Improved methods for determining the concentration of 6-thioguanine nucleotides and 6-methylmercaptopurine nucleotides in blood

The conversion of the cytotoxic and immunosuppressive 6-mercaptopurine (6MP) to the active 6-thioguanine nucleotides (6TGN) is necessary for clinical efficacy of 6MP and its prodrug azathioprine. Another metabolite, 6-methylmercaptopurine nucleotide (6MMPN), is formed via a competing pathway by thiopurine methyl transferase. The concentrations of 6TGN and 6MMPN are measured in washed erythrocytes as a surrogate to the intracellular levels of these metabolites in the target tissues. Analysis of 6TGN and 6MMPN in multi-center clinical studies is more complicated because of the requirement to wash erythrocytes. In this investigation, we found no differences in the concentrations of 6TGN and 6MMPN in blood versus washed erythrocytes in samples obtained from patients taking therapeutic doses of oral 6MP or azathioprine for inflammatory bowel disease. We concluded that whole blood could be used for the analysis of these analytes, thus saving sample preparation time. We also found that the erythrocyte 6TGN concentration in blood at ambient temperature declined 2–4% per day, a loss that can be avoided by shipping blood samples frozen. The loss of 6TGN in blood stored at approximately −80°C was 1% after 1 week and 12% after 24 weeks, indicating the analyte was moderately stable. 6MMPN in blood did not significantly change after 24 weeks of storage at approximately −80°C. In addition, the sensitivity of the 6TGN assay was improved by modifying the HPLC conditions, which made the method more suitable for quantifying low levels of 6TGN in human intestinal biopsy samples and blood.