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
✍ Scribed by Hans-Joachim Breter
- Publisher
- Elsevier Science
- Year
- 1977
- Tongue
- English
- Weight
- 611 KB
- Volume
- 80
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 Gthiopurine compounds and of 2 oxidized 6-thiopurines using high-pressure liquid cationexchange chromatography. A 0.18 x lOO-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-' 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, 6thioguanosine, 6MP, guanine, 6methylmercaptopurine ribonucleoside, 6thioguanine, 6-methylthioguanine, and 6-( l-methyl-4-nitro-imidazole-)MP. Using a 0.18 x 40-cm column at a flow velocity of 8.7 cm.min-I, either the 6thiopurine 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