Reversed-phase ion-pair high-performance liquid chromatographic assay of 5-fluorouracil, 5'-deoxy-5-fluorouridine, their nucleosides, mono-, di-, and triphosphate nucleotides with a mixture of quaternary ammonium ions.

Several quaternary ammonium ions were evaluated in the development of a reversed-phase ion-pair high-performance liquid chromatographic assay for the separation of the known nucleosides and nucleotides of 5-fluorouracil (FU) and its analogue, 5'-deoxy-5-fluorouridine (5'-dFUR). The capacity factors of FU, 5'-dRUR, and their eight anabolites including 5-5-fluorouridjine and its mono-, di-, triphosphate and diphosphoglucose, 5-fluorodeoxyuridine and its mono;- and diphosphate nucleotides, were dependent on the chain length and concentration of the counter ions, pH, type of buffer, as well as the type of bonded stationary phase. Separation of FU, 5'-dFUR, their nucleosides and monophosphate nucleotides was readily achieved using tetrabutylammonium ion alone as the counter ion. However, under these conditions, the di- and triphosphate nucleotides were eluted from the column only after lengthy gradient elution and with poor reproducibility. Optimal conditions for a simultaneous separation of the ten fore-mentioned compounds were achieved using a two-step elution with a mixture of tetraethylammonium (C8) and tetrabutylammonium (C16) ions. The first eluent consisted of C8 and C16 ions in a mixture of acetate--phosphate buffer and methanol, the second eluent contained an additional 30 mM phosphate. FU, 5'-dFUR, their nucleosides and monophosphates and diphosphoglucose were separated by isocratic elution from a microBondapak C18 reversed-phase column using the first eluent; and the di- and triphosphate nucleotides were subsequently eluted, isocratically with the second eluent. This assay does not require gradient elution and can be completed within 50 min with good reproducibility.