Overcoming stability challenges in the quantification of tissue nucleotides: determination of 2'-C-methylguanosine triphosphate concentration in mouse liver
| Autor: | Marita Larsson Cohen, Kusum Sachdev Gupta, Sanjeev Bhadresa, Steven Spencer Good, William Roger Rush, Hassan Rashidzadeh |
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| Rok vydání: | 2015 |
| Předmět: |
Male
Ion chromatography Guanosine Monophosphate Pharmaceutical Science Biology Tandem mass spectrometry Antiviral Agents chemistry.chemical_compound Mice Pharmacokinetics Tandem Mass Spectrometry Freezing Animals Nucleotide Prodrugs Solid phase extraction Trichloroacetic acid Trichloroacetic Acid Pharmacology chemistry.chemical_classification Guanosine Nucleotides Solid Phase Extraction General Medicine Prodrug Chromatography Ion Exchange Hepatitis C chemistry Biochemistry Liver Guanosine Triphosphate Homogenization (biology) Chromatography Liquid |
| Zdroj: | Biologicalpharmaceutical bulletin. 38(3) |
| ISSN: | 1347-5215 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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