Quantitative determination of tautomeric FK506 by reversed-phase liquid chromatography

The quantitative determination of FK506, an immunosuppressant for organ transplants, was studied by using reversed-phase high performance liquid chromatography. There were three peaks corresponding to FK506 and its tautomeric compounds on the chromatogram obtained from aqueous solution. Interconversion among these compounds due to epimerization occurs and then reaches an equilibrium in aqueous solution. An increase in the water content in water-solvent solutions caused by the peak areas of FK506 and Tautomer I to decrease and that of Tautomer II to increase. For quantitative analyses of aqueous solutions this creates a problem because the composition of the mixture at equilibrium varies with the water content. A simple method, using a solution of polyoxyethylene lauryl alcohol ether (Brij-35) as a diluent, has been developed to provide a constant equilibrium. By diluting the sample with the Brij-35 diluent, it is possible to quantify FK506 in aqueous solutions. The reliability of the proposed method was confirmed with samples extracted from fermentation broth.