Incurred sample re-analysis

The most recent bioanalytical Workshop Report (Viswanathan 2007) devotes considerable space to this topic and some recommendations not discussed previously (Section 9.4.7b) are included below. There should be some assessment of both reproducibility and accuracy of the reported concentration. Sufficient data should be generated to demonstrate that the current matrix (i.e. the incurred sample matrix) produces results similar to those previously validated. It is recognized that accuracy of the result generated from incurred samples can be more difficult to assess. It requires evaluation of any additional factors besides reproducibility upon storage, which could perturb the reported concentration. These could include metabolites converted to parent during sample preparation or LC-MS/MS analysis, matrix effects from high concentrations of metabolites, or variable recovery between analyte and internal standard (Viswanathan 2007). Most of these phenomena are those described previously (Jemal 2002) and discussed in Section 9.4.7b. 

Further implications of differences between incurred bioanalytical samples and calibrators and QC samples prepared in accord with accepted practice (FDA 2001) have been discussed (Gallicano 2006). It was emphasized that if the imprecision of data obtained for incurred samples differs from that of spiked samples used in the original validation, this will lead to questioning the estimates of accuracy, LLOQ and stability for the incurred samples. If the incurred sample data are of low precision it will be necessary to analyze more samples to investigate these parameters. It was proposed (Gallicano 2006) that an LLOQ higher than the lowest standard concentration (LSC) should be considered if the incurred samples show more imprecision than spiked samples, because the main criteria for selection of the LSC is its precision on the basis of spiked samples . 

It has also been reported (Gallicano 2007) that matrix effects on a structural analog SIS standard have been observed in incurred samples that were not apparent in spiked samples. It was suggested that the % CV of SIS peak response should be reported for each batch of study samples. If a variable response is observed, the question arises as to whether this is the result of random variability, or the result of spiked and incurred sets of samples 

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As previously discussed (Section 9.4.7b), extracted control matrix is used to assess the selectivity for the method under development. The selectivity of the method is a function of the sample preparation (extraction and clean-up), chromatography and mass spectrometry conditions that are used for the method. Assuming that the control matrix is representative of the sample matrix to be analyzed, and that method blanks have been used to demonstrate that the method is free of exogenous interferences due to solvents, or to containers or other apparatus (a source of interferences that is often overlooked in the method development process), an extracted blank is used to demonstrate that the method has sufficient selectivity for the intended analytical purpose. When interferences at the expected retention time of the analyte being quantified are detected, modifications to the sample preparation and chromatography (and sometimes the ions monitored hy the method) can be made to improve the selectivity of the method. Recall (Section 9.4.7b) that only re-analysis of incurred samples can reveal interferences resulting from metahoUtes or degradates of the analyte with either or both of the analyte and SIS. 

A related problem in biomedical analyses (possibly with analogies in other applications) arises from the presence of metabolites of the analyte in incurred analytical samples, but not in the control matrix spiked with analytical and internal standards used in method development and validation. Problems arising from this distinction are most likely to be important when rate of sample throughput is emphasized at the expense of chromatographic efficiency (Section 9.3.4) and can be addressed by re-analysis of incurred samples as described in Section 9.4.7b. The discussion in the present section is concerned with matrix effects during method development, but these must also be addressed in the context of method validation (Section 10.4.1d). 

Another aspect that is common to matrix interferences (direct contrihutions of matrix components to the signal measured for analyte and/or SIS) and matrix effects (suppression or enhancement of ionization) is that of the consequences of the presence of metabolites or other types of degradates when analyzing incurred analytical samples. Such interferences are in principle absent from the control matrix used for matrix matched calibrators, QC samples etc. Thus use of re-analysis of incurred samples to evaluate and consequent matrix effects was discussed in Section 9.4.7b, and applies equally to matrix interferences arising from presence of metabolites. Variations of metabolite levels among samples (e.g. from different time points in a pharmacokinetic study), which can lead to parallel variations in the extent of both matrix effects and matrix interferences, are an example of how some problems can arise unexpectedly despite prior precantions. 

The previous assay was applied to the analysis of SDZ in salmon tissue, with some modifications. The re-extraction with phosphoric acid solution was replaced by SPE on an SCX cartridge preconditioned with MeCN and phosphoric acid solution. The cartridge was washed with MeCN, and SDZ was eluted with an MeCN phosphoric acid mixture. The eluate was injected directly into the chromatographic system, followed by postcolumn derivatization under similar conditions to the previous assay. The derivatization time was 1.2 min, and the fluorescence intensity was approximately a quarter of that for optimal conditions. However, the postcolumn derivatization was found to be considerably less labor intensive and was easily reproducible (recoveries 83-85% CV < 7%). A significant improvement in the LOD value was obtained (0.2 yug/kg) (160). The SDZ residues from incurred salmon tissue were confirmed by MS detection however, the sample cleanup should be improved due to the lack of sensitivity of MS. Therefore, SDZ residues were eluted from the SCX SPE cartridge with phosphoric acid, and the eluate was concentrated on a C18 SPE cartridge preconditioned with MeOH and water. The residues were eluted with MeCN, and the eluate was evaporated to dryness and reconstituted prior to the analysis. The column effluent was delivered into the atmospheric-pressure ion source, and SIM was chosen for positive ions at m/z 251, 158, 156, and 108, respectively (161). 

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