A Study Utilising High Temperature UPLC with Mass Spectrometric Detection to Quantify the Metabolites of 2-, 3- and 4- Bromobenzoic Acids both In Vitro and In Vivo

The use of radiolabels (14C and 3H) during drug development programmes as a tracer for drug related materials during metabolic profiling is both extremely useful and expensive. An alternative method may be utilised dependent upon the presence of suitable elements within the drug and that is inductively coupled plasma mass spectrometry (ICP-MS) which has been successfully used in excretion pathway [1-3] and metabolic profiling studies [4-5]. Conventional mass spectrometers may also be coupled to provide structural information on compound(s) of interest [4-5]. There are downsides to the use of ICP-MS with the use of organic modifiers during HPLC studies which can lead to problems with the plasma which may result in reduced sensitivity and stability. Some of the problems may be addressed with the use of oxygen to the argon carrier gas but it is not a complete solution as plasma stress then comes into play. Chromatographic theory predicts that as water increases in temperature it behaves in a manner analogous to a mobile phase containing organic modifiers in that its elutropic strength increases. Pure water at high enough temperatures has been shown [6-10] to be a useful mobile phase allowing elution of a wide series of differing polarity compounds than water at room temperature. This technique of HTLC has found use in metabolite profiling [6-10] where a wide range of polarities may be encountered with thermal gradients using pure water to elute the compounds as opposed to the standard practice of gradients based on increasing eluotropic strengths. Here we show how using HTLC with thermal gradients and ICP-MS and MS-MS in parallel as detection systems, isomers of bromobenzoic acid may be studied and analysed under both In Vito and In Vivo conditions thereby further demonstrating the possibilities of this new approach.