Method ruggedness

One of the key aspects in developing a method for regulatory analysis is method ruggedness. The more rugged a method, the less susceptible it is to failure or to excessive variations due to differences in equipment, analyst technique, and other differences that are typically present among laboratories. Several factors contribute to poor method ruggedness insufficient testing by the developer, excessive method complexity, and a failure of the developer to identify and communicate critical points. 

Some other sources have definitions that are different from the one given above [7,14]. The US Pharmacopeia [7] defines ruggedness as The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same sample under a variety of normal test conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, 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 fact this is nearly the definition of reproducibility. This definition is also followed by other authors [15]. 

If the sample preparation is simple (i.e. does not involve extensive shaking, heating or ultrasonification and does not require more than one dilution step or a derivatisation or extraction stage) then it is not usual to test the ruggedness of the sample preparation unless the method is for a strictly controlled regulatory environment. However, if a derivatisation or extraction is performed then it makes sense to test factors from these procedures as they are likely to effect the method ruggedness. Table 5.2 shows some examples of factors that can effect the ruggedness of the sample preparation procedure. 

The standard error reflects the statistical relevance of all the main effects (i.e. a main effect with a smaller value than the standard error is not statistically relevant). In this instance a main effect must be larger than -0.7 to be considered a real effect and not just a reflection of the overall precision of the method. The results for each factor are given in Table 5.18. The largest effect is around 3% and is due to the change in the acid type used to control the pH of the mobile phase. All observed effects were unlikely to cause a lack of method ruggedness as no effect caused a critical reduction in the plate count. 

It is imperative to also consult the vendor to determine an adequate number of valves for spray weight testing. If the metered chamber is plastic, valves totaling at least twice the number of the vendor s mold impressions should be tested to guarantee complete evaluation of the lot of valves. The spray weight methodology conducted on valves can drastically influence the results. Because the valve is a mechanical device, the way in which it is actuated is technique-oriented. Manual actuation versus automatic actuation can cause variation in the results. Method ruggedness is essential in evaluation of the valve performance. 

Sample Preparation/Extraction The process of separating potentially interfering components from a sample prior to LC-MS analysis for the purposes of improving sensitivity, specificity, and/or method ruggedness. Variations include solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation (PPT). Extraction may be performed off-line, in which the cleanup is completely independent from the LC-MS analysis, or on-line, in which the cleanup is integrated directly into the LC-MS analysis. 

Reproducibility encompasses the variation in analytical results between laboratories and provides a second level of method ruggedness. This is becoming an increasingly important part of method validation as the pharmaceutical industry becomes more specialized and diversified. Major manufacturers may develop and validate a method in a corporate research center for use in a foreign manufacturing site or at a contract testing laboratory. It is therefore critical that the validation demonstrates that the method is free of analyst or instrument bias. 

It is generally expected that an analytical method will perform in an acceptable manner each time it is used. While a consideration of method ruggedness is a necessary part of any method s validation, it is a critical issue for compendial methods because of their widespread use in many different laboratories. 

Method development comes into play when an established standard method does not exist for a particular sample constituent. In this case, a set of experimental steps for measuring a known amount of a given constituent in various matrices is developed. For the new method to become accepted by the scientific community, it must be validated. Validation consists of three steps (1) determination of the MDL, (2) analysis of independently prepared standards and (3) determination of the stability of the result produced when steps comprising the new method are varied (method ruggedness). New methods passing these three tests are subjected to collaborative testing prior to becoming a standard method. 

Establishment of ofher controls or restrictions to improve method ruggedness or robustness. Examples of fhis include identification of specific equipment and data processing methods needed to ensure appropriate method performance. 

Borman P, Chatfield MJ, Damjanov I, Jackson P. Method ruggedness studies incorporating a risk based approach a tutorial. Anal Chim Acta 2011 703 101-13. 

The use of LC or GC coupled with MS is the preferred approach given its inherent specificity, sensitivity, fast method development, and broad applicability to many GTIs. LC/MS/MS is a useful technique especially in cases where additional sensitivity or specificity is needed. Fluorescence may also prove to be a highly sensitive and selective detector for some GTIs. Other sensitive detectors may also be useful including UV, electrochemical, or FID for GC. In general, one should use the technique that is most suitable for the GTI of interest that provides the required selectivity, sensitivity, and method ruggedness and robustness. 

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