Validation of analytical methods. Adrian Covaci Toxicological Center, University of Antwerp

Transcription

1 Validation of analytical methods Adrian Covaci Toxicological Center, University of Antwerp

2 Why validate? The objective of validating an analytical method is to demonstrate its suitability for its intended purpose. Need to ensure that quality of analyses is good and stays good = consistent, reliable and accurate data Need to ensure a way of standardization between methods Validation requested by most regulations and QC measures that impact laboratories

3 When validating? Analytical methods need to be validated, verified, or revalidated: Before initial use in routine testing When transferred to another laboratory Whenever the conditions or method parameters for which the method has been validated change (e.g., instrument with different characteristics or samples with a different matrix) and the change is outside the original scope of the method. Identification of problem Method Development Method Validation Quality Control Apply method to real samples Quality control and quality assurance more details in the presentation of Felix

4 Which guidelines to follow for validation? Various validation schemes Harmonized guidelines between International Conference on Harmonization (ICH) of technical requirements for registration of pharmaceuticals for human use (ICH Q2(R1), AOAC International (AOAC), and the IUPAC - for single laboratory method validation. US FDA - guidance document for analytical method validation for bioanalytical methods. European Medicines Agency (EMA, formerly EMEA) - Guideline on bioanalytical method validation.

5 Which guidelines to follow for validation? Eurachem guide The Fitness for Purpose of Analytical Methods and NordVal Guide in Validation of Alternative Proprietary Chemical Methods - not limited to a specific technique. More specialized guidelines, such as validation guidelines for pesticide residue analysis in food and feed by SANCO/12571/2013 Or European Commission Decision 2002/657/EC implementing the Council Directive 96/23/EC, - to ensure the quality and comparability of analytical results generated by official control laboratories. No (official) validation guidelines for wastewater analysis!

6 Which guidelines to follow for validation? Differences between the various guidelines: - In the requirements (nr days/replicates for a parameter) - In the established limits (no limits in ICH gudelines) Choose wisely according to your purpose!

7 Definitions Internal (surrogate) standard: - added in a constant amount to samples, as early as possible in the sample preparation - added also in the blank and calibration - used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte concentration. - is very similar, but not identical to the analyte(s). This results in similar effects during the sample preparation and analysis. Recovery standard: - Added in the final extract before analysis - Used to calculate recovery of IS in samples

8 Quantitation methods - external calibration - calibration with internal standard - standard addition - calibration graph in solvent or in the extract of an appropriate matrix the so-called matrix-matched calibration (the standard addition methods are intrinsically matrix-matched). For matrix-matched calibration, a blank matrix sufficiently similar to the sample matrix is needed

9 Quantitation methods Using an internal standard (IS) can help correcting for sample preparation and to a lesser extent also for ionization suppression. Two types of IS can be distinguished: - structural analogs of the analytes - isotopically labeled internal standards (ILIS). ILIS may either be isotopically labeled analyte or some other isotopically labeled compound

10 Which parameters to validate? Linearity LOD and LOQ Precision Accuracy Recovery Robustness Matrix effects Qualitative aspects

11 Linearity How many calibration points? How are they distributed over the range? Tests for linearity - Plot of residuals (absolute or relative) - Lack of fit (residual variance) - Zero testing - Include or exclude origin? - Weighting of the curve (non, 1/x, 1/x²) - Non-linear calibrations (e.g. quadratic or polynomial) - Range: LLOQ ULOQ Calibrators: in solvent or matrix? Matrix blank!! Not for influent/effluent WW OECD wastewater? Tap water? Milli Q water? Surface water?

12 Linearity

13 Linearity - What to do when measurement is outside the calibration range? - Re-analyze the sample by taking less sample and adding more IS - Dilution will not always help Sensitivity of the analysis slope. Calibration curves on different days (check differences in slopes) Slope should be similar between different calibration batches (e.g. Between weeks, months). For ILIS, a ~ 1 in y = ax+b Where y = Ax/A IS and x = Cx/C IS

14 LOD and LOQ LOD defined as the concentration which leads to S/N = 3 LOQ defined as the concentration which leads to S/N = 10 Instrumental LOQ (pg injected) a measure of the instrument performance, can be done with standards or extracts. Method LOQ (ng/l) a measure of the method performance, done with extracts. EMA/FDA require <20% bias and <20%imprecision at LLOQ More information will be given by Tito

15 Precision - Describes the agreement between repetitive individual measurements and is calculated as the coefficient of variation (CV) or RSD %. - Should be demonstrated for the LLOQ, low (3xLLOQ), medium (around 50% of the calibration curve) and high (75% of ULOQ) concentrations, within-day and between-day. - The imprecision should be <20% for the LLOQ en <15% for the other levels.

16 Precision Repeatability (intra-day precision) - Same operating procedures over a short period of time - Measurements on the same day (n=5 or 7) Intermediate precision (inter-day precision) - Measurements on different/consecutive days (n=5 of 7 per day) - D = 3 to 5 days Reproducibility (overall precision) - Precision between laboratories

17 Trueness/Accuracy - Describes the agreement between the measured value and the acccepted reference value and is expressed as percentual difference from this accepted value. - Should be measured on samples with a known concentration of analyte (QCs). - Should be measured for LLOQ, low, medium and high concentrations, within-run and between-run (similar to precision). - Should be <20% for the LLOQ en <15% for the other levels.

18 Accuracy - Measurements on the same day (n=5 or 7) - Measurements on different/consecutive days (n=5 of 7 per day) D = 3 to 5 days Spiked samples = QC samples? SRMs, CRMs not always available. Can also be QC samples during analysis of real samples. Associated with the uncertainty of the method

19 Recovery - Calculated as the percentage of analyte signal after sample preparation against the blank matrix in which the same concentration analyte was added. - Should be evaluated at at least 2 levels: 3 x LLOQ (low) en 75% ULOQ (high). - For 5 days, the following samples are analyzed: 1 spiked sample and 1 blank sample where the analyte was added after extraction. The IS is always added after the extraction.

20 Recovery Recovery of analytes - Done using spiked (QC) samples Recovery of internal standards - Spiked in each sample or QC - Should be monitored during analysis of sample batches - SD is also an important parameter. A method has to have a low SD (be reproducible and stable), even if recovery is lower. - When using ILIS, recoveries can be also lower, but stable.

21 Matrix effects vs. Recovery

22 Matrix effects - Matrix effects are changes in the signal due to the presence of coeluting analytes or other interferences in the extract. - Supression or enhancement of the analyte is dependent on the matrix, the quality of sample preparation, the additives in the mobile phase and the ionisation type. - Should be assessed for different batches of matrices (n=6?, RSD<15%) - To be assessed for each analyte and internal standard (see next slide) - Absolute matrix factor calculated as on the next slide - Relative matrix factor = ratio between absolute matrix factor of the analyte and the corresponding IS. - For ILIS, relative matrix factor ~1

23 Matrix effects vs. Recovery

24 Robustness (or ruggedness) Ability of an analytical method to remain unaffected by - small variations in method parameters (mobile phase composition, column age, column temperature, etc.) - influential environmental factors (room temperature, air humidity, etc.) It characterizes its reliability during normal usage. Considering robustness as a method development parameter, EMA and FDA guidelines are missing the term.

25 Robustness (or ruggedness) Recommended method parameters to be investigated during robustness studies

26 Selectivity - Rather good to very good for MS (or HRMS) methods Selectivity is the capability of a method to measure ondubbelzinnig the analyte(s) and to differentiate the analyte(s) and the internal standard(s) from components possibly present in the samples. Such components are typically endogeneous matrix components, degradation products, impurities, etc, but can be also other analytes! More information in the presentations of Tania (related to validation of qualitative methods) and Robert (related to identification and confirmation of analytes).

27 Stability - Calibrators (in organic solvent) - Calibrators (in water-organic solvent) - Stability throughout the sample preparation - Extracts in organic solvent - Extracts in initial mobile phase - Different temperatures (+4 C, +20 C - autosampler) Freeze-thaw cycles Short-term temperature stability Long-term stability

28 Stability

29 What to do when you want to add a new compound to a validated method? Partial validation, which parameters to be changed? All parameters for this compound! - Accuracy - Precision - LOQ - Linearity

30

31 Food for thought No official guidelines for WW Which guidelines to follow? Should we propose guidelines for method validation for ww analysis?

32 Suggested literature EMA- ICH- FDA - guidelines Matrix effects: Matusewzki et al., AC 2003