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Summary.A simple and convenient chromatographic method of simultaneous separation, identification, and quantitative determination of thimerosal (TM) (preservative) and aluminum (adjuvant) in vaccines and pharmaceuticals by reversed phase highperformance liquid chromatography (RP-HPLC) with visible (VIS) detection was developed and validated. Due to postcolumn derivatization with dithizone, any interference from matrix was excluded. Similarly, a possibility of on-column decomposition of dithizonates was eliminated. Evaluated detection limits were 0.3 μg TM and 3.0 μg Al, which correspond to the smallest, but possible to recognize, visible peak.

It has been experimentally proven that germinated plant seeds, such as leek, red clover, broccoli, and others contain proteases, which are able to digest animal protein such as gelatin, bovine albumin, casein, and egg albumin. Preliminary tests were carried out with the use of a semi-qualitative gelatin test, which is often applied to prove the presence of fruit enzymes. Quantitative examinations were carried out with the use of a ninhydrin reaction for amino acid determination as well as the Bradford and Smith methods for protein determination. Respective calibration curves were obtained for glycine (amino acid analysis) as well as for egg albumin, bovine albumin, and gelatin (protein analysis), with a full statistical evaluation including Mandel and Lack-of-Fit tests to check for linearity. It has been proven that the selected germinated seeds containing proteases can be applied as an effective aid for animal protein digestion.

Background: In order to organize and give a better understanding of the existing population of protease activity units together with their respective methods of enzymatic activity assessment, there is a need of their clear classification system. Results and Conclusion: The following system has been proposed: Enzyme Centered Units (ECU) equivalent to Enzyme Process Unit notation; Protein Centered Units (PCU) equivalent to Protein Process Unit notation; Legal Authority and Enzyme Centered Units (LAECU) equivalent to Enzyme Centered Units system additionally related to a legal authority or an organization. The suitable ways for the mutual conversion of commonly used units and their conversion into the standard SI units have been included. A convenient gravity/spectrophotometer test of proteolytic activity with the use of three protein types has also been proposed. The test gives high degree of confidence of the experimental determination for a wide spectrum of protease activity in samples of plant origin. The whole paper allows both theoretical and practical orientation in the range of different proteolytic activity units as well as in the methods of their determination.