Validation, method interfering substances

End-point methods are often not based on kinetic-ally optimum conditions. However, an end-point method is often the only convenient one available. In this case, the method should have been validated by showing that the catalysis of the substrate follows well defined kinetics, rate of reaction is proportional to enzyme concentration, blanks and interfering substances are corrected for, and that appropriate standards are available. 

Specific bias effects, which could be large and need to be understood and minimized, should have been studied during method development, e.g. the presence or absence of a potential interfering substance, or the effect of changing a deriva-tization reaction time. The task during validation is to study residual effects and this can be done as part of ruggedness studies, as discussed in Section 4.6.5. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

Two somewhat different types of null hypotheses are tested, one during the development and validation of an analytical method and the other each time the method is used for one purpose or another. They are stated here in general form but they can be made suitably specific for experimentation and testing after review and specification of the physical, chemical and biochemical properties of the analyte, the matrix, and any probable interfering substances likely to be in the same matrix. Further, the null hypotheses of analytical chemistry are cast and tested in terms of electronic signal to noise ratios because modern analytical chemistry is overwhelmingly dependent on electronic instrument responses which are characterized by noise. 

We used a rather simple protocol in our laboratory validation studies. Future studies should include other factors such as temperature effects, the effects of potential interfering substances on recovery and actual field trials. These field trials should be monitored by an independent method in which we have a high degree of confidence. 

Determination of the extent of the effects of interfering substances and the ability of the method to measure the measurand analysis in different matrices covered by the scope of the validation... 

Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication. Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted. Method selectivity should be evaluated during method development and method validation and can continue during the analysis of actual study samples. 

A mandatory requirement when validating an LC-MS(-MS) based method according to the FDA guidelines is to evaluate and attempt to minimize the incidence of matrix effects [58], Matrix effects refer to the direct or indirect alteration or interference in response due to the presence of interfering substances in the sample [52], It can either reduce the analyte response (ion suppression) or enhance it (ion enhancement). Both can considerably compromise the accuracy of quantification and ion suppression may in the worst case even lead to decrease sensitivity and to false negative results. 

Which is better, GC-MS or immunoassay This is a question often asked about plant hormone quantification. GC-MS, which is now more widely available since the Introduction of bench-top instruments, has the advantage that it not only provides quantification of the hormone by the isotope dilution method, but also confirms the identity of the compound concerned by comparison of its spectrum with that of a standard. However, once validated for a particular tissue, immunoassay has the advantage that many samples can be analysed very quickly. Both techniques require sample pre-purification, often by the same methods. A more recent development is a powerful combination of the two technologies which uses the antibody immobilised on a polymer support as a method of affinity-purifying the hormones (together with interfering substances) from plant extracts prior to analysis by GC-MS. Immunoaffinity chromatography is discussed in the next section. 

It is not possible to test for all potential interferences, and there is a possibility that an interfering substance will subsequently be encountered in analysis of field samples from diverse sources. Detection using tandem mass spectrometers, with multiple- or selected-reaction monitoring (MRM or SRM see Chapters 6 and 7), reduces the probability of encountering unexpected interferences, but does not totally eliminate the possibility. When interference is discovered once a validated method has been put into routine use, it may be necessary to modify the method (clean-up or chromatography) to eliminate the interference. Such modifications, if they involve significant change, may require re-validation of the method. 

Latner (1948) described in a preliminary communication the application of the diazo reaction after the treatment of ultrafiltrates of blood or plasma with strong alkali. Touster (1951) made use of the reaction of ergothioneine with bromine water to form inorganic sulfate, which was determined colorimetrically. Quantitative recoveries were not obtained. Ohara et al. (1952) determined the amount of sulfate produced from blood and tissues by oxidation with alkaline peroxide. Jocelyn (1958) has determined free and bound ergothioneine in blood and proteins by a method based on the estimation of trimethylamine released by heating the material with strong alkali. The validity of results obtained with these methods cannot be assessed at this time since the absence of interfering substances has not, in the reviewer s opinion, been adequately demonstrated. 

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