The quantitative determination of metabolites of 6-mercaptopurine in biological materials: I. A separation method for purine and 6-thiopurine bases and nucleosides using high-pressure liquid cation-exchange chromatography

In the course of determining 6-mercaptopurine (6MP) metabolites formed in 6MP-treated cultured cells or in animal tissues, on the base and (deoxy-)-ribonucleoside levels a method has been established for the separation and quantitative determination of picomole amounts of 15 purine and 6-thiopurine compounds and of 2 oxidized 6-thiopurines using high-pressure liquid cation-exchange chromatography. A 0.18 × 100-cm column filled with strongly acidic Beckman M71 exchange resin is eluted with 0.4 m ammonium formate, pH 4.6, at a flow velocity of 5.2 cm·min −1 and at 50°C column-oven temperature. After 75 min, the flow velocity is doubled. A separation is finished within 140 min. The compounds are eluted in the series: 6-thiouric acid + 8-hydroxy-6MP (within the void fraction), uric acid, xanthosine, inosine, xanthine, 6MP ribonucleoside, hypoxanthine, 6MP deoxyribonucleoside, 6-thioguanosine, 6MP, guanine, 6-methylmercaptopurine ribonucleoside, 6-thioguanine, 6-methylthioguanine, and 6-(1-methyl-4-nitro-imidazole-)MP. Using a 0.18 × 40-cm column at a flow velocity of 8.7 cm·min −1, either the 6-thiopurine bases or the 6-thiopurine ribonucleosides are separated from each other within 30 and 80 min, respectively. About 30 pmol of 6MP and 15 pmol of 6-thioguanosine have quantitatively been detected with a variablewavelength uv detector. The most sensitive determination is achieved at 254 nm for common purines, at 291 nm for 6-methylthiopurines, at 322 nm for 6-thiopurines, and at 342 nm for 6-thioguanines.