u.v validation

DEVELOPMENT OF ANALYTICAL METHOD AND VALIDATION OF UV-VISIBLE SPECTROPHOTOMETER :

DEVELOPMENT OF ANALYTICAL METHOD AND VALIDATION OF UV-VISIBLE SPECTROPHOTOMETER Presented by: A.Mounika M.Pharm1st year Pharmaceutical analysis Sarojini Ramulamma college of pharmacy

Validation def::

Validation def: Validation is establishing documented evidences, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics.

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validation is considered a good manufacturing practice (GMP) activity, validation experiments must be properly documented and performed on qualified and calibrated instrumentation and equipment. At this stage, there should be documented evidence that the method is robust.

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A generalized flowchart of the validation process

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Method development by UV- Spectrophotometric methods: Selection of the solvent. Selection of analytical wavelengths. Study of Beer-Lambert’s Law. To perform analysis of standard laboratory mixture and tablet formulations by proposed method. To validate the developed methods by using different statistical parameters

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The USP has published specific guidelines for method validation for compound evaluation . USP defines eight steps for validation: Accuracy Precision Specificity Limit of detection Limit of quantitation Linearity and range Ruggedness Robustness

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Demonstration of the Accuracy of the Method: This is defined as the closeness of agreement between a test result and the accepted reference value (combination of random and systematic errors). The accuracy is usually examined by determination of the trueness of a test result, which is the closeness of agreement between the average value of a large number of test results and the true result or an accepted reference value. The measure of the trueness is expressed by the bias, which is the difference between the expectation of the test results and an accepted reference value. The accuracy of a method can be determined by performing recovery experiments, implementing standard addition calibration procedures, testing reference materials, etc. It is also possible to compare the test results of a new method with those of an existing fully validated reference method through “cross validation” experiments. Accuracy is often determined by recovery studies in which the analytes are spiked into a solution containing the matrix.

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Precision : “The precision of an analytical procedure expresses the closeness of agreement between a series of measurements from multiple sampling of the same homogeneous sample under prescribed conditions. Precision may be considered at three levels: Repeatability, intermediate precision, and reproducibility.” Repeatability :“Repeatability expresses the precision under the same operating conditions over a short interval of time.”

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Intermediate precision: “Intermediate precision expresses within laboratories’ variations: Different days, different analysts, different equipment, etc.” Reproducibility: “Reproducibility expresses the precision between laboratories.”

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Repeatability (r): The expected maximum difference between two results obtained by repeated application of the analytical procedure to an identical test sample under identical conditions. The measure for repeatability (r) is the standard deviation . For series of measurements of a sufficient size (usually not less than 6), the repeatability is defined as r = 2.8 x (confidence level 95%) Repeatability should be obtained by the same operator with the same equipment in the same laboratory at the same time or within a short interval using the same method.

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Within-laboratory reproducibility : The expected maximum difference between two results obtained by repeated application of the analytical procedure to an identical test sample under different conditions but in the same laboratory. The measure for the within laboratory reproducibility (R,) is the standard deviation ( sR ,). For series of measurements of sufficient size (usually not less than 6), the within-laboratory reproducibility is defined as Within-laboratory reproducibility should be obtained by one or several operators with the same equipment in the same laboratory at different days using the same method.

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Reproducibility (R) : The expected maximal difference between two results obtained by repeated application of the analytical procedure to an identical test sample in different laboratories. The measure for the reproducibility (R)is the standard deviation (SR). For series of measurements of sufficient size (usually not less than 6) the reproducibility is defined as Between-laboratory should be obtained by different operators with different instrumentation in different laboratories on different days using the same method. For a given method, the most important factors in the determination of repeatability and reproducibility are Laboratory, Time, Analyst and Instrumentation.

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Standard deviation (s): A measure of the spread in the observed values as a result of random errors These observed values all have the same expected value. The equation to be used is xi = individual measured value, = mean measured value, n = number of measurements.

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Relative standard deviation (s) (RSD) The standard deviation(s) expressed as a percentage of the mean value. The relative standard deviation is defined as:

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Specificity: “Specificity is the ability to assess unequivocally the analyse in the presence of components which may be expected to be present.” Detection Limit: “The detection 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.”

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Quantitation Limit: “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.” 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 the analyte in the sample.”

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Range: “The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of the analyte in the sample (including these concentrations) for which it has been shown that the analytical procedure has a suitable level of precision, accuracy, and linearity.” Robustness: “The robustness of an analytical procedure is the 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.”

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Ruggedness: “the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions, such as different labs, different analysts, different lots of reagents, .... Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst.” Sensitivity: The sensitivity of an analytical method is equal to the slope of the calibration line in a linear system.

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Generalized validation process for an u.v visible assay and purity method

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start Collect active peak and maximum absorbance

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finish

Demonstration of Linearity and Range: Determination of Relative Response Factor:

Demonstration of Linearity and Range: Determination of Relative Response Factor Linearity is the ability to obtain results that are directly or indirectly (by well-defined mathematical transformation) proportional to the concentration of a substance in a sample within a given range. The range is the interval between the upper and the lower levels of the analytical method that have been demonstrated to obtain acceptable accuracy, linearity, and precision.

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The relationship between the sample concentrations and the corresponding instrumental signals for the majority of analytical techniques is one of a straight-line (first order) type. A line that fits best through the coordinates of the measured signals and the corresponding concentrations of the sample represents such a relationship. This line, known as the calibration line, is expressed by an estimated first-order equation: Y = aX + b where Y is the measured signal, X the concentration of the sample, and a and b the linear regression coefficients of the line, of which a is called the slope of the line and b the intercept.

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a. Linearity of the Active Component The linearity can be demonstrated by analyzing five or more concentrations of the active compound in the presence of the matrix: for example, 50%, 75%, 100%, 125%, and 150% of the normal sample concentration for a stability-indicating method (three separate preparations at each level). There are also added advantages to evaluating the linearity over the whole range from LOQ to 150%. Linearity can be established by visual evaluation of a plot of the area as a function of the analyte concentration. Furthermore, the correlation coefficient, y intercept, slope, and RSD for all the generated response ratios (= area/concentration) should be calculated. The y intercept should statistically not differ from 0. Low levels of the active compound (0.05–1.0%) are also examined to determine its LOQ .

Linearity of the active component (0.05–150%).:

Linearity of the active component (0.05–150%). absorbance

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b. Linearity of the Related Compounds The linearity should be demonstrated by analyzing five concentrations in the presence of the matrix: at LOQ, at the specification level, at an upper level above specification, and at two intermediate concentrations (e.g., 0.1%, 0.25%, 0.5%, 0.75%, and 1.0 %). Linearity can be established by visual evaluation of a plot of the area as a function of the analyte concentration. The correlation coefficient, y intercept, slope, and RSD for all the generated response ratios (= area/concentration) should be calculated. The y intercept should statistically not differ from 0. The slopes of these curves are divided by the slope of the active compound curve to determine the RRFs; these are recorded in the method procedure if the method does not prescribe the use of external standards for related compounds.

Linearity of the active component (0.05–1.0%).:

Linearity of the active component (0.05–1.0%). absorbance

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Linearity of specified related substances (0.05–1.0%). concentration absorbance

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Determination of Detection and Quantitation Limits LOD and LOQ can be simply determined from the known amount (concentration) of an analyte that produces such responses when the noise level can be easily measured. In fact, some chromatography data systems can be programmed to report baseline noise. An alternative method of determination is described by the ICH guidelines as follows: . In pharmaceutical analysis of the active drug substance, the target value for the LOQ is typically set at 0.05%.

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Systematic error or bias: The difference between the average observed value, obtained from a large series of observed values and the true value

Acceptance criteria for u.v assay purity method:

Acceptance criteria for u.v assay purity method

REVALIDATION :

REVALIDATION According to the validation life cycle,24 test methods may require additional validation or revalidation when regulatory agencies issue new requirements or when changes are made to the methodology. Method changes and additional validation activities may be required when there are 1. Instrument changes 2. Product changes 3. Method modifications 4. Analyst changes 5. Outdated technology Revalidation may be necessary after changes in (a) drug substance synthesis, (b) drug product composition, and (c) the analytical procedure. The degree of revalidation required depends on the nature of the changes made.

References:

References Handbook of modern pharmaceutical analysis by satinder ahuja and stephen scypinski . Vol 3 pg. no 415 to 438. Valid analytical methods and procedures by charistopher burgess.pg.no 6 to 9.

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Thank u