ORIGINAL PAPER Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development Filip Cuyckens & Lieve I. L. Balcaen & Kenny De Wolf & Bjorn De Samber & Cis Van Looveren & Rob Hurkmans & Frank Vanhaecke Received: 27 August 2007 / Revised: 15 November 2007 / Accepted: 21 November 2007 / Published online: 3 January 2008 # Springer-Verlag 2007 Abstract 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. Keywords HPLC-ICP-MS . HPLC-ESI-MS . Metabolite profiling . Metabolite identification . Bromine . Isotopic-data-dependent scanning Introduction Next to AIDS, tuberculosis (TB) is one of the most important infectious diseases in the world. Although drugs are available that can prevent, treat, and cure TB [1], newer and better drugs are urgently needed to shorten the duration of TB treatment and to reduce the emergence of drug resistance. In 2005, Andries et al. [2] reported the potent antimycobacterial properties of a diarylquinoline, R207910 (also known as TMC207) (Fig. 1). Owing to its novel mech- anism of action, the new compound is active against all multidrug-resistant strains of TB tested so far, and hence, may have the potential to improve and shorten the treatment of TB. Although pharmacokinetic studies of R207910 in mice are promising, a profound study of the compound’s metabolism in humans is required before the prospective drug can enter definitive clinical testing. Since the development of atmospheric pressure ionization (API) sources, mass spectrometry has become the preferred analytical tool for the detection and identification of metabolites [3–6]. Because electrospray ionization (ESI) is the most “soft” ionization technique, limiting fragmentation of analyte ions, it is generally preferred for metabolite identification [7, 8]. The intensity of the MS signal obtained with the API sources strongly depends on the chemical structure analyzed. Therefore, authentic standards should be used for MS quantification, but these are generally not available in metabolism studies. Radiotracer technology ( 14 C or 3 H) is most often used instead and is still the method of choice to study the in vivo disposition of a new Anal Bioanal Chem (2008) 390:1717–1729 DOI 10.1007/s00216-007-1761-6 F. Cuyckens : C. Van Looveren : R. Hurkmans Global Preclinical Development, Johnson & Johnson Pharmaceutical R&D, Turnhoutseweg 30, 2340 Beerse, Belgium L. I. L. Balcaen : K. De Wolf : B. De Samber : F. Vanhaecke (*) Department of Analytical Chemistry, Ghent University, Krijgslaan 281-S12, 9000 Ghent, Belgium e-mail: Frank.Vanhaecke@UGent.be