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Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS/MS) Study for the Identification and Characterization of In Vivo Metabolites of Cisplatin in Rat Kidney Cancer Tissues: Online Hydrogen/Deuterium (H/D) Exchange Study.

Bandu R, Ahn HS, Lee JW, Kim YW, Choi SH, Kim HJ, Kim KP - PLoS ONE (2015)

Bottom Line: A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements.Online HDX experiments have been used to further support the structural characterization of metabolites.The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yong-in City, Republic of Korea.

ABSTRACT
In vivo rat kidney tissue metabolites of an anticancer drug, cisplatin (cis-diamminedichloroplatinum [II]) (CP) which is used for the treatment of testicular, ovarian, bladder, cervical, esophageal, small cell lung, head and neck cancers, have been identified and characterized by using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with on line hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, kidney tissues were collected after intravenous administration of CP to adult male Sprague-Dawley rats (n = 3 per group). The tissue samples were homogenized and extracted using newly optimized metabolite extraction procedure which involves liquid extraction with phosphate buffer containing ethyl acetate and protein precipitation with mixed solvents of methanol-water-chloroform followed by solid-phase clean-up procedure on Oasis HLB 3cc cartridges and then subjected to LC/ESI-HRMS analysis. A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements. Online HDX experiments have been used to further support the structural characterization of metabolites. The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether. This is the first research approach focused on the structure elucidation of biotransformation products of CP in rats, and the identification of metabolites provides essential information for further pharmacological and clinical studies of CP, and may also be useful to develop various effective new anticancer agents.

No MeSH data available.


Related in: MedlinePlus

LC/ESI-MS/MS spectra of (a) Protonated M10 (b) Protonated M12 and (c) Protonated M17 at 28 eV.
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pone.0134027.g008: LC/ESI-MS/MS spectra of (a) Protonated M10 (b) Protonated M12 and (c) Protonated M17 at 28 eV.

Mentions: M10 ([M+H]+; m/z 410.1173): The metabolite M10 at m/z 410.1173 ([M+H]+) with an elemental composition of C5H17N3O4PtS (-4.09 ppm) was detected at 22.5 min, suggesting an addition of an acetyl group to M8. The HRMS data also revealed the lack of chlorine atom and inclusion of hydroxy group in M10 when compared to M9. The LC-MS/MS spectrum of protonated M10 gives an abundant peak at m/z 392.1688 (C5H15N3O3PtS; -4.09 ppm) (Fig 8A) by the loss of H2O, confirms the presence of Pt-OH in M10. This is also supported by the fact that protonated M9 loses HCl to form m/z 392.1688. The protonated M10 also yields m/z 368.1261 ion (Protonated M8) which is formed by the loss of C2H2O (Fig 7), confirms the presence of acetyl group in M10. Further, it can be seen from Fig 7 that the loss of CH3CONH2 (m/z 351.1388) from the protonated molecular ion (m/z 410.1173) of M10, also confirms the presence of N-acetyl group in its structure (Fig 7), as this is not the case for protonated M7 and M8 which eliminates NH3 due to the presence of-NH2 group (Fig 7). The LC-MS/MS spectrum of protonated M10 also shows other characteristic fragment ions at m/z 246.5177 which involves the loss of C5H10NO3S and m/z 164.0544 (protonated acetylcysteine) (Fig 7), authenticates the presence of Pt+(NH3)2(OH) moiety (discussed in M2, M6 and M8) and acetylcysteine moiety, respectively, in its structure. This was further supported by the MS/MS spectrum of deuterated M10 in which m/z 246.5177 and m/z 164.0544 ions got shifted to higher masses by one and four units, respectively, due to exchange of labile protons with deuterium atoms.


Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS/MS) Study for the Identification and Characterization of In Vivo Metabolites of Cisplatin in Rat Kidney Cancer Tissues: Online Hydrogen/Deuterium (H/D) Exchange Study.

Bandu R, Ahn HS, Lee JW, Kim YW, Choi SH, Kim HJ, Kim KP - PLoS ONE (2015)

LC/ESI-MS/MS spectra of (a) Protonated M10 (b) Protonated M12 and (c) Protonated M17 at 28 eV.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526507&req=5

pone.0134027.g008: LC/ESI-MS/MS spectra of (a) Protonated M10 (b) Protonated M12 and (c) Protonated M17 at 28 eV.
Mentions: M10 ([M+H]+; m/z 410.1173): The metabolite M10 at m/z 410.1173 ([M+H]+) with an elemental composition of C5H17N3O4PtS (-4.09 ppm) was detected at 22.5 min, suggesting an addition of an acetyl group to M8. The HRMS data also revealed the lack of chlorine atom and inclusion of hydroxy group in M10 when compared to M9. The LC-MS/MS spectrum of protonated M10 gives an abundant peak at m/z 392.1688 (C5H15N3O3PtS; -4.09 ppm) (Fig 8A) by the loss of H2O, confirms the presence of Pt-OH in M10. This is also supported by the fact that protonated M9 loses HCl to form m/z 392.1688. The protonated M10 also yields m/z 368.1261 ion (Protonated M8) which is formed by the loss of C2H2O (Fig 7), confirms the presence of acetyl group in M10. Further, it can be seen from Fig 7 that the loss of CH3CONH2 (m/z 351.1388) from the protonated molecular ion (m/z 410.1173) of M10, also confirms the presence of N-acetyl group in its structure (Fig 7), as this is not the case for protonated M7 and M8 which eliminates NH3 due to the presence of-NH2 group (Fig 7). The LC-MS/MS spectrum of protonated M10 also shows other characteristic fragment ions at m/z 246.5177 which involves the loss of C5H10NO3S and m/z 164.0544 (protonated acetylcysteine) (Fig 7), authenticates the presence of Pt+(NH3)2(OH) moiety (discussed in M2, M6 and M8) and acetylcysteine moiety, respectively, in its structure. This was further supported by the MS/MS spectrum of deuterated M10 in which m/z 246.5177 and m/z 164.0544 ions got shifted to higher masses by one and four units, respectively, due to exchange of labile protons with deuterium atoms.

Bottom Line: A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements.Online HDX experiments have been used to further support the structural characterization of metabolites.The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yong-in City, Republic of Korea.

ABSTRACT
In vivo rat kidney tissue metabolites of an anticancer drug, cisplatin (cis-diamminedichloroplatinum [II]) (CP) which is used for the treatment of testicular, ovarian, bladder, cervical, esophageal, small cell lung, head and neck cancers, have been identified and characterized by using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with on line hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, kidney tissues were collected after intravenous administration of CP to adult male Sprague-Dawley rats (n = 3 per group). The tissue samples were homogenized and extracted using newly optimized metabolite extraction procedure which involves liquid extraction with phosphate buffer containing ethyl acetate and protein precipitation with mixed solvents of methanol-water-chloroform followed by solid-phase clean-up procedure on Oasis HLB 3cc cartridges and then subjected to LC/ESI-HRMS analysis. A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements. Online HDX experiments have been used to further support the structural characterization of metabolites. The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether. This is the first research approach focused on the structure elucidation of biotransformation products of CP in rats, and the identification of metabolites provides essential information for further pharmacological and clinical studies of CP, and may also be useful to develop various effective new anticancer agents.

No MeSH data available.


Related in: MedlinePlus