The mono oxidation of the methyl group in the piperazine ring of M12 was

The mono oxidation of the methyl group in the

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[16]. The mono-oxidation of the methyl group in the piperazine ring of M12 was predicted to generate the specific product ions at m/z 461.1955 and m/z 113.0711, indicating the loss of CO and piperazine-1-carbaldehyde moiety, respectively ( Figure 4B ). The M7 and M12 metabolites have been reported to be formed under simulated sunlight in humans [17]. M8 was observed at m/z 436.1645 (C 19 H 26 O 5 N 5 S), indicating demethanaime, N , N - deethylation, and mono-hydroxylation at retention times of 16.8 min ( Table 1 ). The generated major product ions at m/z 311.1494 and m/z 283.1183 were not changed compared ACCEPTED MANUSCRIPT
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12 to those of the parent fragments. M9 was observed at m/z 392.1382 (C 17 H 22 O 4 N 5 S) as an end product of the oxidative breakdown of the piperazine ring, 83 Da less than the protonated form at a retention time of 16.9 min ( Table 1 ) [17]. The characteristic ions of M9 at m/z 311.1495 and 283.1183 were the same as those of the parent compound ( Supplemental Figure 4J ). There were no characteristic ions involving loss of CO from the ethoxy group. M10 and M11 were observed at m/z 477.1911 (C 21 H 29 O 5 N 6 S) and 477.1912 (C 21 H 29 O 5 N 6 S) at retention times of 17.2 and 17.7 min, respectively, indication mono-oxidation in the piperazine ring after N -demethylation ( Table 1 ). The ions at m/z 311.1495 for M10 and m/z 311.1494 for M11 indicated heterolytic cleavage of the S-N bond and loss of the piperazinesulfinic acid. The characteristic ions at 459.1799 (loss of water) and m/z 418.15348 (loss of hydroxyl-ethanamine) strongly suggested mono-hydroxylation in the aliphatic group of the piperazine ring of M10 ( Supplemental Figure 4K ). M11 was suggested as an N - hydroxyl metabolite after N -demethylation in the piperazine moiety ( Supplemental Figure 4L ). The elemental composition of M11 was the same as M10; even the product ion due to water was not detected in MS 2 spectra of protonated M11. 3.3. CYP-dependent formation of sildenafil metabolites To further determine which CYP is primarily responsible for the generation of phase 1 metabolites of sildenafil in humans, sildenafil was incubated with human recombinant CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP3A5. The formation of each metabolite is displayed as relative percent (%) compared to maximum generated level of each metabolite (Table 2). All metabolites, except M1, M6, and M8, were generated by CYP3A5 as the main CYP responsible for the metabolism of sildenafil in humans. CYP2C9 mainly generated M1, and CYP3A4 clearly formed M6 and ACCEPTED MANUSCRIPT
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13 M8. These results show that all metabolites can be generated by CYP isoforms in human liver after sildenafil administration. The results for the postulated metabolic pathway of sildenafil in human liver are summarized in Figure 5. ACCEPTED MANUSCRIPT
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14 4. Discussion In this study, phase 1 metabolism of sildenafil was revisited using LC-HR-MS-based metabolomics approaches. We identified twelve phase 1 metabolites in HLMs, including seven novel metabolites (M6-M12) and five previously reported metabolites (M1-M5) [11, 12]. LC-MS-based metabolomics in the study of xenobiotic metabolism was first proposed by Plumb et al. [18] and has been reviewed recently [3, 4]. Combining this with other techniques
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