Sample results, troubleshooting

Immunoassay troubleshooting is a sequential, cumulative process whereby one first looks at the calibration curve, then QC s, and finally the sample results. If the curve is not acceptable, the assay fails. If the curve is acceptable, then one reviews QC performance if QC performance is unacceptable, the assay fails. If QCs are acceptable, then one examines individual sample results. If the assay fails as the result of an apparent technical error, the error is corrected and the assay is repeated. If the cause for the failure is not apparent or the assay has failed consistently, then a troubleshooting process should be initiated prior to further analysis (and depletion) of study samples. A summary of the calibrator/QC/sample result troubleshooting process is depicted in Fig. 3.14. 

Stratified sampling is more likely to reveal uniformity problems than if less discriminating methods were used. A tool was developed and published (84) to aid formulation and process development scientists in troubleshooting content uniformity problems. The approach is that if stratified sample results were available for blend and/or product samples, the data will point the troubleshooter towards potential causes for uniformity problems in rank order of likelihood. Once potential root causes have been identified, areas for further investigation and possible corrective actions are also presented. 

Validation provides confidence in the quality of products manufactured as the over quality of a particular process cannot be established due to the limited sample size. Validation leads to less troubleshooting within routine production. As a result, it reduces the number of customer complaints and drug recalls. 

If the pipettor does not deliver exactly 100 pi (check by weighing an aliquot of water see Critical Parameters and Troubleshooting), it should not affect the final result since the same amount will be used in all three portions. The same micropipettor must be used for the glucose standard and samples. Do not use oversized tips (e.g., 500 or 1000 pi), since this leads to increased error. Precise pipetting is critical for accuracy of results (see Critical Parameters and Troubleshooting). 

These examples are used for illustration purposes only, and we do not mean to imply that the explanations given will always fit scenarios you will see in your own processes. Certainly, as stated earlier, process changes could have resulted ill the time plots shown. However, the point we wish to make emphatically is that sampling should be on any checklist for troubleshooting process problems and should be taken seriously as a possible cause for problems. 

The most common cause of extra peaks in the chromatogram is the sample itself. The source of the sample may be different, it may have decomposed or aged otherwise. It should be easy to verify whether a particular sample is the source of the extra peak by comparison with samples from other sources or the injection of standards or blanks. If the injection of standards or blanks results in extra peaks, we should next troubleshoot the injector and the mobile phase. 

While one is engaged in troubleshooting, it is necessary to consider the source of the problem and to examine how the problem might have arisen. Additional problems might arise from the sample composition and the mobile phase, and these also need to be considered. It should be noted that problems can present themselves in many different ways, and what might appear to be a pump-related problem can quite easily be a result of something else entirely. 

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