Cidofovir is a pyrimidine-based phosphate used as a viral agent in pharmaceutical industry. Molecule is polar and acidic in nature, and can also form a cyclic by-product. Cidofovir and cyclo-cydofovir were separated on a Primesep B column based on their weak hydrophobic and strong acidic properties. Selectivity of separation can be controlled by the amount of acetonitrile and amount of buffer in the mobile phase. Elution can be monitored by UV, ELSD, CAD and LC/MS. Primesep D column can be used as alternative to Primesep B. Direct biofluid analysis is possible due to repulsion effect occurring on the column. Proteins from biological fluids (urine, blood, serum, etc.) and stationary phase both carry positive charges at lower pH and repel each other. Most of the proteins will come close to the void on Primesep B and Primesep D columns. Method does not require ion-pairing reagent to retain polar pyrimidine-based phosphate.