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Analytical Method Validation - HPLC (ICH Q2 and B parameters (PALL-SLayeR…
Analytical Method Validation - HPLC
Regulatory
21 CFR 211.194
The suitability of all testing methods used shall be verified under actual conditions of use.”
Guidelines
FDA 1987 - Method Validation Guidance
ICH Q2A and B Analytical Method Validation (1995)
USP, EP and BP Pharmacopeia
ICH Q2 and B parameters (PALL-SLayeR-Robot -System
Precision
extend of agreement between individual test results
Intermediate repeatability
2 or more operators, 2 HPLC, multiple days
minimum 3 concentrations ranges
RSD of ≤ 2%
Reproducibility
precision obtained between different laboratorities
Repeatability
1 operator, 1 piece of equipment, short interval
Minimum of 9 terminations over a concentration (3 conc, 3 reps)
Minimum of 6 determinations on 100% of test concentration
FDA requirements - 1% RSD for drug substance and product, 2% for bulk or finished products
Minor components can be RSD 5%, may reach 10% RSD at LOQ
Accuracy
degree of agreement between the result obtained from the method to that of the true value, or a conventional true value
Drug substance - use a reference standard of known purity
Drug product - use of synthetic mixture containing components of the drug products in known quantities
Impurities - use method on samples of impurities of known concentration
9 determinations - 3 concentrations over 3 replicates over a specified range
reported as percentage recovery - regulated product acceptance criteria 100% ± 2%
LOD
lowest amount of analyte which can be detected but not necessarily quantified as an exact value
Visual evaluation - analysis of samples with known concentration. Establish minimum level at which it can be detected.
Signal-to-Noise - comparing signals from low concentration of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected.
LOQ
lowest amount of analyte which can be quantitatively determined with accuracy, precision and reliability
A. Visual evaluation - minimum level of analyte that can be quantified with acceptable accuracy and precision
B. Signal-to-noise - comparing signals from samples with known low concentrations with those of blank samples to establish which can be accurately and precisely quantified
Specificity
The ability to detect the analyte of interest in the presence of interfering substances
injecting extracted placebo to demonstrate the absence of interference with the elution of analyte
Linearity
ability of the analytical method to obtain result directly proportional to the concentration
visual inspection of a plot of signals as a function of concentration. 6 concentrations and 3 replicates.
correlation coefficient ≥ 0.999 for the range of 80-120%
y-intercept must ≤ 2%
Range
upper and lower concentration which can be precisely and accurately detected.
assessed with the obtained linearity and accuracy data
assess the precision of the three replicate samples analysed at each level in the accuracy studies
must yield precision of ≤ 3% RSD.
System Suitability
verifies that the system is suitable for its intended purpose
Test and results are based on the relevant pharmacopeia.
Robustness
measure of the capacity to remain unaffected by small (deliberate) variations in the method parameters
influence of pH in the mobile phase, mobile phase composition, columns, temperature and flow rate
methanol content in mobile phase adjusted by ±2%, mobile phase pH adjusted by ± 0.1 pH units, column temperature adjusted by ± 5˚C. Changes must be within limits
Tested using Homogenous and authentic samples
Signal of 3 or 2:1 considered acceptable
