A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The (79)Br/(81)Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like "bromatograms". The combination of accurate mass measurements and the fragmentation behavior in the MS(2) spectra obtained using the Q-Tof Ultima mass spectrometer or MS(n) spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0-24 h post-dose.

In this study, a novel method was developed for tracing down reactive drug metabolites, the formation of which in the human body constitutes an important health risk as a result of their capability to bind to body proteins and DNA. Clozapine was used as a model because this drug forms both reactive and stable metabolites. Glutathione, which forms complexes with reactive metabolites, was added in order to trap reactive species of clozapine, formed by degradation in an electrochemical cell, thereby mimicking the real drug metabolism process. The method developed is based on the use of reversed-phase HPLC as a chromatographic separation technique and inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring of Cl and S. Via Cl-monitoring, all metabolites of the Cl-containing clozapine can be detected, whereas S-monitoring allows for the detection of the S-containing molecule glutathione and its conjugates with reactive metabolites. The spectral overlap of the signals of S-32(+) and S-34(+) with those of (OO+)-O-16-O-16 and (OO+)-O-16-O-18, respectively, was tackled in 2 ways. On one hand, a quadrupole-based ICP-MS instrument, equipped with a dynamic reaction cell, was used. O-2 was used as a reaction gas to convert the S+ ions to a sufficient extent into the corresponding SO+ species. This did not yield optimal results, due to pronounced ArC+ signals at mass 48 and 50 upon introduction of methanol into the ICP. On the other hand, a sector-field ICP-MS instrument operated at medium mass resolution permitted interference-free monitoring of the S+-signals. A new type of skimmer cone - termed X-skimmer - was evaluated and its use resulted in a 4-fold increase in the sensitivity in a methanolic environment, providing a limit of detection of 1 mu g L-1 for S. The chromatograms obtained via HPLC-SF-ICP-MS permitted differentiation between reactive and stable metabolites. As a result, the method developed looks very promising for the detection of glutathione conjugates and shows potential for their quantification in early stages of drug development when a radiolabeled compound is not yet available

In this study, a high-performance liquid chromatography (HPLC) inductively coupled plasma (ICP) mass spectrometry (MS) method was developed intended for use in metabolism studies of bromine-containing drugs, administered to test animals (or test persons). As a case study, the method was applied to a new antituberculosis compound, the bromine-containing diarylquinoline R207910. A method has been proposed to overcome the incompatibilities between the high organic solvent content (45%CH3OH and 45% CH3CN) used in the reverse-phase liquid chromatography (LC) separation on one hand and the limitations of the ICP on the other hand. Therefore, several instrument modifications had to be made. For the introduction of the column effluent, a combination of a perfluoroalkoxy LC nebulizer with a PC(3) Peltier-cooled inlet system (operated at 2 degrees C) was used. Additionally, the standard injector tube (internal diameter 2 mm) was replaced by an injector tube with an internal diameter of 1 mm and to avoid carbon build-up on the interface cones and the torch, the nebulizer gas was admixed with 6% v/v of oxygen. After optimization of the method, HPLC-ICP-MS was applied for metabolite profiling of faeces samples after dosing of (14)C-radiolabelled R207910 to dogs and rats. To evaluate the method developed, the HPLC-ICP-MS results were compared with those of HPLC with UV spectrophotometric and (14)C radiochemical detection. As the HPLC-ICP-MS method gave rise to a higher selectivity than HPLC with UV detection and to a better detection limit (5 ng R207910) than the method with radiochemical detection (65 ng R207910), it can be concluded that ICP-MS can be used as a good alternative to the more traditional detection methods, even when a mobile phase with high organic solvent content has to be used in the LC separation.