[Evaluation of assay methods for isepamicin sulfate (HAPA-B) in body fluids. Bioassay, HPLC and EIA methods]

Assay methods including microbiological assay (bioassay), high performance liquid chromatography (HPLC) and enzyme immunoassay (EIA) for isepamicin sulfate (HAPA-B), a new aminoglycoside antibiotic, in body fluids were studied. The most suitable bioassay method was double layer agar-well method using Bacillus subtilis ATCC 6633 as the test organism on plate consisting of a seed-layer of nutrient agar at pH 8.0 and a base-layer of nutrient agar supplemented with 0.4% sodium chloride. Sensitivities in bioassay, HPLC and EIA methods for plasma concentration were 0.08 microgram/ml, 0.20 microgram/ml and 0.05 microgram/ml, respectively. Plasma and urinary concentrations after intramuscular administration of HAPA-B at the dose of 200 mg to healthy volunteers were measured with these 3 methods. The HPLC and the EIA methods yielded values which compared favorably to the bioassay method. Using the bioassay method, HAPA-B levels in human plasma and urine samples were found to be stable at least for 15 days at -20 degrees C.