Ruggedness and robustness

There is a certain lack of uniformity and certainly a degree of confusion in the literature and in the various compendia and regulatory guidelines regarding the definitions of ruggedness and robustness [10, 35, 36, 39, 40], Whereas in some circles the ruggedness and robustness are synonymous, the USP defines ruggedness [35] as "the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions, such as different laboratories, different analysts, different instruments, different reagent lots, different elapsed assay times, different assay temperatures, different days, etc. Ruggedness is normally expressed as the lack of influence on test results of operational and environmental variables of the analytical method. Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst". In the USP the robustness of an analytical procedure is defined as "a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability in normal usage".

log x

Figure 2.23

Sensitivities (dyi/dxi) for non-linear (upper) and linear (lower) response functions x

Figure 2.23

Sensitivities (dyi/dxi) for non-linear (upper) and linear (lower) response functions

The USP goes on to indicate that assay ruggedness should be determined by "analysis of aliquots of homogenous lots in different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within the specified parameters of the assay. The degree of reproducibility of test results is then determined as a function of the assay variables. This reproducibility may be compared to the precision of the assay under normal conditions to obtain a measure of ruggedness of the analytical method".

The detailed definitions of ruggedness and robustness described in the USP are in contrast to the Guidelines on Validation of Analytical Procedures for Pharmaceuticals published in 1994 by the International Conference on Harmonization (ICH) [41], which defines the robustness of an assay "as a measure of its capacity to remain unaffected by small, deliberate variations in method parameters and provides an indication of reliability during normal usage". No specific mention is made in the ICH Guidelines to assay ruggedness, which is alluded to in the definitions of precision: "Repeatability expresses the precision under the same operating conditions over a short period of time. Repeatability is also termed inter-assay precision. Intermediate precision expresses within laboratory variations: different days, different analysts, different equipment etc. Reproducibility expresses the precision between laboratories (collaborative studies)".

Notwithstanding the present ambiguity of definitions, it is convenient to apply the term assay ruggedness to the variation or errors in assay results arising from different operation conditions and to apply the term assay robustness to the ease with which the critical parameters of the assay may be reproduced. It also follows that the ruggedness of an assay is influenced by its robustness. For example, an LC assay demonstrating very good precision in one laboratory might yield very poor precision in a second laboratory if the separation obtained by the analyst in the second laboratory gave very tailed peaks. Such examples of the robustness in LC assays are commonplace because of the number of environmental and operational factors that can affect the reproducibility of the desired separation.

Degradation of chromatography columns is a common cause of assay deterioration and lack of robustness [40], Thus, any chromatographic method should include a description of column washing and storage conditions as well as the inclusion of appropriate system suitability tests (SSTs) for the determination of column performance. In addition, the biggest cause of poor inter-laboratory reproducibility of chromatographic separations is large batch-to-batch variability of chromatography columns. An additional problem is the difference in the chromatographic performance of stationary phase purported to have the same chemical composition [40], Thus during the method development the influence of different columns, even from the same manufacturer, should be examined and the effects clearly described in any published report.

Other factors that will affect the reproducibility of separations include the effects of mobile phase composition and temperature. The need to document the sensitivity of an assay to small changes in environmental and operational conditions is particularly important if that method is submitted in support of a New Drug Application (NDA). In 1986, Sheinin [40] described examples of problems encountered by scientists at the FDA in attempts to reproduce LC methods submitted in support of various NDAs. These problems included:

• Inadequate column specifications - separation could not be reproduced using column specified

• Variations in the mobile phase - separation was very sensitive to small changes in mobile phase composition (organic modifier)

• Inadequate method development - separation on column described in method was poor, other columns tried by FDA laboratories were more suitable

• Column deterioration - resolution of critical peak pairs degraded within a few hours after initiation of assay

• Lack of samples - authenticated samples of related compounds not supplied

The degree of ruggedness and robustness of a particular analytical method will depend largely on the intended application. For example, the degree of ruggedness and robustness needed for an assay of very limited application to study a new chemical entity in the very early stages of development is much less than would be required to control the quality of a marketed product being manufactured at several different locations throughout the world. In the first case it may only be necessary to examine the effects of changing the chromatography column and making small changes in mobile phase composition on the resolution of the critical pair of peaks in the chromatogram. In the second case an extensive muti-laboratory collaborative study would be required.

The Canadian Guidelines on assay validation [42] define three levels of ruggedness testing depending on the anticipated future application of a new method (Table 2.2). However, it should be noted that the Canadian Guidelines do not distinguish between the ruggedness and the robustness of analytical methods. As well as defining the types of test to be conducted, the description of ruggedness testing in the Canadian Guidelines indicate that the means obtained in inter-analyst or inter-laboratory studies should be within 1% and 2% for raw materials and finished formulations, respectively. However, the acceptance criteria described in the Canadian Guidelines do not consider the precision of the methods used and an alternative approach is to set the acceptance criteria for the qualification of additional laboratories using an appropriate statistical approach such as that described by Westlake [43].

In 1977, Horwitz [44-46] showed that for well-behaved analytical methods the between-laboratory RSD is approximately related to the fraction of analyte in the sample (fa) (Fig. 2.24) by the empirical expression:

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