Analyte Stability during Sample Processing Analysis

6 Analyte Stability during Sample Processing (Analysis) 

It must be established that the analyte remains stable throughout the analysis. There have been some reports in the literature of conversion of the analytes to apparent metabolites or degradation products, such as in the case of tetracyclines, and this can certainly confuse the interpretation of the analytical results. This has led some jurisdictions to amend MRLs to take account of this phenomenon. For example, in the EU, the MRL for the various tetracyclines is expressed as the sum of the parent tetracycline and the 4-epimer of the veterinary drug.  

The pure standard in solution should be taken through the complete analysis once the method has been developed. If this experiment demonstrates significant loss of analyte, the experiment should be repeated for each step in the analysis to determine where loss of analyte occurs, and this step should be modified to eliminate the loss. The cause of the loss or degradation of the analyte should be determined to ensure that similar steps are not included in future methods involving the analyte. Potential causes of degradation or ioss include poor solubility or instability in a solvent, instability or lack of solubility under certain conditions of pH, interactions with a chromatographic support material, and adsorption onto glass or other contact materials. 

When information on the stability of analyte in matrix during typical conditions of storage is unavailable from the literature or from proficiency testing (PT) providers, such as the food analysis proficiency assessment scheme (PAPAS), this information shouid be developed in the laboratory as part of the method development and validation. However, a study to obtain information on stability of the analyte first requires the availability of a validated method if results are to be considered reliable. When a method is being developed to introduce a test capability for a new analyte into the laboratory, the new 

---

The method should be assessed using the same or similar calibration standards, QCs and blanks that will be used for validation. In addition to a final assessment of sensitivity, linearity, accuracy and precision, additional experiments designed to assess analyte stability during the sample preparation and analysis processes, method robustness and ruggedness, carryover, and potential... 

The major quality parameters to be addressed during sample preparation are listed in Table 1.4. These are accuracy, precision, extraction efficiency (or recovery), and contamination control. These quality issues also need to be addressed during the analysis that follows sample preparation. Accuracy is determined by the analysis of evaluation samples. Samples of known concentrations are analyzed to demonstrate that quantitative results are close to the true value. The precision is measured by running replicates. When many samples are to be analyzed, the precision needs to be checked periodically to ensure the stability of the process. Contamination is a serious issue, especially in trace measurements such as environmental analysis. The running of various blanks ensures that contamination has not occurred at any step, or that if it has, where it occurred. As mentioned before, the detection limits, sensitivity, and other important parameters depend on the recovery. The efficiency of sample preparation steps such as extraction and cleanup must be checked to ensure that the analytes are being recovered from the sample. 

The stability of the reference substance in a stock solution made from a particular solvent and stored nnder a given set of conditions should be established prior to use (Section 10.2.7). Method development studies can commence prior to establishing known stability but the analyst must be aware that the analyte could be degrading in solution (or during all phases of sample processing) and take that into consideration especially when unusual or unexpected results are obtained during development. In no instance should solutions without established analyte stabihty be used for sample analysis. Also, additional stability data should be generated for any other solutions that are prepared in a different solvent and/or stored under... 

The validation process begun in Phase I is extended during Phase II. In this phase, selectivity is investigated using various batches of drugs, available impurities, excipients, and samples from stability studies. Accuracy should be determined using at least three levels of concentration, and the intermediate precision and the quantitation limit should be tested. For quality assurance evaluation of the analysis results, control charts can be used, such as the Shewart-charts, the R-charts, or the Cusum-charts. In this phase, the analytical method is refined for routine use. 

Postpreparative Stability (Autosampler Stability) Stability of processed samples should be determined at the autosampler temperature that will be used during study sample analysis and for a duration of time equal to or greater than the batch size expected to be used for routine study sample analyses. If sample extracts are to be stored (e.g., in a refrigerator or freezer) before analysis, then the stability of the analyte in the reconstituted sample extract under these conditions should be investigated. In postpreparative stability assessment, the mean concentration value obtained from each QC concentration level after storage in autosampler is compared with the respective nominal concentration value. The mean bias (%) between the values should be within +15%. If the stability is demonstrated, then an LC-MS/MS run can be restarted after an unexpected interruption as long as system suitability has been established and documented. 

---