Analytical method validation interference studies

Untreated (control) soil is collected to determine the presence of substances that may interfere with the measurement of target analytes. Control soil is also necessary for analytical recovery determinations made using laboratory-fortified samples. Thus, basic field study design divides the test area into one or more treated plots and an untreated control plot. Unlike the treated plots, the untreated control is typically not replicated but must be sufficiently large to provide soil for characterization, analytical method validation, and quality control. To prevent spray drift on to the control area and other potential forms of contamination, the control area is positioned > 15 m away and upwind of the treated plot, relative to prevailing wind patterns. 

With respect to method application, once validation has been satisfactorily completed, there is little question that use of the analytical method in worker safety and re-entry studies falls under the full requirements of the GLP Standards. In addition, there should be an adequate level of quality control measurements taken in conjunction with the specimens so as to provide for a meaningful assessment of accuracy and precision, as well as verification of freedom from artifactual interferences. Along with these measurements there needs to be reasonably rigid data acceptance criteria in place (usually established during validation) which are consistently applied during the course of the specimen analytical phase of the study. 

The stress studies should demonstrate that impurities and degradants from the active ingredient do not interfere with the quantitation of the API [12]. Stress testing of the API, in addition to validating the stability-indicating power of the analytical method, can also help establish the degradation pathways and the intrinsic stability of the molecule [7]. 

How analytical methods deal with interferences is one of the more ad hoc aspects of method validation. There is a variety of approaches to studying interference, from adding arbitrary amounts of a single interferent in the absence of the analyte to establish the response of the instrument to that species, to multivariate methods in which several interferents are added in a statistical protocol to reveal both main and interaction effects. The first question that needs to be answered is to what extent interferences are expected and how likely they are to affect the measurement. In testing blood for glucose by an enzyme electrode, other electroactive species that may be present are ascorbic acid (vitamin C), uric acid, and paracetamol (if this drug has been taken). However, electroactive metals (e.g., copper and silver) are unlikely to be present in blood in great quantities. Potentiometric membrane electrode sensors (ion selective electrodes), of which the pH electrode is the... 

A new requirement of the final CLIA regulations is that laboratories must perform method validation studies oh all new tests introduced after April 24, 2003. Before this, laboratories that implemented new methods and analytical systems that had been cleared by the Food and Drug Administration (FDA) could simply follow manufacturers directions for operation and assume that the manufacturer s performance claims were valid. With the issuance of the final rule, the performance of all new tests must be validated in each laboratory to document the reportable range, precision, accuracy, and reference intervals. For some methods, it may also be necessary to determine the detection limit and to test for possible interferences. 

To avoid bias in the evaluation of the actual precision and accuracy of the bioanalytical method, the results of all QC samples assayed within accepted analytical runs should be reported and taken into consideration in the descriptive statistical analysis. Exclusion of values should be considered only in the case of a documented analytical problem (e.g. chromatographic interference) and the reason for the exclusion should be reported. This applies to both the pre-study validation of the method and the study phase itself. 

It should be mentioned that, at first, most of the analytical methods desalbed previously in Section 3.1 would be valid to perform photostability studies, provided that the forming byproducts do not interfere in the determination of the parent UV filters. 

Validate routine methods, i.e., define the conditions under which the assay results are meaningful.115 To do that, one must select samples that are truly representative of the product stream. This may be a difficult task when the process is still under development and the product stream variable. The linearity of detector response should be defined over a range much broader than that expected to be encountered. Interference from the sample matrix and bias from analyte loss in preparation or separation often can be inferred from studies of linearity. Explicit detection or quantitation limits should be established. The precision (run-to-run repeatability) and accuracy (comparison with known standards) can be estimated with standards. Sample stability should be explored and storage conditions defined. 

HPLC with column switching and mass spectrometry was applied to the online determination and resolution of the enantiomers of donepezil HC1 in plasma [38]. This system employs two avidin columns and fast atom bombardment-mass spectrometry (FAB-MS). A plasma sample was injected directly into an avidin trapping column (10 mm x 4.0 mm i.d.). The plasma protein was washed out from the trapping column immediately while donepezil HC1 was retained. After the column-switching procedure, donepezil HC1 was separated enantioselectivity in an avidin analytical column. The separated donepezil HC1 enantiomers were specifically detected by FAB-MS without interference from metabolites of donepezil HC1 and plasma constituents. The limit of quantification for each enantiomer of donepezil HC1 in plasma was 1.0 ng/ml and the intra-and inter-assay RSDs for the method were less than 5.2%. The assay was validated for enantioselective pharmacokinetic studies in the dog. 

Accuracy is often determined by recovery studies in which the analytes are spiked into a solution containing the matrix. The matrix (placebo in formulations) should be found not to interfere with the assay of the compound(s) of interest. For stability-indicating HPLC methods, it is necessary to determine the accuracy of the active ingredient and that of all related compounds. It is possible to determine the accuracy of each related compound separately, but it is more efficient to validate these related compounds in a combined spiked mixture of all the related compounds at their appropriate levels. The analyst should be certain that the impurity standards used to spike the solutions are pure and do not contain significant impurities s that would effect the results. 

Nonspecific nonspecificity results from interference of matrix components that are structurally unrelated to the analyte of interest. Examples of such interfering matrix components would include serum proteins, lipids, heterophilic antibodies, rheumatoid factor, proteases, and so on. Nonspecific nonspecificity is often referred to as matrix effect. Figure 4.3 depicts the impact of matrix on the assay performance. Matrix interference is one of the chief reasons that LB As often require more method development and validation prior to switching from one species matrix to another or even within the same species. In addition, we recommend during clinical study support that matrix from the relevant disease populations be tested for matrix effects as soon as that matrix becomes available. Matrix effects should be evaluated by comparing the concentration response relationship of both spiked and unspiked samples of the biological matrix (recommendation is 10 or more lots of individual sources) to a comparable buffer solution. It is recommended that the spiked sample... 

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