Example of Method Validation

H. A. (1999), Some practical examples of method validation in the analytical laboratory, Trends Anal. Chem., 18, 584-593. 

Additional examples of method validation were published by M. Blanco et al. [32] in the implementation of a NIR model for active prediction across pharmaceutical processes or by Peinado et al. [33], who proposed a method validation approach for in-line measurements. 

Examples are generally easier to apprehend than theoretical explanations so this section will present a concrete example of method validation. The described procedure is not universal or exhaustive, but it follows the recommendation of the Societe Fran aise des Sciences et Techniques Pharmaceutiques (SFSTP, French Society of Science and Pharmaceutical Techniques) and has proven its efficiency. The number of analyses conducted per day and the number of days during which experiments are conducted are indicative and determined by the person in charge of validation. 

The sensitivity achieved (LOD) is not normally presented. It is recognized that different laboratories determine dissimilar values for this parameter and even within a laboratory the repeatability of the LOD is low. Most often, the lowest validated concentration gives an impression about the lowest levels that can be analyzed generally with acceptable results. A measure of selectivity is the intensity of blank results. This intensity is discussed by the participants of inter-laboratory validation studies. However, results are not reported and limits are not defined by CEN TC 275. The results of method validations of the several multi-residue/multi-matrix methods are not reported in the same way, but newer methods with limited scope generate analogous tables with validation results (as an example, see Table 7). 

An indication of the minimum size of a subsample can be obtained by using the concept of a sampling constant. For example, in the laboratory, the sampling constant can be used to estimate the minimum size of the test portion. However, the suitability of the chosen test portion size must be confirmed as part of method validation. The sampling constant Ks has units of mass. This is the mass of the test portion necessary to ensure a relative subsampling error of 1% (at the 68% confidence level) in a single determination. The value of /Ks is numerically equal to the coefficient of variation, CV (see Chapter 6, Section 6.1.3) for results obtained on 1 g subsamples in a procedure with insignificant analytical error. 

This chapter focuses on approaches to the validation of high-performance liquid chromatography methods based on regulatory guidance documents and accepted industry practices. The information in this chapter gives a brief review of the reasons for performing method validation and the regulations that describe this activity. Individual validation parameters are discussed in relation to the type of method to be validated. Examples of typical validation conditions are presented with references to additional information on individual topics. This chapter was written to help analysts responsible for method validation. 

As an identity (ID) test, per ICH guidelines, only selectivity is required in method qualification and validation. Repeatability and intermediate precision are often included to ensure reliability of p7 determinations. Additionally, method robustness should be tested to assure that the assay performance is suitable for QC environment. Quantitative parameters such as LOD/LOQ are not required for an ID assay. If a cIEF method is used for purity determination, then all the purity parameters shown in Section 4 should be qualified. The following sections illustrate an example of method development and qualification procedures for cIEF. 

System suitability is part of method validation. Experience gained during method development will give insights to help determine the system suitability requirements of the final method. An example is the hydrolysis of acetylsalicylic acid to salicylic acid in acidic media. Separation of this degradation peak from the analyte could be one criterion for the system suitability of an acetylsalicylic acid assay. 

Cross-Validation. Cross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as the reference and the revised bioanalytical method is the comparator. The comparisons should be done both ways. When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory... 

High-performance liquid chromatographic methods are the most common form of analytical technique used to support drug product registration. An example of a validation protocol for an HPLC assay and related compounds method is provided in Example 2. 

EXAMPLE 2 AN EXAMPLE OF A VALIDATION PROTOCOL FOR A CHROMATOGRAPHIC METHOD... 

An LC-MS/MS method [34] will be described in detail in order to serve as an example of method development and validation. This method allows the determination of some of the most common antidepressants used in clinical practice and some of their main metabolites in oral fluid and plasma samples. 

Table 1 contains examples of what needs to be checked in the process of method validation and the procedures that could be followed. This list is not meant to be comprehensive. 

One of the imderlying assumptions of the least squares method is that there is no error in the measurement of the independent variable (the y values). This assumption is often not valid, and one of the most obvious cases of this is found in method-comparison analysis. A typical example of method-comparison analysis involves the comparison of two different instruments, a current production instrument and an improved model. Measurements made on a series of samples with the two instruments, and plotted by using current instrument readings as the x values and new instrument readings as the y values, should ideally result in a straight line of unit slope and zero intercept. The actual slope and intercept of the line can provide estimates of the proportional and constant error between the two methods. 

It is essential to identify and separate these two types of errors to avoid confusion. If numerical errors are not isolated, they may lead to undesirable spurious model calibration exercises. It is, therefore, necessary to devise systematic methods to quantify numerical errors. The basic idea behind error analysis is to obtain a quantitative measure of numerical errors, to devise corrective measures to ensure that numerical errors are within tolerable limits and the results obtained are almost independent of numerical parameters. Having established adequate control of numerical errors, the simulated results may be compared with experimental data to evaluate errors in physical modeling. The latter process is called model validation. Several examples of model validation are discussed in Chapters 10 to 14. In this section, some comments on error analysis are made. 

Typical example of analytical validation in the QC laboratory method validation of a robotic analysis system for ketotifen 1 mg capsules and tizanidine hydrochloride 1 mg tablets... 

When identifying suitable testing labs, the main emphasis should be on the method application requirements, for example, sample analysis for preclinical or clinical studies, testing in conformance with GLP guidelines and, last but not least, the extent and philosophy of method validation between the laboratories. The selection of a compatible laboratory as the receiving laboratory is pivotal to the success of the method transfer process. 

Other examples of cleaning validation have been reported, but identities of the compounds were withheld due to proprietary considerations. " Nevertheless, quantitative determination of residual APIs and intermediates on equipment surfaces were described in detail. The linear dynamic ranges for the compounds investigated were 0.1-1.0 pg/mL for one compound and 1-10 pg/mL for the others. With IMS technology, they were able to evaluate 30 samples in less than 2 h. In another study, analysis required only 0.5 min/sample compared with 15-30 min/sample using the HPLC method. ... 

Table 4 Examples of the validated capillary electrophoresis methods for the determination of the chiral purity of drugs...

In each case a referee laboratory is responsible for providing the sample and organizing its distribution and the collation and analysis of the results. For the majority of studies the purpose of the study is to test the laboratory (proficiency testing), the test material (material certification), or the method (method validation study). The purpose determines some of the study parameters. For example, a method validation study requires all the laboratories to use the same method, but for other studies the method may not be prescribed. For materials certification the laboratories are chosen for their demonstrated proficiency in analysis of the material. It would not be appropriate if there was any dubiety concerning the assigned quantity value because of a suspect result. 

Analytical method The most serious errors are those in the method itself. Examples of method errors include (1) incomplete reaction for chemical methods, (2) unexpected interferences from the sample itself or reagents used, (3) having the analyte in the wrong oxidation state for the measnre-ment, (4) loss of analyte during sample preparation by volatilization or precipitation, and (5) an error in calculation based on incorrect assumptions in the procedure (errors can arise from assignment of an incorrect formula or MW to the sample). Most analytical chemists developing a method check all the compounds likely to be present in the sample to see if they interfere with the determination of the analyte unlikely interferences may not have been checked. Once a valid method is developed, an SOP for the method should be written so that it is performed the same way every time it is run. 

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