In the previous paper⁽¹⁾, we reported chemical and biological properties of kanamycin B. In that paper, we also described the necessity to establish a differential assay method of kanamycins A and B. After degradation by 40% sulfuric acid⁽²⁾, kanamycin A yielded a furfural-like substance, showing a specific absorption at 287 mμ⁽³⁾, and this substance was determined to be yielded from 6-glucosamine moiety of kanamycin A when treated with 4 N hydrochloric acid. On the other hand, kanamycin B having no 6-glucosamine did not show this property. Kanamycins A and B have been known to show an equal antimicrobial activity by agar diffusion assay. These properties were expected to be useful for the differential assay of these two substances. However, this method did not give a good reproducibility. Fluctuation of the results was considered to be due to difference of their dose-response relations.

Lamoy and Lannon⁽⁴⁾ reported that rates of diffusion of A and B were different and the result obtained by means of agar diffusion assay was invalid. They found, however, dose-response curves of A and B in turbidimetric assay were similar in the form using Staphylococcus aureus. Thus, they presented “two point turbidimetric” assay method.

The authors found a different characteristic between kanamycins A and B after acid degradation. Kanamycin B gave a microbiologically active substance after refluxed with 6 N hydrochloric acid for 30 to 60 minutes, but kanamycin A lost completely the activity after this treatment. This microbiological activity of B after the treatment could be easily determined by agar diffusion assay and gave an accurate value of B in a sample tested.

In this paper is presented a differential assay method of kanamycin B.