Determination of clonazepam in tablets by dc polarography.

A simple and convenient method for the routine determination of clonazepam in tablets by dc polarography is described. Clonazepam is extracted from the sample by ethanol and diluted with pH 4.15 acetate buffer in a volumetric flask. The filtered solution is then polarographed at the dropping mercury electrode versus the saturated calomel electrode. The polarographic wave is well developed; the determination is quantitative, precise, and accurate.

Biotransformation of antibiotics. II. Investigation of the chloramphenicol acetyltransferase in Streptomyces griseus.

Additional parameters for the chloramphenicol acetyltransferase (CAT) activity in spores of S. griseus are substantiated. A linear increase in activity was observed with increasing spore number up to a concentration of 5 x 10(10) spores/ml. Similarly an increase of the chloramphenicol concentration up to 500 mug/ml increased the activity. However, a drastic decrease in activity was noted above this level suggesting inhibition of the enzyme by the substrate. The CAT activity in the spores was highly influenced by the pH of the medium reaching a maximum at pH 6.5. This may suggest that CAT is apparently located to the outer surface of the spores and therefore very sensitive to variations in pH of the medium. The CAT showed a marked specificity for D-threo and D-erythrochloramphenicol, while no activity was observed with L-isomers. The enzyme acetylates D,L-erythrodechlor-chloramphenicol with a yield of 45% as compared to the D-threo parent antibiotic. While the tyrosinase characteristics (melanin formation) of S. griseus was eliminated by acriflavine or ethidium bromide treatment the CAT characteristic was persistent. The melanin negative variants retained all otherproperties of the parent strain including the production of antimicrobial agents; and revertants were not detected. The results suggest that the tyrosinase determinant gene is apparently located on an extrachromosomal element (plasmid). On the other hand, the location of the gene for CAT is not assigned yet. The nature of CAT in growing cells and the spores of S. griseus was investigated. The results show that CAT accumulated during the sporulation phase or the vegetative growth is inducible in nature; therefore the morphogenetic sequence in the strain bears no influence on CAT induction.

Biotransformation of antibiotics. I. Acylation of chloramphenicol by spores of Streptomyces griseus isolated from the Egyptian soil .

Incubation of spores, washed mycelium or whole cultures of a Streptomyces sp. with chloramphenicol (I) resulted in the loss of in vitro bioactivity of the antibiotic. Gas chromatographic estimation of an appropriate extract revealed that more than 95% of the antibiotic was inactivated under the specified conditions. The spores inactivated chloramphenicol in an inorganic buffer solution, or in distilled water, without the addition of carbohydrate or external co-factor. However, addition of certain carbon sources to the spores showed a pronounced effect on the chloramphenicol transformation process and on the relative concentration of the inactivated products. Time-course studies on the spore-catalyzed chloramphenicol transformation activity showed a maximum activity at 12-hour incubation. Addition of glucose or acetate at this point maintained maximum activity. The transformation products were identified as: chloramphenicol-1-acetate (IIa); chloramphenicol-3-acetate (IIb); chloramphenicol-3-propionate (III); CHLORAMPHENICOL-O-ISOBUTYRATE (IV); chloramphenicol-3-butyrate (V); and chloramphenicol-3-isovalerate (VI), by techniques of TLC, CPC, GC, UV, IR, MS and NMR. The microbial characteristics of the isolated strain include the formation of flexuous gray aerial mycelium with smooth to rough spores, irregular in size. It is an H2S and melanin former, non-chromogenic, and was inhibited by a streptomycin-producing strain of Streptomyces griseus (Krainsky 1914) Waksman and Henrici(1948).