title: Overcoming stability challenges in the quantification of tissue nucleotides: determination of 2'-C-methylguanosine triphosphate concentration in mouse liver.
abstract: A conventional, rapid and high throughput method for tissue extraction and accurate and selective LC-MS/MS quantification of 2'-C-methylguanosine triphosphate (2'-MeGTP) in mouse liver was developed and qualified. Trichloroacetic acid (TCA) was used as the tissue homogenization reagent that overcomes instability challenges of liver tissue nucleotide triphosphates due to instant ischemic degradation to mono- and diphosphate nucleotides. Degradation of 2'-MeGTP was also minimized by harvesting livers using in situ clamp-freezing or snap-freezing techniques. The assay also included a sample clean-up procedure using weak anion exchange solid phase extraction followed by ion exchange chromatography and tandem mass spectrometry detection. The linear assay range was from 50 to 10000 pmol/mL concentration in liver homogenate (250-50000 pmol/g in liver tissue). The method was qualified over three intraday batches for accuracy, precision, selectivity and specificity. The assay was successfully applied to pharmacokinetic studies of 2'-MeGTP in liver tissue samples after single oral doses of IDX184, a nucleotide prodrug inhibitor of the viral polymerase for the treatment of hepatitis C, to mice. The study results suggested that the clamp-freezing liver collection method was marginally more effective in preventing 2'-MeGTP degradation during liver tissue collection compared to the snap-freezing method.
authors.affiliation: Idenix Pharmaceuticals, a wholly-owned subsidiary of Merck & Co.
authors.forename: Hassan
authors.initials: H
authors.lastname: Rashidzadeh
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Bhadresa
authors.forename: Steven Spencer
authors.initials: SS
authors.lastname: Good
authors.forename: Marita
authors.initials: M
authors.lastname: Larsson Cohen
authors.forename: Kusum Sachdev
authors.initials: KS
authors.lastname: Gupta
authors.forename: William Roger
authors.initials: WR
authors.lastname: Rush
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000493:pharmacokinetics; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D005615:Freezing; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006526:Hepatitis C / Q000188:drug therapy; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D009711:Nucleotides / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism* / Q000493:pharmacokinetics; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: C116616:8-methylguanosine; D000998:Antiviral Agents; C557801:IDX184; D009711:Nucleotides; D011355:Prodrugs; D006151:Guanosine; C016578:7-methylguanosine; D014238:Trichloroacetic Acid; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate