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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 M5 (b) Protonated M6 and (c) Protonated M8 at 33 eV.
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pone.0134027.g005: LC/ESI-MS/MS spectra of (a) Protonated M5 (b) Protonated M6 and (c) Protonated M8 at 33 eV.

Mentions: M5 ([M+H]+;m/z 414.1479): The metabolite M5 at m/z 414.1479 ([M+H]+; C5H18ClN3O2PtS; 3.22 ppm) got eluted at 25.2 min. The appearance of diagnostic product ion at m/z 264.4732 (base peak) (Fig 5A) corresponding to the loss of C5H12NO2S (Fig 6) in the LC-MS/MS spectrum of protonated M5, which is also a characteristic product ion observed in the LC-MS/MS of protonated CP, M1 and M4 (Fig 4), clearly indicating the presence of Pt+(NH3)2(Cl) moiety [two ammine groups, one chlorine atom and platinum atom] in its structure. The LC-MS/MS spectrum of protonated M5 shows diagnostic product ion at m/z 150.0587 corresponding to protonated methionine, confirms the presence of methionine moiety in its structure. This was further substantiated by MS/MS experiments on deuterated derivative M5 in which the m/z 150.0587 ion got shifted to m/z 154.0816 due to incorporation of four deuterium atoms (-NH2,-COOH and mobile proton). It can be noted that the neutral loss fragments of C5H12NO2S (m/z 264.4732) and HCl (m/z 378.1751) (Fig 6) from the protonated molecular ion, also substantiates the presence of methionine moiety and chlorine atom, respectively in M5. Besides, the LC-MS/MS spectrum of protonated M5 shows other fragment ions at m/z 368.1872 (loss of HCOOH), m/z 351.2663 (loss of NH3 from m/z 368.1872) and m/z 74.0231 (C2H4NO2+), supports the presence of methionine moiety in its structure. Based on these data, M5 was identified as a methionine 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)

LC/ESI-MS/MS spectra of (a) Protonated M5 (b) Protonated M6 and (c) Protonated M8 at 33 eV.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134027.g005: LC/ESI-MS/MS spectra of (a) Protonated M5 (b) Protonated M6 and (c) Protonated M8 at 33 eV.
Mentions: M5 ([M+H]+;m/z 414.1479): The metabolite M5 at m/z 414.1479 ([M+H]+; C5H18ClN3O2PtS; 3.22 ppm) got eluted at 25.2 min. The appearance of diagnostic product ion at m/z 264.4732 (base peak) (Fig 5A) corresponding to the loss of C5H12NO2S (Fig 6) in the LC-MS/MS spectrum of protonated M5, which is also a characteristic product ion observed in the LC-MS/MS of protonated CP, M1 and M4 (Fig 4), clearly indicating the presence of Pt+(NH3)2(Cl) moiety [two ammine groups, one chlorine atom and platinum atom] in its structure. The LC-MS/MS spectrum of protonated M5 shows diagnostic product ion at m/z 150.0587 corresponding to protonated methionine, confirms the presence of methionine moiety in its structure. This was further substantiated by MS/MS experiments on deuterated derivative M5 in which the m/z 150.0587 ion got shifted to m/z 154.0816 due to incorporation of four deuterium atoms (-NH2,-COOH and mobile proton). It can be noted that the neutral loss fragments of C5H12NO2S (m/z 264.4732) and HCl (m/z 378.1751) (Fig 6) from the protonated molecular ion, also substantiates the presence of methionine moiety and chlorine atom, respectively in M5. Besides, the LC-MS/MS spectrum of protonated M5 shows other fragment ions at m/z 368.1872 (loss of HCOOH), m/z 351.2663 (loss of NH3 from m/z 368.1872) and m/z 74.0231 (C2H4NO2+), supports the presence of methionine moiety in its structure. Based on these data, M5 was identified as a methionine 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