Introduction to validation of analytical methods

INTRODUCTION TO VALIDATION OF ANALYTICAL METHODS

The philosophy behind process of analytical method validation should demonstrate that the method is best fit for its purpose. The validation should follow a plan that must cover the scope of the method, its performance characteristics and acceptance limits.

Introduction

Method validation is defined by the process that establishes the performance characteristics of the method and meets the requirements for the intended analytical applications. Methods need to be validated or revalidated before their introduction for routine use. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use has developed a formal literature on the validation of analytical procedures. The United States Food and Drug Administration (USFDA) have certain guidelines on submitting samples and analytical data for methods validation.

Stages in Method Validation

1. Development of validation protocol or procedure for the Validation

2. Purpose of application, purpose and scope of the method

3. Various performance parameters and acceptance criteria

4. Execution of validation experiments

5. Verification of relevant performance characteristics of equipments used

6. Approved materials e.g. standards and reagents

7. Evaluation of pre-validation experiments

8. Adjustment of various method parameters or/and acceptance criteria

9. Apply full internal and external validation experiments set up

10. Development of standard operating procedures (SOPs) for execution of the method

11. Explanation of criteria for revalidation

12. Type and frequency of system suitability tests for the routine checks

13. Documentation of validation experiments and results obtained in the validation

Parameters for Method Validation:

The parameters as defined by the ICH and by other regulatory body are Specificity, selectivity, precision, repeatability, intermediate precision, reproducibility, accuracy, linearity range, limit of detection, limit of quantization, robustness and ruggedness.

Selectivity / Specificity

The terms selectivity and specificity are often used interchangeably. The term specific generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method which gives responses for a number of chemical entities that may or may not be distinguished from each other. If the response is notable from all other responses, the method is said to be selective.

Determination

In the case of qualitative determination, the ability to select between compounds of closely related structure that are likely to be present should be established. This should be confirmed by obtaining desired results from samples containing the substance, coupled with negative results from samples that do not contain the substance and by confirming that a positive response is not obtained from materials structurally similar to the substance.

Precisions and Reproducibility

The precision of a method is the extent to which the individual test results of multiple samples of a series of standards having same opinion. The measured standard deviation can be further divided into three sub categories: repeatability, intermediate precision and reproducibility.

Repeatability is obtained by using one piece of equipment over a relatively short time-span analysis in one laboratory. At least 5 or 6 determinations of three different samples at two or three different concentrations should be done to calculate relative standard deviation.

Intermediate precision is defined as the long-term variability of the measurement process and is determined by comparing the results of particular method performance within a single lab over a number of weeks. Objective of this validation is to verify that in the same laboratory the method will provide the same output once the development phase is over.

Reproducibility defined as precision obtained between labs. Objective is to verify that the method will provide the same results in different labs.

Accuracy and recovery

The accuracy of any analytical method is the extent up to which test results generated by the method and the true value satisfy.

Linearity

The linearity of an analytical method is to judge its ability to elicit test results that are (directly or by means of well-defined mathematical transformations) proportional to the concentration of substance in samples within a given range. Linearity is determined by a series of two to six samples of five or more standards whose concentrations span 80-130 percent of the desired concentration range.

Range

The range of an analytical method is the gap between the upper and lower levels that have been established to be resolute with precision, accuracy and linearity using the method.

Limit of Detection and Quantitation

Limit of detection: It is the lowest concentration of substance in a sample that can be detected but not necessarily quantified.

Limit of quantitation: Itis the minimum amount that gives precise measurements.

Ruggedness

Ruggedness is assessment of reproducibility test results under the variation conditions normally expected from lab to lab and from analyst to analyst.

Robustness

Robustness is a measurement of its capacity to remain unchanged by small but deliberate variations in method process and parameters and provides a sign of its consistency during normal practice.

???????