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

Uchida M, Kanazawa M, Ogiwara A, Sezaki H, Ando A, Miyamoto Y - Int J Mol Sci (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan. Youhei_Miyamoto@nts.toray.co.jp.

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.

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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.
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f4-ijms-14-19716: 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.

Mentions: Pioglitazone, pioglitazone-d4, flurbiprofen and flurbiprofen-d3 were incubated in human liver microsomes, individually, and these reaction mixtures were analyzed by LC-TOF-MS separately. Pioglitazone-d4 and furbiprofen-d3 used were shown in Figure 1. The data were processed with Signpost MS software, which we developed. The data from the deuterium-labeled and un-labeled compounds were aligned, and then particular ions that had the same retention times, a similar signal intensity and also a specific mass-shift of 4.00 ± 0.04 Da and 3.00 ± 0.03 Da were captured in pioglitazone and flurbiprofen, respectively. The concept of detecting drug metabolites using mass-shift filtering is illustrated in Figure 2. In addition, the software also could process the data in a different manner in which the data were derived from a mixture of deuterated and non-deuterated compounds. Eight ion pairs were detected in pioglitazone and seven in flurbiprofen (Figures 3 and 4), as listed in Table 1. The ions were automatically filtered and visualized using the Signpost MS software (Figures 3 and 4). The retention times of deuterium-labeled ions were all shorter than those of un-labeled compounds. These differences due to the deuterium isotope are well known [16]. The signal intensity of the labeled compound was not constant per ion pair. Wang has reported the deuterium isotope’s effects in LC/MS and these effects affected the signal intensities of analytes due to a different degree of ion suppression between deuterium-labeled compound and its parent compound [17]. In the study, the deuterium effects of the LC/MS analyses were in a range, ±0.1 min for retention time and ±50% for signal intensity. In addition, we carried out low-resolution analyses and set a mass-shift to 4 ± 0.5 Da for pioglitazone and pioglitazone-d4, and 3 ± 0.5 Da for flurbiprofen and flurbiprofen-d3, and then detected four more ion pairs that were not metabolites but unknown signals from the matrix in both pioglitazone and flurbiprofen analyses (Figure 5). Furthermore, when signal intensity was also neglected in the mass-shift filtering, then four and ten more ion pairs, which originated from background noise were detected (data not shown).


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

Uchida M, Kanazawa M, Ogiwara A, Sezaki H, Ando A, Miyamoto Y - Int J Mol Sci (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821582&req=5

f4-ijms-14-19716: 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.
Mentions: Pioglitazone, pioglitazone-d4, flurbiprofen and flurbiprofen-d3 were incubated in human liver microsomes, individually, and these reaction mixtures were analyzed by LC-TOF-MS separately. Pioglitazone-d4 and furbiprofen-d3 used were shown in Figure 1. The data were processed with Signpost MS software, which we developed. The data from the deuterium-labeled and un-labeled compounds were aligned, and then particular ions that had the same retention times, a similar signal intensity and also a specific mass-shift of 4.00 ± 0.04 Da and 3.00 ± 0.03 Da were captured in pioglitazone and flurbiprofen, respectively. The concept of detecting drug metabolites using mass-shift filtering is illustrated in Figure 2. In addition, the software also could process the data in a different manner in which the data were derived from a mixture of deuterated and non-deuterated compounds. Eight ion pairs were detected in pioglitazone and seven in flurbiprofen (Figures 3 and 4), as listed in Table 1. The ions were automatically filtered and visualized using the Signpost MS software (Figures 3 and 4). The retention times of deuterium-labeled ions were all shorter than those of un-labeled compounds. These differences due to the deuterium isotope are well known [16]. The signal intensity of the labeled compound was not constant per ion pair. Wang has reported the deuterium isotope’s effects in LC/MS and these effects affected the signal intensities of analytes due to a different degree of ion suppression between deuterium-labeled compound and its parent compound [17]. In the study, the deuterium effects of the LC/MS analyses were in a range, ±0.1 min for retention time and ±50% for signal intensity. In addition, we carried out low-resolution analyses and set a mass-shift to 4 ± 0.5 Da for pioglitazone and pioglitazone-d4, and 3 ± 0.5 Da for flurbiprofen and flurbiprofen-d3, and then detected four more ion pairs that were not metabolites but unknown signals from the matrix in both pioglitazone and flurbiprofen analyses (Figure 5). Furthermore, when signal intensity was also neglected in the mass-shift filtering, then four and ten more ion pairs, which originated from background noise were detected (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan. Youhei_Miyamoto@nts.toray.co.jp.

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.

Show MeSH
Related in: MedlinePlus