Analytical Method Validation

PRESENTATION ON ANALYTICAL METHOD VALIDATIONBY AVADHOOT RAMCHANDRA SUPAL : 

PRESENTATION ON ANALYTICAL METHOD VALIDATIONBY AVADHOOT RAMCHANDRA SUPAL

Definition : 

Definition “Validation of Analytical Procedures is the process of determining the suitability of a given methodology for providing useful analytical data. A method that is valid in one situation could well be invalid in another.”

Common misconceptions : 

Common misconceptions Assay Validation  Assay Optimization  Assay Qualification A Validated Method is NOT necessarily a “tight” method Repeating an assay a number of time does not constitute validation

How an assay evolves : 

How an assay evolves Optimization Validation Implementation Development Revalidation Pre-Validation

Assay Validation Parameters:USP and ICH : 

Assay Validation Parameters:USP and ICH Method Validation Accuracy Precision Limit of Detection Limit of Quantitation Linearity and Range Specificity System Suitability Ruggedness/Robustness

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Table

Specificity/Selectivity : 

Specificity/Selectivity Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure (s). Selectivity describes the ability of an analytical method to differentiate various substances in a sample.

Impurities are available (1) : 

Impurities are available (1) For the assay , this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients; practically, this can be done by spiking pure substances (drug substance or drug product) with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials (by comparison with the assay result obtained on unspiked samples).

Impurities are available (2) : 

Impurities are available (2) For the impurity test, the discrimination may be established by: spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the sample matrix.

Impurities are not available : 

Impurities are not available If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g.: pharmacopoeial method or other validated analytical procedure (independent procedure). As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation. for the assay, the two results should be compared. for the impurity tests, the impurity profiles should be compared.

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Linearity The Linearity of an analytical 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 For the establishment of Linearity, a minimum of 5 concentrations is recommended. Correlation coefficient. Y- intercept. Slope of Regression line. Intercept NMT ±2% of response of 100% the working concentration for Assay Intercept NMT ±10% of response of 100% the working concentration for related substances.

Limits of Quantitation : 

Limits of Quantitation . The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. . A typical acceptable signal-to-noise ratio is 1:10

Limits of Detection : 

Limits of Detection The Limit of Detection (LOD) of a method may be defined as the concentration of analyte which gives rise to a signal that is significantly different from the negative control or blank. The LOD is the lowest concentration of analyte that can be distinguished from background. The results obtained at the Limit of Detection are not necessarily Precise or Accurate LOD can be determined as signal to noise ratio – 3:1

Accuracy : 

Accuracy Accuracy is the measure of exactness of an analytical method, or the closeness of agreement between the measured value and the value that is accepted as a conventional true value or an accepted reference value. The determination of Accuracy usually requires a “gold standard” or an accepted method to which a new method can be compared -For Assay Limit – 98.0% - 102.0% For Dissolution – 95.0% - 105.0 % For Content of Uniformity – 85.0 % – 115.0 % For Related substances – 90.0 % - 110.0%

Range : 

Range The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and Linearity. Within the range of the method, results are accurate, precise and “linear” for the assay of a drug substance or a finished (drug) product: normally from 80 - 120 % of the test concentration for content uniformity, covering a minimum of 70 - 130 % of the test concentration

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for dissolution testing: +/-20 % over the specified range; e.g., if the specifications for a controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claim or the determination of an impurity: from the reporting level of an impurity to 120% of the specification; for impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the detection/quantitation limit should be commensurate with the level at which the impurities must be controlled. if assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification

Precision : 

Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.

Repeatability (1) : 

Repeatability (1) Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Repeatability (2) : 

Repeatability (2) Repeatability should be assessed using: a) a minimum of 9 determinations covering the specified range for the procedure (e.g. 3 concentrations/3 replicates each) or b) a minimum of 6 determinations at 100% of the test concentration.

Intermediate precision : 

Intermediate precision Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged.

Reproducibility : 

Reproducibility Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility should be considered in case of the standardization of an analytical procedure, for instance, for inclusion of procedures in pharmacopoeias. These data are not part of the marketing authorization dossier.

ROBUSTNESS : 

ROBUSTNESS The robustness of 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. The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. 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. One consequence of the evaluation of robustness should be that a series of system suitability parameters (e.g., resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.

System Suitability in Chromatography : 

System Suitability in Chromatography To verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done The resolution, R, is specified to ensure that closely eluting compounds are resolved from each other Replicate injections of a standard preparation are compared to ascertain whether requirements for precision are met The tailing factor, T, has to meet a certain requirement, because as peak asymmetry increases, integration, and hence precision, becomes less reliable

Standards : 

Standards What will be the working standard? Will there be enough of this standard to perform all pre-validation work, execute the validation protocol and run all the pre-clinical, clinical samples or other samples? What is the stability of the standard? Will you be preparing the standards every time you run an assay or will you be using pre-prepared frozen standards?

Controls : 

Controls Aliquots of the analyte that have been made in the sample matrix separately from the standard curve and which are run in every assay Should be at the low, medium and high end of the standard curve. Their exact values (concentration) is not important and will be determined by repeat assaying Preferably can be added directly to the assay without dilution.

Assay Validation protocolAcceptance Criteria : 

Assay Validation protocolAcceptance Criteria You must have acceptance criteria specified in the protocol for each parameter The acceptance criteria are determined by the Study Director based his/her best scientific judgement and on the purpose of the assay The acceptance criteria will be different for different assays The acceptance criteria must be set prospectively!