Simultaneous determination of thiopental and its metabolite, pentobarbital, in blood by high-performance liquid chromatography and post-column photochemical reaction

A novel approach for the simultaneous high-performance liquid chromatographic (HPLC) analysis of thiopental and its major metabolite, pentobarbital, in blood plasma is described. On-line irradiation of the column eluate with UV-Iight leads to a significant bathochromic shift in the absorbance spectrum of pentobarbital, allowing now sensitive UV-detection of both barbiturates at 270 nm. At this longer wavelength, plasma matrix constituents are less interfering in the analysis, thus less sample preparation is necessary and blood plasma can be directly injected into the HPLC system only after protein precipitation with acetonitrile. Because of the minimal sample handling, the described HPLC method has good accuracy and reproducibility and thiopental and pentobarbital can be determined in small plasma volumes down to 0.2 μg ml⁻¹.