Determination of equilibrium constant of amino carbamate adduct formation in sisomicin by a high pH based high performance liquid chromatography

• Examine a high pH based RP-HPLC to separate sisomicin from its amino carbamate adduct.
• Determine equilibrium constant of sisomicin and its amino carbamate adduct.
• Interpret charge state of sisomicin at physiological conditions.
• Discuss mechanism of the aminoglycoside nephroprotection of sodium bicarbonate.

Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3 × 10−6 and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides.

Fraction of amino carbamate adduct formation in aminoglycoside.Figure optionsDownload as PowerPoint slide