A high-performance liquid chromatographic method for the determination of 6-thioguanine residues in DNA using...

An HPLC method is described for the determination of 6-thioguanine (6-TG) residues in DNA. The assay is based on the release of 2'-deoxy-6-thioguanosine 5'-monophosphate (S6dGMP) by P1-nuclease digestion and its derivatization with the thiol-reactive fluorophore monobromobimane (mBBr). Following treatment with alkaline phosphatase, the resultant 2'-deoxy-6-thioguanosine-mBBr adduct is resolved by isocratic elution from a C18 reversed-phase support and quantified fluorometrically. The chromatographic procedure provides good adduct resolution without interference from reagent peaks or endogenous components present in the DNA. Recoveries of S6dGMP following DNA digestion were quantitative and the assay displayed a linear response from 18 pmol 6-TG bases/microgram DNA down to the lowest concentration tested (0.56 pmol 6-TG bases/microgram DNA). Within-run coefficients of variation were 2.6 and 3.1% for samples containing 18 and 0.9 pmol 6-TG bases/microgram DNA, respectively. Between-day coefficients of variation were 3.1% at 18 pmol and 4.4% at 0.9 pmol 6-TG bases/microgram DNA. In the standard procedure, derivatized sample corresponding to 5 micrograms of DNA (approximately 5 x 10(5) cells) was injected per analysis. This allowed the quantification of < 2.8 pmol adduct and permitted an assessment of 6-TG base incorporation into the DNA of cells exposed to 6-mercaptopurine concentrations as low as 30 nM. This method may be useful in clarifying the relationship between drug metabolite uptake into DNA and the anticancer effect mediated by 6-thiopurines. In addition it may form the basis of improved methods for clinical monitoring during pharmacotherapy with these agents.