Fig. 1.41 (A) Product ion spectrum of remikiren obtained on a QqTOF. (B) Software-predicted fragments (Mass Frontier, HighChem) for the ion at m/z 282.

with much smaller mass windows than for unit mass resolution mass analyzers eliminating therefore background interferences. In MS/MS mode on hybrid systems (LIT-orbitrap, QqTOF, IT-TOF, FT-ICR) high resolution improves the interpretation of product ion spectra. As an example, in the product ion spectrum recorded at unit mass resolution spectra of bosentan and its phenol metabolites display an ion at m/z 280. When performing the accurate mass measurements of this ion on a QqTOF it was found that bosentan generates an ion at m/z

280.0835 and its phenol metabolite at m/z 280.0628 [68]. It was shown that both ions were formed through a different cyclisation mechanism involving either the phenol or the amine substituant. The mass difference of 20.7 milliunits corresponds to the mass difference betwenn NH2 and O.

The understanding of the fragmentation mechanism of the parent drug is very important for the metabolite assignment. The product ion spectrum of remikiren is illustrated in Fig. 1.41. Conventional spectra interpretation is time-consuming and the use of predictive fragmentation software such as Mass Frontier (High-Chem) can help to rationalize spectra interpretation [83]. In the case of the fragment at m/z 282, three different fragments are proposed by the software. Only accurate mass measurement with an accuracy better than 10 ppm allowed selection of the right fragment (Fig. 1.41B, middle structure).

A similar approach using accurate mass measurements and predictive fragmentation software was also applied for the examination of the human microsomal metabolism of nefazodone using a linear ion trap-orbitrap hybrid mass spectrometer. Based on a single LC-MS run, using data-dependant acquisition, 15 metabolites of nefazodone could be identified in MS and MS/MS with a mass accuracy better than 3 ppm.

Zhang et al. [84] reported a strategy using a software mass defect filter to improve the detection of expected and unexpected metabolites in accurate mass LC-MS. Metabolic structural changes in the parent drug have an effect on the mass defect of the metabolites compared to the parent drug. As an example hydroxla-tion changes the mass defect by -5 milliunits, demethylation by —23 milliunits and glucuronation by +32 milliunits. In fact most phase I and phase II metabolites have a mass defect window within 50 milliunits. It is therefore possible to apply a software filter which includes ions within a mass defect window relatively close to the parent drug and exclude ions, generally matrix interferences, which are outside the specified window. The application of the mass defect filter to a plasma sample spiked with omeprazole metabolites is illustrated in Fig. 1.42 [85].

For spectra interpretation and metabolite characterization accurate mass measurements become a must while it remains complementary to MSn, precursor and neutral loss for identifying metabolites in complex biological matrices.

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