Overcoming Stability Challenges in the Quantification of Tissue Nucleotides: Determination of 2′-<i>C</i>-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.Key words LC-MS/MS; nucleotide; liver tissue analysis; 2′-C-methylguanosine triphosphate; hepatitis C Hepatitis C virus (HCV) infection is an important global healthcare concern with approximately 150 million individuals infected worldwide and an estimated 4 million newly infected patients added each year.1-3) For many years, pegylated α-interferon in combination with ribavirin (PegIFN/RBV) was the standard of care therapy for HCV patients. This combination therapy boosts patient's immune response against the HCV infection to achieve a sustained virologic response (SVR). [4][5][6] In 2011, protease inhibitors were the first directacting antivirals (DAAs) introduced in combination with PegIFN/RBV, resulting in increased SVR rates and shorter duration of therapy for the majority of HCV patients. 7)The development of nucleoside and nucleotide analogue molecules have also been key to the evolution of HCV treatment as inhibitors of the important viral replication step catalyzed by HCV NS5B RNA polymerase. [8][9][10][11][12] For this class of compounds, the active entity is nucleoside triphosphate (NTP). The NTPs have poor cellular uptake and they undergo rapid enzymatic degradation. The conversion of a nucleoside to its monophosphate is a rate limiting first step towards the formation of the active NTP. To overcome these difficulties prodrug versions of these molecules have been developed to deliver and target nucleosides to the liver for HCV and address bioavailability and stability issues. [7][8][9]11,13,14) 2′-C-Methylguanosine triphosphate or 2′-MeGTP is a nucleotide analog which effectively disrupts RNA formation by the viral po...

Mass Spectrometry for the Sensitive Analysis of Intracellular Nucleotides and Analogues

“…The pre-analytical phase, including sampling, transport of material and sample preparation, plays an important role in the whole analytical process. The treatment of the sample is based on the material used for analysis, such as whole blood [12], erythrocytes [13][14][15], mononuclear cells [14,[16][17][18][19][20][21][22][23][24], cultured cells [25][26][27][28][29][30][31][32][33][34][35], plasma [12,19,20,36,37], urine [38], dry blood spots [15] and others [36,[39][40][41][42][43][44][45][46][47]. The enzymes involved in purine and pyrimidine metabolism can fundamentally change the nucleotide pool during the pre-analytical process and this should be stopped as soon as possible.…”

“…Protein precipitation (PP) is usually performed with a strong acid such as perchloric acid (PCA) [17,25,36,41,62,80] or trichloroacetic acid (TCA) [42,75] and most often by organic solvents such as methanol (MetOH) [12, 18, 26-28, 39, 43, 55, 60, 81, 82] or ethanol (EtOH) [19] or acetonitrile (AcCN) [37]. Because of the instability of nucleotides at a low pH, a neutralization step after the PP with strong acid is required.…”

“…The effect of ion suppression on the peaks before and after SPE was also compared and the results indicated that SPE extraction allows the matrix effect to be reduced [81]. An additional sample clean-up with WAX-SPE after PP was introduced in several studies [39,42,83]. Comparison of extraction efficiency in protein-precipitated samples with and without consequent WAX SPE clean-up procedure was described by Kamčeva et al Extraction recovery for the procedure without SPE was achieved in the range 59.2-91.9% and with SPE in the range from 94.2 to 121.8%.…”

“…Because of their hydrophilicity, triphosphate nucleosides have very poor retention when separated on traditional reverse-phase high-performance liquid chromatography columns, and thus another chromatographic approach is required. The possibility is using ion-pairing [12, 16, 19-21, 23, 26, 31, 32, 41, 47] or anion exchange chromatography [13,17,18,27,42]. These approaches enhance the retention on RP columns, but they are, however, compromised by the high levels of additives in mobile phases (ammonium salts and alkylamines), which cause significant ion suppression, poor robustness and instability of the ion exchange columns, and also source pollution, which remains a major problem [10,16,60,76].…”

“…The total run time was 10 min per sample. The assay was linear over a 50-10,000 pmol mL −1 concentration range in liver homogenate[42].In a paper by Derissen et al, the development of an LC-MS/MS assay for the quantification of the widely used chemotherapeutic capecitabine with the active component 5-fluorouracile (FU) and its active metabolites FUTP, FdUTP and FdUMP was described. Because of the low concentrations of the analytes, the optimization of the sample preparation, including cell lysis and nucleotide extraction and subsequent chromatographic separation, was necessary.…”