Evolving bioanalytical methods for the cardiovascular drug bosentan

The history of the development of analytical methods for the determination of the cardiovascular agent bosentan, and its three major metabolites in biological fluids is described. Before the advent of LC-MS-MS, conventional HPLC-UV methodology was adequate for the determination of the drug alone in samples generated from toxicology studies. The progressive changes in the methods described reflect improvements in MS instrumentation and software, the need for measuring metabolites, as well as the availability of deuterated internal standards. Finally, three LC-MS-MS methods were chosen as being suitable for routine use. The most sophisticated of these (E), involving protein precipitation, liquid-liquid extraction, and on-line solid phase extraction by automated column switching, allowed the simultaneous and sensitive determination of the drug and three metabolites in a variety of biological matrices from several species to support pharmacokinetic studies. Two further, simpler, MS methods were developed (F and G) which, compared with the sophisticated method, focussed on reduced manual effort and reduced instrumental run cycle times. The limits of quantitation were between 1 and 2 ng mL−1 for bosentan and the three metabolites.