Validation of analytical methods

 

11 Principles

The analytical methods used for batch testing must be validated in order to ensure that the results obtained are "correct and precise". As illustrated in chapter 15.C.4 Testing procedures and test protocol, a series of points are to be described. The last and most important point is the "Validation" part.

Validation is defined in accordance with USP </225> (Sucker, 1983)

Corresponding definitions can be found in USP (USP <1225>), the WHO-GMP Guidelines and in FDA/ICH (Sickmüller, 1995) (Throm, 1997) (ICH Q2A/B).

Four groups of techniques are distinguished in terms of their field of use:

• Identity test
• Quantitative determination of impurities
• Limit tests of impurities
• Assy determination

A further group includes the testing of special parameters (performance characteristics), e.g. testing the dissolution rate (DR) or the content uniformity (CU). Depending on the application group, the following parameters must be validated:

• Accuracy,
• Precision,
• Specificity/selectivity,
• LOD = Limit of Detection,
• LOQ = Limit of Quantitation,
• Linearity, (linear) range.

Figure 12 summarises the groups of techniques and the corresponding parameters to be validated

12 Definitions of the parameters

The parameters to be validated are listed below with their definitions. In addition, the method for validating the corresponding parameters is also specified.

12.1 Precision

Precision expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same sample under prescribed conditions. It is expressed as the variance, standard deviation or coefficient of variations and may be performed at three levels: repeatability, intermediate precision and reproducibility. The following are distinguished:

Repeatability: n determinations are executed by a person quickly one after the other, under conditions as similar as possible. At least 6 100% determinations are required (note: "100%" corresponds to the concentration of the test solution if the content of the analyte to be investigated holds exactly 100% of the declaration), or at total of at least 9 determinations with 3 different concentrations (e.g. 3 determinations each at 80%, 100%, 120%).

Intermediate precision: Variation of the determinations within a lab if executed by several persons, on different days using different equipment.

Reproducibility: Reproducibility from ring trials with the participation of several labs (cf. Determination of content of standards of Ph. Eur., for more information: see chapter 14.C Standards and reference substances).

The standard deviation, the relative standard deviation (coefficient of variation) and the confidence area must be specified.

12.2 Accuracy

Accuracy expresses the closeness of agreement between the value which is accepted either as a conventional or an accepted reference value and the value found. It can be determined with a known accuracy from a second technique, or through determination of the recovery rate. Here, different, known quantities of analyte (API) are added to the sample to be investigated and the recovery is determined (spiked placebo method). The accuracy can also be derived from the regression lines of the linearity. If zero is included in the confidence area of the axis section, the technique is considered to be accurate, i.e. not tainted with a systematic error.

Validation should ensure that the results obtained are correct and precise. For clarification, please refer to the model of the target (see figure 13).

12.3 LOD = Limit of Detection

The limit of detection is the smallest quantity of a substance in a sample (matrix), that can be detected but not necessarily quantitated as an exact value. This can be determined in various ways. One possibility is the visual procedure, where the minimum detectable quantity is derived from samples of a known concentration (dilution series). Often the signal/noise ratio (S/N) is used. Here, the noise of a blanks is measured over a certain time and the limit of detection is established from this with S/N = 3:1. Another approach uses the standard deviation of the calibration lines ("3s-Procedure", cf. Band 3 ICH Q2B).

12.4 LOQ = Limit of Quantitation

The limit of quantitation is the smallest quantity of a substance in a sample (matrix), that can be quantified as an exact value with acceptable precision and accuracy. This can be determined in various ways. As with the limit of detection, it can be determined visually or via the signal/noise ratio. Here, the limit of quantitation is established with S/N = 10:1.

Another approach uses the standard deviation of the calibration lines ("10s Procedure", cf. Band 3 ICH Q2B).

Note: The relevant chromatograms must be added to determine the limit of detection and the limit of quantitation.

12.5 Selectivity

Selectivity is the ability of the technique to detect an analyte that is free from any interference in the presence of other components, such as by-products and degradation products, excipients (matrix) and other impurities.

During development of the technique for API characterisation, forced decomposition tests are often executed. These aim to degrade the API in order to be able to develop the technique optimally on this basis.

12.6 Linearity, Range

The aim of checking the linearity is to derive a direct proportionality between the detector signal and the concentration of a substance in the sample over a certain range. The correlation coefficient, the slope and the intercept with the confidence level must be specified. In addition, a graphical represantation must be enclosed.

12.7 Robustness

Robustness is a measure to establish that (deliberate) small changes in the parameters of the method do not influence the result. It is an indicator of the reliability of a method in normal routine use. Possible influence factors are listed in figure 14.

In practice it has been shown that these investigations are usually very time-consuming, which can lead to the temptation to do without the corresponding explanations. However, the benefit of routine use is quickly apparent and pays for itself many times over. At the latest when "another person" carries out analyses, it becomes clear where possible problems might lie. Problems caused by methods that are not robust can lead to false analytical results and, as a result, lead to extensive OOS investigations. Above all, the things to be avoided should be described in the method validation. In the current environment where methods often have to be transferred, robust methods are a requirement to be ready for use at the new site more quickly and more reliably.

13 Documentation

Before starting to validate an analytical method, a protocol must also be created. As the validation parameters can be well structured, many companies do not write project-specific validation protocols, but have created a manual for validation. This includes the principles according to which validation should be carried out.

The results of the validation can be given in the form of a report which contains the validation of all methods for a product. This corresponds to the method description (see chapter 15.C.4 Testing procedures and test protocol).

Alternatively, it may be of use to supplement the testing procedure with the validation section. This brings the advantage that the corresponding documents are always present with the method.

14 Revalidation

If changes are made to the method, partial or complete revalidation must be carried out. This includes, for example, the conversion of an assay determination from UV measurement to HPLC/UV determination. If the sample preparation of a tablet is changed from extraction/filtration to Solid Phase Extraction (SFE), this must be revalidated. Changes in equipment or modifications in manufacturing procedures are further reasons for revalidation. The method must stay abreast of the new circumstances, for current GMP demands that the methods be state-of-the-art.

This is a very ambitious objective, where benefits and costs must be weighed up. For products that have already been on the market for a long time, it is often not worth the effort. However, it is clearly the responsibility of the companies to bring only faultless goods to the market. To this end, adequate analytical methods are required. One advantage of revising methods is also to curb the historical "uncontrolled growth of methods" and to establish uniform methods for a product for the various administration forms, dosages and markets.