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HPLC Method and Validation 
basics 
Regulatory guidelines— 
Shreekant Deshpande 
Senior Scientist 
Eutech Sci Ser Inc
Outline 
• HPLC methodology 
Content of HPLC test procedure 
System Suitability Testing (SST) 
Relative Response Factor (R...
Information Sources 
• FDA CDER reviewer guideline for validation of 
chromatographic methods (1994) 
• WHO TRS 937 Append...
High Performance Liquid Chromatography (HPLC) 
HPLC is a separation technique based on a solid stationary phase 
and a liq...
A flow scheme for HPLC
Content of HPLC test procedure 
Any analytical procedure submitted should be described in 
sufficient detail, includes: 
•...
Content of HPLC test procedure 
• Elution procedure: isocratic or gradient elution 
• Preparation of standards and samples...
Compendial methods 
When claim a compendial method, there should be no change in: 
• The type of column i.e the stationary...
System suitability testing (SST) 
• Precision: 
• Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5 
• Impurities: in general, RSD...
System suitability testing (SST) 
• Tailing factor/peak asymmetry: (≤ 2)
System suitability testing (SST) 
• Number of theoretical plates (N): column efficiency ≥ 2000 
• Gradient elution is one ...
System suitability testing (SST) 
A SST should contain: 
• For Assay: 
precision + one or more other parameter 
• For impu...
Relative Response Factor (RRF) 
Quantitation of Impurities/ derivatives 
• Where the loss of analyte is inevitable, Use IS...
Relative Response Factor (RRF) 
• Response factor: the response (e.g. peak area) of drug 
substance or related substances ...
Relative Response Factor (RRF) 
Rifampicine: 
y =31.312 x + 4.963 
Rifampicine Quinone: 
y = 26.198 x + 1.154 
RRF= 26.198...
Relative Response Factor (RRF) 
• To review: 
a) RRF calculation, and 
b) if RRF is properly used in the final calculation...
Review points for HPLC method 
• is the analytical procedure described in detail including all the parameters ? 
• is SST ...
Validation – compendial methods 
Assay – API 
No validation generally required. Exception: specificity for major 
impuriti...
Non-compendial methods 
Full validation is required for purity, assay and dissolution methods 
(HPLC, UV) : 
Specificity 
...
Specificity 
• Blank solution to show no interference 
• Placebo to demonstrate the lack of interference from excipients 
...
Linearity / Range 
• The working sample concentration and samples tested for 
accuracy should be in the linear range (conc...
Linearity / Range 
• Assay : 80-120% of the theoretical content of active 
• Content Uniformity: 70-130% 
• Dissolution: ±...
Linearity / Range 
Correlation coefficient (r) 
API: ≥ 0.998 
Impurities: ≥ 0.99 
y-Intercept and slope should be indicate...
Accuracy 
Assay 
API: against an RS of known purity, or via an alternate method of 
known accuracy; analysis in triplicate...
Accuracy 
Impurities: API/FPP spiked with known impurities 
Experienced in PQ: 
Across the range of LOQ to150% of the targ...
Precision 
• System precision: 
• by multiple injections (n ≥5) of a homogeneous sample 
(standard solution). 
• RSD ≤ 1% ...
Precision 
• Repeatability (method precision) 
• Multiple measurements of a sample by the same analyst 
• A minimum of 6 d...
Precision 
• Intermediate precision (part of ruggedness) 
• Test a sample on multiple days, analysts, equipments 
• Repeat...
LOD/LOQ 
• signal to noise ratio: LOD 3:1 , LOQ 10:1 
• May vary with lamp aging, model/manufacturer of detector, column 
...
LOD/LOQ 
• LOD: below the reporting threshold 
• LOQ: at or below the specified limit 
Not required for assay/dissolution ...
Robustness 
• The method's capability to remain unaffected by small but 
deliberate variations in method parameters 
• Inf...
Robustness 
:Parameters 
Change in column temperature ± 5°C 
Change in flow rate ± 0. 2ml /min 
Change in mobile phase Buf...
Conclusion 
• HPLC methods play a critical role in analysis of 
pharmaceutical product 
• Validation of HPLC should demons...
Thank you
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Method-Validation-HPLC-case-study

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Method-Validation-HPLC-case-study

  1. 1. HPLC Method and Validation basics Regulatory guidelines— Shreekant Deshpande Senior Scientist Eutech Sci Ser Inc
  2. 2. Outline • HPLC methodology Content of HPLC test procedure System Suitability Testing (SST) Relative Response Factor (RRF) • Validation of HPLC method • Case study
  3. 3. Information Sources • FDA CDER reviewer guideline for validation of chromatographic methods (1994) • WHO TRS 937 Appendix 4 “Analytical Method Validation 2006 • ICH Q2(R1) 2005 • Compendial General Chapters • Methods and Validation presentation–Lynda Paleshnuik • Methods and Validation basics–Hua Yin
  4. 4. 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 detection is most commonly used form of HPLC, is selected to illustrate the parameters of HPLC method and validation.
  5. 5. A flow scheme for HPLC
  6. 6. Content of HPLC test procedure Any analytical procedure submitted should be described in sufficient detail, includes: • Materials/Chemicals • Preparation of mobile phase • Chromatographic condition: • Column: type (e.g., C18 or C8), dimension (length, inner diameter), particle size (10μm, 5 μm) • Detector: wavelength • Injection volume • column Temp • flow rate,
  7. 7. 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.2 analytical procedures and validation summaries
  8. 8. 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, column temp, dimension of column, particle size (reduction only), may be necessary to achieve the system suitability criteria. The allowable variations for each parameter, see Int.Ph 1.14.4 or USP general chapter <621>.
  9. 9. 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
  10. 10. System suitability testing (SST) • Tailing factor/peak asymmetry: (≤ 2)
  11. 11. System suitability testing (SST) • Number of theoretical plates (N): column efficiency ≥ 2000 • Gradient elution is one way to increase the N
  12. 12. 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
  13. 13. Relative Response Factor (RRF) Quantitation of Impurities/ derivatives • Where the loss of analyte is inevitable, Use IS and RRF! • Against impurity RS’s: when reference standard available • Against API itself Relative response factor should be considered
  14. 14. Relative Response Factor (RRF) • Response factor: the response (e.g. peak area) of drug substance or related substances per unit weight. RF= peak area / concentration (mg/ml) • Relative response factor (RRF): RRF=RF impurity / RF API, OR, RRF=slope impurity / slope API
  15. 15. Relative Response Factor (RRF) Rifampicine: y =31.312 x + 4.963 Rifampicine Quinone: y = 26.198 x + 1.154 RRF= 26.198 / 31.312 =0.84
  16. 16. 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
  17. 17. Review points for HPLC method • is the analytical procedure described in detail including all the parameters ? • is SST well defined to ensure the consistency of system performance? • The preparation of solutions: • assay: concentration of reference standard should be close to the sample solution • Quantitation: Sample concentration should fall under standard curve • impurities: concentration of the reference standards should be close to the limit • The way of quantitation of impurities In case API is used as the reference, RRF should be used or justification of exclusion should be provided. To check the determination of RRF, check the correction of calculation of impurities • confirm/complete the QOS 2.3.R.2
  18. 18. Validation – compendial methods Assay – API No validation generally required. Exception: specificity for major impurities not in the monograph. Assay – FPP Specificity, accuracy and precision (repeatability). Purity – API and FPP Full validation for specified impurities that are not included in the monograph (specificity, linearity, accuracy, repeatability, intermediate precision, LOD/LOQ) Validation of the limit for individual unknowns, if tighter than that in the monograph: LOQ of the API should be below the limit for individual unknowns
  19. 19. 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)
  20. 20. 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
  21. 21. Linearity / Range • The working sample concentration and samples tested for accuracy should be in the linear range (concentrations Vs. Peak areas) • Minimum 5 concentrations • Dilute of stock solution or separate weighings
  22. 22. 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 should cover 0-110% of l.c (Label Claim). • Impurities: reporting level to 120% of shelf life limit • Assay/Purity by a single method: reporting level of the impurities to 120% of assay limit
  23. 23. Linearity / Range Correlation coefficient (r) API: ≥ 0.998 Impurities: ≥ 0.99 y-Intercept and slope should be indicated together with plot of the data
  24. 24. Accuracy Assay API: against an RS of known purity, or via an alternate method of known accuracy; analysis in triplicate. FPP: samples/placeboes spiked with API, across the range of 80- 120% of the target concentration, 3 concentrations, in triplicate each. Report per cent recovery (mean result and RSD): 100±2% ICH Q2 states: accuracy may be inferred once precision, linearity and specificity have been established.
  25. 25. Accuracy Impurities: API/FPP spiked with known impurities Experienced in PQ: Across the range of LOQ to150% 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
  26. 26. Precision • System precision: • by multiple injections (n ≥5) of a homogeneous sample (standard solution). • RSD ≤ 1% is recommended for assay; • RSD ≤ 5% is recommended for related substances (reference standards at the limit) • Indicates the performance of the HPLC system • As a system suitability test
  27. 27. 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%
  28. 28. 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
  29. 29. 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 calibration curve at levels approximating the LOD /LOQ σ = 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.
  30. 30. 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
  31. 31. Robustness • The method's capability to remain unaffected by small but deliberate variations in 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
  32. 32. Robustness :Parameters Change in column temperature ± 5°C Change in flow rate ± 0. 2ml /min Change in mobile phase Buffer pH ± 0. 2units %Change in organic composition ± 2.0 :Acceptance Criteria The system suitability parameters should pass for all the ,conditions All known and Unknown impurities shall be separated from .each other; in sample spiked with impurities
  33. 33. Conclusion • HPLC methods play a critical role in analysis of pharmaceutical 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
  34. 34. Thank you

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