A sensitive method for measuring 6-thioguanine incorporation into DNA and RNA utilizing a dual column system is presented. The measurement of the 6-thioguanine deoxyribo-or ribonucleosides and deoxyadenosine or adenosine is made simultaneously, thereby allowing for direct calculation of the incorporation per nucleic acid base. The separation utilizes a strong anion-exchange column connected in series with an octadecylsilane column. Prior to high pressure liquid chromatography, the sample is partially purified and oxidized with potassium permanganate. Following a 10-min delay, a 10-min linear gradient from 2 to 20 methanol in 30 mM NH4H2PO4, pH 3.7, is employed. Detection of eluting material is by fluorescence and by UV absorbance at 254 nm. Recovery of the 6-thioguanine nucleosides was determined using 8-14C-6-thioguanine. The sensitivity of the method for the oxidized 6-thioguanine compounds is approximately 1 pmole (fluorescence) whereas that for the adenine nucleosides (UV absorbance) is about 100 pmoles. This sensitivity is adequate to determine the incorporation in less than 106(about 1 mg) Chinese hamster ovary cells exposed to a cytotoxic concentration of 6-thioguanine.
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