The fraction of sisomicin in the form of the carbamate over a pH range is calculated as described by Matthew et al.  using pK1 and pK2 values of 6.20 and 9.82 respectively and the Kc value determined here.
3. Materials and methods
3.1. Chemicals and reagents
Water, acetonitrile, and 28–30% ammonium hydroxide solutions were HPLC grade and were purchased from VWR International, USA (EMD brand). Sisomicin sulfate salt, ammonium bicarbonate, and sodium chloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). One N hydrochloric SW033291 and 1 N sodium hydroxide were obtained from VWR International, USA (J.T. Baker). The standard pH buffer solutions were obtained from VWR International (Radnor, PA, USA).
3.2. Instrumentations and conditions
The 1100 Series HPLC system from Agilent Technologies (Santa Clara, CA, USA) was equipped with a binary pump, autosampler, column heater, and diode array detector (DAD). Data were acquired and analyzed using Agilent ChemStation software. An XBridge C18, 2.1 mm × 50 mm, 2.5 μm, P/N 186003085, column was purchased from Waters, Inc. (Milford, MA, USA). Solvent A and Solvent B for the gradient elution mobile phase were prepared by adding 16.7 mL of 28–30% of ammonium hydroxide solution into 1 L of water and into 1 L of acetonitrile, respectively. The flow rate was 0.4 mL/min, the column temperature 40 °C, the injection volume 40 μL, and the detection wavelength was 214 nm. The gradient was linear from 0% to 25% B in 8 min for separation followed re-equilibration to 0% B. The total run time was 15 min.