Use of periodate and methylamine for the quantitation of intracellular 5-fluoro-2'-deoxyuridine-5'-monophosphate by high-performance liquid chromatography.

High-performance liquid chromatography was used for the quantitation of [3H]fluorouracil and metabolites in L1210 cells with or without pre-treatment with methotrexate. Ribonucleotide pools were evaluated on a chemically bonded anion-exchange column. Deoxynucleotide pools were determined following periodate and methylamine treatment which eliminated the ribonucleotides (greater than 99.9% complete) and allowed for a rapid quantitation of the acid-soluble 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) pools. FdUMP levels as low as 10 fmol/10(6) cells were detectable by polystyrene anion-exchange chromatography. Confirmation of these FdUMP values was performed by rechromatography on a reversed-phase ion-pair column. The use of periodate and methylamine as a means for the reliable elimination of the ribonucleotides will allow accurate isolation of deoxynucleotides by high-performance liquid chromatography.