ratio
Increase Flow rate
Decrease column length
Increase the column temperature

12. 3. Low Retention time with low Theoretical Plates?
Increase Buffer ratio
Increase column length
Decrease Flow rate
Decrease column Temperature
4. High Tailing?
Injection volume must be reduced
Change the diluent
Wash the column
Change the column (new column)

13. 5. Low resolution?
Decrease the Particle size
Increase the Buffer ratio
Increase the column length
Gradient Elution
6. Peak splitting?
Column washing
Take new column

14. ICH
An agreement between
-European union
-Japan
-United states
ICH was created in the year 1990, April in Brussels.

15. ICH
Safety Efficacy Quality Multidisciplinary
Quality has been divided into Q12
Q1: Stability
Q2: Analytical Method Validation
Q3: Impurities
Q4: Pharmacopoeias
Q5: Biotechnological quality
Q6: Specifications
Q7: GMP
Q8: Pharmaceutical Development
Q9: Quality Risk Management
Q10: Pharmaceutical Risk Management
Q11: Development and Manufacture of Drug substance
Q12: Technical and Regulatory considerations for Pharmaceutical drug
product life cycle Management

16. Q2 Analytical Method Validation
Validation:
Parameters:
1. System suitability
2. Accuracy
3. Precision
4. Specificity
5. Robustness
6. Ruggedness
7. LOD
8. LOQ
9. Linearity
10. Range

17. 1. System suitability: The purpose of system suitability is
to ensure that the instrument, method , analyst are
suitable for the intended application.
Acceptance criteria: All the system suitability parameters
must be within the limits.
2. Accuracy/Recovery: The closeness of agreement
between the true value and found value.
The method is said to be accurate if individual recovery
is between 97-103%.
3. Precision/Repeatability: Closeness of agreement
between a series of measurements obtained from
multiple sampling of the same homogeneous sample.
The %RSD must be NMT 2%.

18. 4. Specificity: is the ability to assess unequivocally the
analyte in the presence of components which may be
expected to be present
Blank
Placebo
Standard
Sample
There should be no peak of Blank, Placebo, Impurity
chromatograms at Analyte retention time.

19. 5. 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 its reliability during normal usage.
Mobile phase composition Variation
pH in mobile phase variation
Flow rate variation
Column temperature variation
Filter validation
All the system suitability parameters should be within
the limits.

20. 6. Ruggedness:
System to system variation
Column to column variation
Analyst to analyst variation
Bench top stability of Std. and Sample solution
Refrigerator stability of Std. and Sample solution
Bench top stability of Mobile phase
7. Limit of Detection (LOD): Minimum concentration of
the standard compound in which the peak of the
standard get merged with noise.
8. Limit of Quantification (LOQ): Minimum
concentration of the standard compound in which the
peak of the standard get detected and quantified.

21. 9. Linearity: To demonstrate the linearity range of 50%-
150% of target concentration.
6 points: Plot the graph b/n Conc. Vs Peak Area
R2 = NLT 0.999
10. Range: Range of concentration in which the method
is linear, precise & accurate.
Method Transfer QC