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Method and Validation basics
HPLC case study
Hua YIN(Assessor)
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The prequalification programme --Assessor's training | 19-20 January 20112|
Outline
HPLC methodology
- Content of HPLC test procedure
- System Suitability Testing (SST)
- Relative Response Factor (RRF)
Validation of HPLC method
case study
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Information Sources
FDA CDER reviewer guideline for validation ofchromatographic methods (1994)
WHO TRS 937 Appendix 4 Analytical Method Validation
2006
ICH Q2(R1) 2005
Compendial General Chapters
Methods and Validation presentationLynda Paleshnuik
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High Performance Liquid Chromatography (HPLC)
HPLC is a separation technique based on a solid stationary phase and
a liquid mobile phase. Separations are achieved by partition,
adsorption, or ion-exchange processes, depending upon the type of
stationary phase used.
Chiral
Ion--exchange
Ion--pair/affinity
Normal phase
Reversed phase
Size exclusion
The reversed-phase HPLC with UV detectionis most commonly usedform of HPLC, is selected to illustrate the parameters of HPLC
method and validation.
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A f low scheme for HPLC
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Content of HPLC test procedure
Any analytical procedure submitted should be described in
sufficient detail, includes:
Preparation of mobile phase
Chromatographic condition:
Column: type (e.g., C18 or C8), dimension (length, innerdiameter), particle size (10m, 5 m)
Detector: wavelength
Injection volume
column T
flow rate,
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Content of HPLC test procedure
Elution procedure: isocratic or gradient elution
Preparation of standards and samples
Operation procedure: sequence of injections
System suitability testing (SST) and criteria
Calculations
QOS 2.3.R.2analytical procedures and validation summaries
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Compendial methods
When claim a compendial method, there should be no change in:
The type of column i.e the stationary phases
Detector wavelength
Components in Mobile phase
System suitability testing and criteria
Adjustments to ratio of components in mobile phase, flow rate, columntemp, dimension of column, particle size (reduction only), may be
necessary to achieve the system suitability criteria. The allowablevariations for each parameter, see Int.Ph 1.14.4 or USP generalchapter .
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System suitability testing (SST)
Precision: Assay: RSD 1% (API) or 2% (FPP), n 5
Impurities: in general, RSD 5% at the limit level, up to 10% or
higher at LOQ, n 6
Resolution (R): >2
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System suitability testing (SST)
Tailing factor/peak asymmetry: ( 2)
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The prequalification programme --Assessor's training | 19-20 January 201111|
System suitability testing (SST)
Number of theoretical plates (N): column efficiency 2000
Gradient elution is one way to increase the N
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System suitability testing (SST)
A SST should contain:
For Assay:
precision + one or more other parameter
For impurity test:
resolution + precision+ one or more other parameter
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Relative Response Factor (RRF)
Quantitation of Impurities
Against impurity RSs: when reference standard available
Against API itself
Relative response factor should be considered
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Relative Response Factor (RRF)
Response factor: the response (e.g. peak area) of drugsubstance or related substances per unit weight.
RF= peak area / concentration (mg/ml)
Relative response factor (RRF):
RRF=RFimpurity/ RFAPI,OR,
RRF=slopeimpurity/ slopeAPI
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Relative Response Factor (RRF)
Rifampicine:
y =31.312x + 4.963
Rifampicine Quinone:
y = 26.198x + 1.154
RRF= 26.198/ 31.312
=0.84
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Relative Response Factor (RRF)
To review:
a) RRF calculation, and
b) if RRF is properly used in the final calculation for %
impurity
If RRF within 0.8-1.2, correction may not be necessay
Correction factor= 1/RRF, the reciprocal of the RRF
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Review points for HPLC method
is the analytical proceduredescribed in detail including all the parameters ? is SSTwell defined to ensure the consistency of system performance?
The preparation of solutions:
assay: concentration of reference standard should be close to thesample solution
impurities: concentration of the reference standards should be close tothe limit
The way of quantitation of impurities
In case API is used as the reference, RRF should be used or justification ofexclusion should be provided.
To check the determination of RRF, check the correction of calculation ofimpurities
confirm/complete the QOS 2.3.R.2
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Validationcompendial methods
AssayAPI
No validation generally required. Exception: specificity for majorimpurities not in the monograph.
AssayFPP
Specificity, accuracy and precision (repeatability).PurityAPI and FPP
Full validation for specified impurities that are not included in themonograph (specificity, linearity, accuracy, repeatability,intermediate precision, LOD/LOQ)
Validation of the limit for individual unknowns, if tighter than that inthe monograph: LOQ of the API should be below the limit forindividual unknowns
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Non-compendial methods
Full validation is required for purity, assay and dissolution methods(HPLC, UV) :
Specificity
Linearity
Accuracy
Repeatability
Intermediate precision
LOD/LOQ (not required for assay, dissolution)
Robustness (recommended)
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Specificity
Blank solution to show no interference
Placebo to demonstrate the lack of interference from excipients
Spiked samples to show that all known related substances are
resolved from each other
Stressed sample of about 10 to 20% degradation is used to
demonstrate the resolution among degradation products
Check peak purity of drug substance by photodiode array detector (PDA): eg
purity angle is lower than the purity threshold.
Representative chromatograms should be provided with time scale
and attenuation indicated
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Linearity / Range
The working sample concentration and samples tested foraccuracy should be in the linear range (concentrations Vs.
Peak areas)
Minimum 5 concentrations
Dilute of stock solution or separate weighings
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Linearity / Range
Assay : 80-120% of the theoretical content of active
Content Uniformity: 70-130%
Dissolution: 20% of limits; eg if limits cover from
20% to 90% l.c. (controlled release), linearity shouldcover 0-110% of l.c.
Impurities: reporting level to 120% of shelf life limit
Assay/Purity by a single method: reporting level ofthe impurities to 120% of assay limit
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Linearity / Range
Correlation coefficient (r)
API: 0.998
Impurities: 0.99
y-Intercept and slope should be indicated together with
plot of the data
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Accuracy
Assay
API: against an RS of known purity, or via an alternate method ofknown accuracy; analysis in triplicate.
FPP: samples/placeboes spiked with API, across the range of 80-120% of the target concentration, 3 concentrations, in triplicateeach.
Report per cent recovery (mean result and RSD): 1002%
ICH Q2 states: accuracy may be inferred once precision, linearity
and specificity have been established. (Demonstration preferred).
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Accuracy
Impurities: API/FPP spiked with known impurities
Experienced in PQ:
Across the range ofLOQ-150%of the target concentration
(shelf life limit), 3-5 concentrations, in triplicate each.(LOQ, 50%, 100%, 150%)
Per cent recovery: in general, within 80-120%,depends
on the level of limit
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Precision
System precision: by multiple injections (n 5) of a homogeneous sample
(standard solution).
RSD 1% is recommendedfor assay;
RSD 5% is recommendedfor related substances (referencestandards at the limit)
Indicates the performance of the HPLC system
As a system suitability test
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Precision
Repeatability (method precision) Multiple measurements of a sample by the same analyst
A minimum of 6 determinations at the test concentration (6
times of a single batch), or
3 levels (80%, 100%, 120%) , 3 repetitions each (combined
with accuracy)
For Assay: RSD 2.0%
For individual impurity above 0.05%, in general, RSD 10%
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Precision
Intermediate precision (part of ruggedness) Test a sample on multiple days, analysts, equipments
Repeat the method precision by different analyst in
different equipment using different lot of column on
different days
RSD should be the same requirement as method precision
Reproducibility (inter-laboratory trial)
Not requested in the submission
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LOD/LOQ
signal to noise ratio: LOD 3:1 , LOQ 10:1 May vary with lamp aging, model/manufacturer of detector, column
standard deviation of the response and the slope of the calibrationcurve at levels approximating the LOD /LOQ
= the standard deviation of the response, base on the standard deviation of the blank
The calibration curve
should be validated by analysis of samples at the limits.
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LOD/LOQ
LOD: below the reporting threshold
LOQ: at or below the specified limit
Not required for assay/dissolution methods.
Applicant should provide
the method of determination
the limits,
chromotograms
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Robustness
The method's capability to remain unaffected by small butdeliberate variationsin method parameters
Influence of variations of pH in a mobile phase
Influence of variations in mobile phase composition
Different columns (different lots and/or suppliers)
Temperature
Flow rate
Evaluate the System suitability parameters
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Robustness
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Conclusion
HPLC methods play a critical role in analysis ofpharmaceutical product
Validation of HPLC should demonstrate that the method
is suitable for its intended use
Review the information in dossier against QOS 2.3.R.2
Data for acceptance, release, stability will only be
trustworthy if the methods used are reliable
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Case Study
Case Study 1--HPLC Method.doc
QOS 2.3.R.doc
Case Study 2 -- Validation of HPLC Method.doc
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