A computational drug metabolite detection using the stable isotopic mass-shift filtering with high resolution mass spectrometry in pioglitazone and flurbiprofen

Abstract

The identification of metabolites in drug discovery is important. At present, radioisotopes and mass spectrometry are both widely used. However, rapid and comprehensive identification is still laborious and difficult. In this study, we developed new analytical software and employed a stable isotope as a tool to identify drug metabolites using mass spectrometry. A deuterium-labeled compound and non-labeled compound were both metabolized in human liver microsomes and analyzed by liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS). We computationally aligned two different MS data sets and filtered ions having a specific mass-shift equal to masses of labeled isotopes between those data using our own software. For pioglitazone and flurbiprofen, eight and four metabolites, respectively, were identified with calculations of mass and formulas and chemical structural fragmentation analysis. With high resolution MS, the approach became more accurate. The approach detected two unexpected metabolites in pioglitazone, i.e., the hydroxypropanamide form and the aldehyde hydrolysis form, which other approaches such as metabolite-biotransformation list matching and mass defect filtering could not detect. We demonstrated that the approach using computational alignment and stable isotopic mass-shift filtering has the ability to identify drug metabolites and is useful in drug discovery.

Figure 1

Chemical structures of pioglitazone-d₄ (A) and flurbiporfen-d₃ (B).

Figure 2

The concept of drug metabolite detection using stable isotopic mass-shift filtering. LC-MS data sets for un-labeled (A) and labeled (B) compounds were aligned, and the ions having a specific mass-shift equal to the mass of the labeled isotope were computationally filtered and visualized using the Signpost MS software (C). Metabolite ions, derived from the un-labeled compound and the labeled compound, are shown in red and blue, respectively.

Figure 3

Ions detected by stable isotope mass-shift filtering between deuterium-labeled and un-labeled pioglitazone metabolized in human liver microsomes. The ions derived from un-labeled and labeled pioglitazone are shown by red and blue spots, respectively. By comparing the un-labeled (red) and labeled (blue) compounds, the particular ions having a specific mass-shift equal to 4 ± 0.04 Da were computationally filtered and visualized by being circled in black. Extended figures of captured ions are shown above.

Figure 4

Ions detected by stable isotopic mass-shift filtering between deuterium-labeled and un-labeled flurbiprofen metabolized in human liver microsomes. The ions derived from un-labeled and labeled flurbiprofen were described in red and blue based on the mass spectrometric data, respectively. By comparing un-labeled (red) and labeled (blue) compounds, the particular ions having a specific mass-shift equal to 3 ± 0.03 Da were computationally filtered and visualized by being circled in black. Extended figures of captured ions are shown above.

Figure 5

Ions detected by stable isotopic mass-shift filtering between deuterium-labeled and un-labeled compounds in pioglitazone (A) and flurbiprofen (B) metabolized in human liver microsomes. The ions derived from un-labeled and labeled compounds are shown in red and blue, respectively. By comparing un-labeled (red) and labeled (blue) compounds, the particular ions having a specific mass-shift equal to 4 ± 0.5 Da for pioglitazone and 3 ± 0.5 Da for flurbiprofen were computationally filtered and visualized by being circled in black. The ions, estimated as non-metabolites, were manually marked in red circle.

Figure 6

Representative MS/MS spectra of eight pioglitazone metabolites and their estimated chemical structures. Precursor ions were m/z 315 (A), 389 (B), 373 (C), 272 (D), 373 (E), 373 (F), 332 (G) and 355 (H) with the positive ion mode. Each arrow indicates a possible site of fragmentation, with the corresponding ion.

Figure 7

Representative MS/MS spectra of four flurbiprofen metabolites and their estimated chemical structures. Precursor ions were m/z 435 (A), 275 (B), 259 (C), and 419 (D) with the positive ion mode. Each arrow indicates a possible site of fragmentation, with the corresponding ion.