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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

Proposed fragmentation mechanism for metabolites M7-M10.
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pone.0134027.g007: Proposed fragmentation mechanism for metabolites M7-M10.

Mentions: M7 ([M+H]+; m/z 386.2316): The metabolite M7 at m/z 386.2316 ([M+H]+; C3H14N3O2PtSCl; 3.15 ppm) was eluted at 23.5 min. Similarly to protonated CP, M1, M4 and M5, the LC-MS/MS spectrum of protonated M7 displays the diagnostic product ion at m/z 264.4732 (base peak) corresponding to the loss of C3H8NO2S (Fig 7), authenticating the presence of Pt+(NH3)2(Cl) moiety in its structure. The protonated M7 yields diagnostic product ion at m/z 122.0275 corresponding to protonated cysteine, confirms the presence of cysteine moiety in its structure. This was further confirmed by the MS/MS of deuterated M7 in which m/z 122.0275 ion got shifted to m/z 127.0576 due to incorporation of five deuterium atoms (-NH2,-COOH,-SH and mobile proton). Further, the neutral loss fragments C3H8NO2S (m/z 264.4732) and HCl (m/z 350.1422) from the protonated molecular ion (Fig 7), substantiate the presence of cysteine moiety and chlorine atom, respectively, in M7. Besides, the spectrum also shows other ions at m/z 340.1688 (loss of HCOOH) and m/z 323.1365 (loss of NH3 from m/z 340.1688) which are consistent with the proposed structure. Based on these data, M7 was identified as a cysteine metabolite of CP.


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)

Proposed fragmentation mechanism for metabolites M7-M10.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134027.g007: Proposed fragmentation mechanism for metabolites M7-M10.
Mentions: M7 ([M+H]+; m/z 386.2316): The metabolite M7 at m/z 386.2316 ([M+H]+; C3H14N3O2PtSCl; 3.15 ppm) was eluted at 23.5 min. Similarly to protonated CP, M1, M4 and M5, the LC-MS/MS spectrum of protonated M7 displays the diagnostic product ion at m/z 264.4732 (base peak) corresponding to the loss of C3H8NO2S (Fig 7), authenticating the presence of Pt+(NH3)2(Cl) moiety in its structure. The protonated M7 yields diagnostic product ion at m/z 122.0275 corresponding to protonated cysteine, confirms the presence of cysteine moiety in its structure. This was further confirmed by the MS/MS of deuterated M7 in which m/z 122.0275 ion got shifted to m/z 127.0576 due to incorporation of five deuterium atoms (-NH2,-COOH,-SH and mobile proton). Further, the neutral loss fragments C3H8NO2S (m/z 264.4732) and HCl (m/z 350.1422) from the protonated molecular ion (Fig 7), substantiate the presence of cysteine moiety and chlorine atom, respectively, in M7. Besides, the spectrum also shows other ions at m/z 340.1688 (loss of HCOOH) and m/z 323.1365 (loss of NH3 from m/z 340.1688) which are consistent with the proposed structure. Based on these data, M7 was identified as a cysteine metabolite of CP.

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