Summary and Conclusions

Accuracy
Precision
Specificity
Limit of Detection
Limit of Quantitation
Linearity and Range
Ruggedness
Robustness

Method validation is completed to ensure that an analytical methodology is accurate, specific, reproducible and rugged over the specified range that an analyte will be analyzed. Method validation provides an assurance of reliability during normal use, and is sometime referred to as "the process of providing documented evidence that the method does what it is intended to do." Regulated laboratories must perform method validation in order to be in compliance with FDA regulations. In a 1987 guideline (Guideline For Submitting Samples And Analytical Data For Methods Validation), the FDA designated the specifications in the current edition of the United States Pharmacopoeia (USP) as those legally recognized when determining compliance with the Federal Food, Drug, and Cosmetic Act.

For method validation, these specifications are listed in USP Chapter <1225>, and can be referred to as the "Eight Steps of Method Validation," as shown in Figure 3:

Figure 3: The USP Eight Steps of Method Validation

These terms are referred to as "analytical performance parameters", or sometimes as "analytical figures of merit." Most of these terms are familiar and are used daily in the laboratory. However some may mean different things to different people. Therefore, in order to continue the discussion of method validation, it is necessary to have a complete understanding of the terminology and definitions.

In response to this situation, one of the first harmonization projects taken up by the ICH was the development of a guideline on the "Validation of Analytical Methods: Definitions and Terminology." ICH divided the "validation characteristics" somewhat differently, as outlined in Figure 4:

Figure 4: ICH Method Validation Parameters

The differences in the USP and ICH terminology is for the most part one of semantics, however, with one notable exception. ICH treats system suitability as a part of method validation, whereas the USP treats it in a separate chapter (<621>). Since this guideline has reached step 5 of the ICH process, the FDA has begun to implement it, and it is anticipated that the ICH definitions and terminology will eventually be published in the USP. What follows then is a discussion of current USP definitions of the analytical performance parameters, compared and contrasted to the ICH definitions. Where appropriate, methodology is also presented according to the ICH guideline on this subject.

Accuracy

Accuracy is the measure of exactness of an analytical method, or the closeness of agreement between the value which is accepted either as a conventional, true value or an accepted reference value and the value found. It is measured as the percent of analyte recovered by assay, by spiking samples in a blind study. For the assay of the drug substance, accuracy measurements are obtained by comparison of the results with the analysis of a standard reference material, or by comparison to a second, well-characterized method. For the assay of the drug product, accuracy is evaluated by analyzing synthetic mixtures spiked with known quantities of components. For the quantitation of impurities, accuracy is determined by analyzing samples (drug substance or drug product) spiked with known amounts of impurities. (If impurities are not available, see specificity.)

To document accuracy the ICH guideline on methodology recommends collecting data from a minimum of nine determinations over a minimum of three concentration levels covering the specified range (for example, three concentrations, three replicates each).

The data should be reported as the percent recovery of the known, added amount, or as the difference between the mean and true value with confidence intervals.

Accuracy can be documented through the use of control charts, an example of which is shown in Figure 5.

Figure 5: Documenting Accuracy with Waters Millennium Chromatography Manager Control Charts

Precision

Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the percent relative standard deviation for a statistically significant number of samples. According to the ICH, precision should be performed at three different levels: repeatability, intermediate precision, and reproducibility. Repeatability is the results of the method operating over a short time interval under the same conditions (inter-assay precision). It should be determined from a minimum of nine determinations covering the specified range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration. Intermediate precision is the results from within lab variations due to random events such as different days, analysts, equipment, etc. In determining intermediate precision, experimental design should be employed so that the effects (if any) of the individual variables can be monitored.

Figure 6: Documenting Precision With Waters® Millennium Chromatography Manager Summary Graphics Bar Plot Custom Report

Reproducibility refers to the results of collaborative studies between laboratories. Documentation in support of precision studies should include the standard deviation, relative standard deviation, coefficient of variation, and the confidence interval. Figure 6 illustrates how custom graphics in the form of bar charts can be used to document precision.

Specificity

Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix. It is a measure of the degree of interference from such things as other active ingredients, excipients, impurities, and degradation products, ensuring that a peak response is due to a single component only. i.e. that no co-elutions exist. Specificity is measured and documented in a separation by the resolution, plate count (efficiency), and tailing factor. Specificity can also be evaluated with modern photodiode array detectors that compare spectra collected across a peak mathematically as an indication of peak homogeneity. ICH also uses the term specificity, and divides it into two separate categories: identification, and assay/impurity tests.

For identification purposes, specificity is demonstrated by the ability to discriminate between compounds of closely related structures, or by comparison to known reference materials. For assay and impurity tests, specificity is demonstrated by the resolution of the two closest eluting compounds. These compounds are usually the major component or active ingredient and an impurity. If impurities are available, it must be demonstrated that the assay is unaffected by the presence of spikedmaterials (impurities and/or excipients). If the impurities are not available, the test results are compared to a second well-characterized procedure. For assay, the two results are compared. For impurity tests, the impurity profiles are compared head-to-head.

Limit of Detection

The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantitated. It is a limit test that specifies whether or not an analyte is above or below a certain value. It is expressed as a concentration at a specified signal-to-noise ratio, usually two- or three-to-one. The ICH has recognized the signal-to-noise ratio convention, but also lists two other options to determine LOD: visual non-instrumental methods and a means of calculating the LOD. Visual non-instrumental methods may include LOD’s determined by techniques such as thin layer chromatography (TLC) or titrations. LOD’s may also be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula: LOD = 3.3(SD/S). The standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines. The method used to determine LOD should be documented and supported, and an appropriate number of samples should be analyzed at the limit to validate the level.

Limit of Quantitation

The Limit of Quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. Like LOD, LOQ is expressed as a concentration, with the precision and accuracy of the measurement also reported. Sometimes a signal-to-noise ratio of ten-to-one is used to determine LOQ. This signal-to-noise ratio is a good rule of thumb, but it should be remembered that the determination of LOQ is a compromise between the concentration and the required precision and accuracy. That is, as the LOQ concentration level decreases, the precision increases. If better precision is required, a higher concentration must be reported for LOQ. This compromise is dictated by the analytical method and its intended use. The ICH has recognized the ten-to-one signal-to-noise ratio as typical, and also, like LOD, lists the same two additional options that can be used to determine LOQ, visual non-instrumental methods and a means of calculating the LOQ. The calculation method is again based on the standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula: LOQ = 10(SD/S). Again, the standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines.

Like LOD, the method used to determine LOQ should be documented and supported, and an appropriate number of samples should be analyzed at the limit to validate the level. One additional detail should also be considered; both the LOQ and the LOD can be affected by the chromatography. Figure 7 shows how efficiency and peak shape can affect the signal-to-noise ratio.

Figure 7: Effect of Peak Shape on LOD/LOQ

Sharper peaks result in a higher signal-to-noise ratio, resulting in lower LOQs and LODs. Therefore, the chromatographic determination of LOQ/LOD should take into account both the type and age of the column, which is usually determined over the course of time as experience with the method grows.

Linearity and Range

Linearity is the ability of the method to elicit test results that are directly proportional to analyte concentration within a given range. Linearity is generally reported as the variance of the slope of the regression line. Range is the interval between the upper and lower levels of analyte (inclusive) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as the test results obtained by the method. The ICH guidelines specify a minimum of five concentration levels, along with certain minimum specified ranges. For assay, the minimum specified range is from 80-120% of the target concentration. For an impurity test, the minimum range is from the reporting level of each impurity, to 120% of the specification. (For toxic or more potent impurities, the range should be commensurate with the controlled level.)

For content uniformity testing, the minimum range is from 70-130% of the test or target concentration, and for dissolution testing, +/- 20% over the specified range of the test. That is, in the case of an extended release product dissolution test, with a Q-factor of 20% dissolved after six hours, and 80% dissolved after 24 hours, the range would be 0-100%. Figure 8 is an example of a linearity (calibration) plot.

Figure 8: Waters Millennium Chromatography Manager Calibration Plot

Ruggedness

Ruggedness, according to the USP, is the degree of reproducibility of the results obtained under a variety of conditions, expressed as %RSD. These conditions include different laboratories, analysts, instruments, reagents, days, etc. In the guideline on definitions and terminology, the ICH did not address ruggedness specifically. This apparent omission is really a matter of semantics, however, as ICH chose instead to cover the topic of ruggedness as part of precision, as discussed previously.

Robustness

Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters. The robustness of a method is evaluated by varying method parameters such as percent organic, pH, ionic strength, temperature, etc., and determining the effect (if any) on the results of the method. As documented in the ICH guidelines, robustness should be considered early in the development of a method. In addition, if the results of a method or other measurements are susceptible to variations in method parameters, these parameters should be adequately controlled and a precautionary statement included in the method documentation.