Wednesday, 4 June 2014

Analytical Method Validation


Overview of Analytical Method Validation Process

The purpose of Analytical Method Validation is to get accurate, reliable and consistent data. As we all know that incorrect measurement can lead to tremendous costs. Activity involving in this complete process includes: (1) Sampling (2) Sample Preparations (3) Analysis and (4) Data Evaluation reporting. Some of the considerations prior to Analytical Method Validation are: (1) Suitability of instrument in which status of qualification and calibration of instrument is checked. (2) Suitability of materials in which Status of reference standards, reagents, placebo etc are checked. (3) Suitability of analyst in which status of training and qualification records of analyst are checked. (4) Suitability of documentation , Written analytical procedure and proper approved protocol with pre-established acceptance criteria are checked.

Steps in Analytical Method validation process:-

Some of the steps which are to be followed during analytical method validation are as follows, so let us check it out one by one: (1) Define the application, purpose and scope of the method. (2) Analyst? Concentration? Sample Matrics? (3) Develop an analytical method (4) Develop a validation protocol. (5) Qualification of instrument. (6) Qualify/trained operator. (7) Qualification of material. (8) Perform pre-validation experiments. (9) Adjust method parameters and/or acceptance criteria if necessary. (10) Perform full validation experiments. (11) Develop SOP for executing the method in routine analysis. (12) Document validation experiments and results in the validation report.

Terms used in Analytical method validation:-

There are specific guidelines for "Analytical method Validation" , you will find this guidelines from ICH, USP etc., Some of the validation requirements as per ICH/USP are : Specificity, Linearity, Range, Accuracy, Precision, Repeatability, Limit of detection, Limit of quantification, Ruggedness and Robustmess. In order to get accurate, reliable and consistent result or date it is necessary to have sound knowledge of all this terms given above. So let us check it out this terms one by one with brief explanation.

Specificity is the ability of an process in Analytical method validation to measure the analyst free from interference from other components.An investigation of specificity should be conducted during the validation of an identification test, an impurities assay and a potency assay. Procedures used will depend on the intended objective of the analytical procedure. If a method cannot completely discriminate, two or more procedures are recommended. Identification of Specificity: Should be able to discriminate between closely related compounds. Confirmed by obtaining negative results for samples with spiked related compounds and positives results of samples with analyte. Choice of potential interfering substance should be based on sensible scientific judgement considering substances that could likely occur. Specificity Impurities/Assay: Now at this stage in "Analytical method validation: following are some of the point which are taken into consideration. (1) Chromatography methods which demonstrates resolution. (2) Impurities/Degradants available which spike with impurities/degradants, Which show resolution and lack of interference. (3) Impurities/Degradants not available , as a result It stress samples, For assay, stressed and unstressed samples are compared. For impurity test, impurity profile should be compared. Specificity Potential Interference: Placebo, Drug substance Degradants, Drug product Degradants, Related substances, packaging extractables etc.,

Forced Degradation is also performed in which parameters such as  Heat, Humidity, Acid hydrolysis, Base hydrolysis, Oxidation and Light (Intense UV/Visible light) are taken into consideration.

Linearity and Range are also important parameter in Analytical Method Validation, to develop accurate and reliable method. Linearity : It as an ability of an assay to elicit direct and proportional response to changes in analyte concentration. Linearity should be evaluated by visual inspection of plot of signals against analyte concentration. By appropriate statistical methods such as linear regression correlation coefficient etc., Range : It is the interval between upper and lower concentration of analyte in the sample that have been demonstrated to have a suitable level of precision, accuracy and linearity. Range is normally derived from linearity studies. Specific range dependent upon intended application of the procedure.

Accuracy is another important term in Analytical Method Validation. Accuracy is the closeness of the test results obtained by the method to the true value. Accuracy should be established across specified range of analytical procedure. Should be accessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations). Accuracy should be reported as : percent recovery of known amount added or the difference between the mean assay result and the accepted value.

Precision is also carried out in Analytical method Validation which can be defined as: The closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. Precision should be investigated using homogeneous, authentic samples. Precision is generally divided into 3 levels. (1) Repeatability, (2) Intermediate precision and (3) Reproducibility. Let us check it all three points one by one. Repeatability : Express the precision under the same operating conditions over a short interval of time. Also referred to as intra-assay precision. Should be assessed using minimum of 9 determinants or minimum of 6 determinants with 100% accuracy. The next term of Precision in Analytical method Validation is Intermediate Precision : Expressed within-laboratory variations. Expressed in terms of standard deviation, relative deviation and confidence interval. It Depends on the circumstances under which the procedure is intended to be used. Studies should varying days, analysts, equipment etd., Reproducibility :Ability to reproduce data with the predefined precision .Also expressed in terms of standard deviation, relative standard deviation and confidence interval. 

Limit of detection and limit of quantification also plays important role during Analytical method Validation, So let us see the definition of: Limit of Detection is the lowest amount of analyte in a sample that can be detected but not necessarily quantified. Estimated by signal to Noise ratio. Limit of Quantificatiaon : Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision. Estimated by signal to noise ratio.

Robustness is the capacity to remain unaffected by small but deliberate variations in method parameters. In Analytical method validation robustness can be determined by comparison of results under differing conditions with precision under normal conditions. Variations may include stability of analytical solutions, variation of pH in a mobile phase, different column, temperature, flow rate etc. While in Ruggedness : Variations are such as different laboratories, different analyst, different instruments, days , reagents etc.,

So in order to get better, accurate and reliable data or results this all factors should be taken into consideration during Analytical method Validation process. Various pharma industries follow ICH and other standard guidelines to carry out above mentioned process effectively.

Related Articles on Analytical Method Validation:

  1. What is Analytical Method Validation Definition
  2. Steps in Analytical Method Validation
  3. What are Analytical Method Validation Parameters?
  4. Ruggedness in Analytical Method Validation
  5. Robustness in Analytical Method Validation
  6. Limit of Quantification in Analytical Method Validation
  7. Limit of Detection in Analytical Method Validation
  8. Range in Analytical Method Validation

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