Colorimetric Microtiter Plate Assay of Polycationic Aminoglycoside Antibiotics in Culture Broth Using Amaranth

We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR^3-). Under acidic conditions, at which NEO exists in fully protonated form (NEOH⁶⁺), the AR^3- anion associates with the NEOH⁶⁺ cation to form a precipitate, NEOH(AR)₂. The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR^3- anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.