REGULATORY CONSIDERATIONS FOR THE CHROMATOGRAPHER

After the method file and the sequence file have been set up, the analytical run is started and data are collected. A data file containing the A/D data slices will be obtained for each chromatographic run and sample injected. It is important from scientific and regulatory considerations that the data files must not be capable of alteration. 

Later, Lawrence and Niedzwiadek [37] modified the method to improve its performance (repeatability, ruggedness, recovery) for routine regulatory purposes. Earlier chromatographic conditions (gradient and flow) were changed to reduce the analysis time. The oxidation reaction conditions were modified to achieve the best compromise possible for all the PSP toxins, since it has been shown that the optimum pH for the oxidation of individual toxins varies considerably [38]. A matrix modifier (blank oyster extract) was added to improve the yield and repeatability for the periodate derivatization of standards and sample extracts. Finally, the performance of the second cleanup with SPE-COOH cartridges, which enables the separation of the sulfocarbamoyl toxins from their carbamoyl counterparts, was reevaluated. 

The enzymes of the common aromatic pathway, other than DAHPS, show relatively little response to repression control in S. typhimurhm [76] and E. coli [108,110] under most test conditions. However, considerable variation in the level of the shikimate kinase of E. coli has been reported [3] for aromatic auxotrophs under certain growth conditions. The possibility that this enzyme may play an important regulatory role is also suggested by the evidence for two chromatographically separable shikimate kinase enzymes in S. typhimurium [119]. The presence of two isoenzymes of shikimate kinase in this organism would explain the apparent lack of occurrence of strains with mutational defects in this enzyme. 

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