Reversed-phase high-performance liquid chromatographic method for the determination of the 11-hydroxythromboxane B2 anomers equilibrium.

An improved reversed-phase HPLC method for the separation and detection of both hemiacetalic 11-hydroxy anomers of thromboxane B2 (TXB2) is described. Water-acetonitrile mixtures have served as mobile phases. By diminishing stepwise the temperature of the chromatographic system from 40 to 0 degrees C, the UV-absorbance profile of TXB2 changed from one broad peak to two clearly separated narrow peaks corresponding to the two anomers existing in equilibrium. Modification of the mobile phase pH from 1.6 to 6.9 (0 degrees C) resulted in different concentration ratios of the anomers. The equilibrium constant and the Gibbs free energy were calculated. The intermediate open aldehyde form of TXB2 is unstable and, therefore, cannot be observed either by HPLC or by 1H NMR measurements.