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![Regression equation Y = aX + b
Where “a” is the slope of the line and “b” is the
intercept on the y-axis. When X = 0, “a” & “b” will be
according to equation 1.2.
( )∑ ∑
∑ ∑ ∑∑
−
−
= 22
2
xixiN
xiyixiyixi
b
( )∑ ∑
∑ ∑ ∑−
= 22
xixiN
yixixiyiN
a
( )[ ] ( )[ ]∑ ∑∑ ∑
∑ ∑ ∑
−−−
−
=
yiyiNxixiN
yixixiyi
r
22](https://image.slidesharecdn.com/analyticalmethodvalidation-180314102218/85/Analytical-method-validation-15-320.jpg)
![Numberoftimeseachvalueoccurs
Valuesσ2σ3σ
σ 2σ 3σ
Normal distribution, probability function [P(x)] and confidence
interval [CI]](https://image.slidesharecdn.com/analyticalmethodvalidation-180314102218/85/Analytical-method-validation-29-320.jpg)
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Analytical method validation
- 1. Analytical Method Validation Dr.ARTI R. THAKKAR This presentation was made to solely for students to make them aware/ understand basics of “Validation”. These slides are part of lectures delivered in M. Pharmacy Curriculum & taken up from various books and websites
- 2. Analytical Method Development& Validation
- 3. Analytical Method Development& Validation
- 4. Analytical Method Development& Validation
- 5. Analytical Method Development& Validation
- 6. Method Validation Thus, Methodvalidation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the drug substances and drug products.
- 7. Method Validation Published Guidances –ICH-Q2A “Text on Validation of Analytical Procedure:(1994) – ICH-Q2B “Validation on Analytical Procedures: Methodology: (1995) – CDER “Reviewer Guidance: Validation of Chromatographic Method” (1994) – CDER “Submitting Samples and Analytical Data for Method Validations” (1987) – CDER Draft “Analytical Procedures and Method Validation” (2000) – CDER “Bioanalytical Method Validation for Human Studies” (1999) – USP<1225> “Validation of Compendial Methods” (current revision)
- 8. Considerations Prior toMethod Validation • Suitability of Instrument – Status of Qualification and Calibration • Suitability of Materials – Status of Reference Standards, Reagents, Placebo Lots • Suitability of Analyst – Status of Training and Qualification Records • Suitability of Documentation – Written analytical procedure and proper approved protocol with pre-established acceptance criteria
- 9. Examples of MethodsThat Require Validation Documentation • Chromatographic Methods – HPLC, GC, TLC, GC/MS, etc. – Pharmaceutical Analysis – In support of CMC. – Bioanalytical Analysis – In support of PK/PD/Clinical Studies. • Spectrophotometric Methods – UV-VIS, IR, NIR, AA, NMR, XRD, MS, etc. • Capillary Electrophoresis Methods – Zone, Isoelectric Focusing, Isotachophoresis, etc. • Particle Sizer Analysis Methods – Laser, Microscopic, Photozone, Sieving, SEC, etc. • Dissolution Methods – Method of Analysis – HPLC, UV, Automated, etc. • Titration Methods. • Automated Analytical Methods – Robots, Automated Analysis.
- 10. Specificity & Selectivity Specificrefers to a method that produces a response for a single analyte only selective refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. If the response is distinguished from all other responses, the method is said to be selective. Since there are very few methods that respond to only one analyte, the term selectivity is usually more appropriate.
- 11. Specificity & Selectivity Identitytesting – To ensure the identity of an analyte Purity testing – To ensure the content of impurities of an analyte Assay – To allow the content of an analyte in a sample
- 12. Specificity & Selectivity •Specificity: Overlay chromatogram of an impurity solution with a sample solution
- 13. Analytical Method Development SpecificityandSpecificity and stabilitystability • Stress stability testing to ensure theStress stability testing to ensure the stability indicating potentialstability indicating potential of anof an analytical methodanalytical method – Apply diverse stress factors to the APIApply diverse stress factors to the API – Apply diverse stress factors to the FPPApply diverse stress factors to the FPP Assure that the API can be assessed specifically in the presence of knownAssure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impuritiesand unknown (generated by stress) impurities Assure that known impurities/degradants can be specifically assessed inAssure that known impurities/degradants can be specifically assessed in the presence of further degradantsthe presence of further degradants ByBy peak purity assessmentpeak purity assessment and (overlay of)and (overlay of) chromatogramschromatograms
- 14. Linearity of ananalytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample – If there is a linear relationship test results should be evaluated by appropriate statistical methods • Correlation coefficient (r) • Y-intercept • Slope of regression line • Residual sum of squares • Plot of the data
- 15. Regression equation Y= aX + b Where “a” is the slope of the line and “b” is the intercept on the y-axis. When X = 0, “a” & “b” will be according to equation 1.2. ( )∑ ∑ ∑ ∑ ∑∑ − − = 22 2 xixiN xiyixiyixi b ( )∑ ∑ ∑ ∑ ∑− = 22 xixiN yixixiyiN a ( )[ ] ( )[ ]∑ ∑∑ ∑ ∑ ∑ ∑ −−− − = yiyiNxixiN yixixiyi r 22
- 16. Analytical Method Development •Usual acceptance criteria for a linear calibration curve – r > 0.999; y-intercept a < 0 to 5% of target concentration RSD (wrt calibration curve) < 1.5-2% r > 0.997 r < 0.997
- 18. Range The range ofan analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
- 19. Accuracy • The accuracyof an analytical procedure expresses the closeness of true value and the found value.
- 20. • AccuracyAccuracy – Expressesthe closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found • Sometimes referred to as TRUENESS truetruemeanmean
- 21. To find outwhether a method is accurate: • Drug substance (assay) – Application of the method to an analyte of known purity (e.g. reference substance) – Comparison of the results of one method with those of a second well- characterised method (accuracy known) • Drug product (assay) – Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added i.e. spiked • Drug product may exceptionally be used as matrix • Drug substance/Drug product (Impurities) – Application of the method to samples spiked with known amounts of impurities
- 22. • Accuracy: Applicationof the method to synthetic mixtures of the drug product components to which known quantities of the analyte have been added • Recovery reduced by ~10 – 15%
- 23. • When toexpect Accuracy problems – Insufficient selectivity of the method • Impurity peaks are not resolved and account for assay value – Recovery is < 100% • Irreversible adsorption of analyte to surfaces of the system – Incorrect assay value of a reference standard • Due to decomposition of reference standard – Incorrect assay value due to change in matrix • Analytical laboratory still uses the preceding matrix as standard
- 24. ( ) ()( ) ( ) ( ) 100covRe% 22 × − − = ∑∑ ∑∑∑ xxN yxxyN ery 100× = ionConcentratExpected ionConcentratmeasuredMean Accuracy
- 25. • Precision – Expressesthe closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample – Is usually expressed as the standard deviation (S), variance (S2 ) or coefficient of variation (RSD) of a series of measurements – Precision may be considered at three levels • Repeatability (intra-assay precision) • Intermediate Precision (variability within a laboratory) • Reproducibility (precision between laboratories)
- 26. Precision • Repeatability expressesthe precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. • Intermediate Precision expresses within- laboratories variations: different days, different analysts, different equipment, etc. • Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
- 27. Repeatability ( )∑ −−1/ 2 NXXiS D = 100× X S R S D =
- 29. Numberoftimeseachvalueoccurs Valuesσ2σ3σ σ 2σ 3σ Normaldistribution, probability function [P(x)] and confidence interval [CI]
- 30. • Relationship ofvariability, probability and reliability of data – High variability of data (large σ) generate large confidence intervals and thus lower the reliability of the mean – Low variability of data (small σ) generate small confidence intervals and thus increase the reliability of the mean
- 31. • Reproducibility – Expressesthe precision between laboratories • Collaborative studies, usually applied to standardisation of methodology – Transfer of technology – Compendial methods
- 32. Detection Limit • Thedetection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
- 33. • Limit ofDetection (LOD, DL)Limit of Detection (LOD, DL) • Determination is usually based on – Signal to noise ratio (~3:1) (baseline noise) or – Standard deviation of response (σ) and Slope (S) • 3.3 σ/S
- 34. • Limit ofQuantitation (LOQ, QL)Limit of Quantitation (LOQ, QL) – The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy • The quantitation limit is used particularly for the determination of impurities and/or degradation products • Determination is usually based on – Signal to noise ratio (~10:1) (baseline noise) or – Standard deviation of response (σ) and Slope (S) • 10 σ/S
- 35. NoiseNoise LODLOD Signal toSignal toNoiseNoise = 3:= 3:11 LOQLOQ Signal toSignal to NoiseNoise = 10:= 10:11 LOD, LOQ and Signal to Noise Ratio (SNR)LOD, LOQ and Signal to Noise Ratio (SNR)
- 36. • LOQ – Quantitationby SNR is accepted – Quantitation by Standard deviation of response (σ) and Slope (S) (10 σ/S) is more adequate as it involves the response of the actual analyte
- 37. • LOQ andimpurities – In determination of impurities in APIs the LOQ should be determined in the presence of API • LOQ should be NMT reporting level • LOQ should be given relative to the test concentration of API – Specificity of impurity determination should always be demonstrated in the presence of API at API specification levels • Spiking of test concentration (API) with impurities at levels of their specification range
- 38. • Spiking – APItest concentration (normalised) • 0.1 mg/ml (100%) – Impurity spiking concentrations • 0.001 mg/ml (1%) – specification limit • 0.0001 mg/ml (0.1%) – limit of quantitation (minimum requirement) API at test concentrations API below test concentrations
- 39. Impurities (Quantitation) • Accuracyshould be assessed on samples (substance / product) spiked with known amounts of impurities.
- 40. Robustness • The robustnessof an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
- 41. • Robustness – Robustnessof an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters – The evaluation of robustness should be considered during the development phase – If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure
- 42. • Influence ofbuffer pH and buffer concentration in mobile phase on retention times of API and impurities • Conclusion: The buffer composition should be maintained in a range of 85 ± 0.5% – Missing: Acceptance criterion for maximal deviation of retention time should be defined unless justified API Impurity A Impurity B Impurity C As is 10.46 3.86 7.43 8.26 buffer pH 5.9 10.45 3.94 7.51 8.38 buffer pH 6.9 10.46 3.94 7.49 8.34 Buffer conc. 83% 7.84 3.43 6.16 6.66 Buffer conc. 87% 15.26 4.77 9.61 11.18
- 43. System Suitability Testing •System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated.
- 44. • Method stability –System suitability over time • Sample solution stability – A solution of stavudine is stable for ~ 2 h, then it starts to degrade to thymine • Impurity-spiked sample solution stability A solution containing stavudine spiked with its impurity thymine does not allow to clearly distinguish between degradation and spike Should be analysed immediately
- 45. • When tobe „surprised“ about validation data: – Precision of impurity determination – Precision of API determination – Method precision of released API (dissolution) System precision % RSD 0.33 – 2.25 Method precision % RSD 0.0 Average peak area % RSD 0.08 Acceptance criterion % RSD ≤ 2.0 Average peak area % RSD 0.4 Acceptance criterion % RSD ≤ 10.0
- 46.