Determination of tobramycin and impurities using high-performance anion exchange chromatography with integrated pulsed amperometric detection

Tobramycin is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that tobramycin and its major impurities, including kanamycin B and neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.00 mM) at a column temperature of 30 °C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between tobramycin and kanamycin B ranged from 5.71 and 6.06 over 7 days of consecutive analysis (5.92 ± 0.07, n = 590 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (50 days, 2368 injections) retention time stability with R.S.D.s of 0.4% and 0.3% for tobramycin and kanamycin B, respectively. Peak area R.S.D.s for tobramycin and kanamycin B (10 μM each, 20 μL injection) over 7 days (572 injections) were 2.3% and 1.9%, respectively. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. Based on the results of these evaluations, this method can be used for tobramycin identity, assay, and purity.