Studies have shown that cytosine can increase the content of white blood cells, uracil can increase myocardial contractility and enhance vasoconstriction. The determination of their changes in body fluids can help us predict diseases. In this abstract, an analytical method was established for the determination of cytosine and uracil by using a new HPLC method with a new chitosan derivatized calix[4]arene column.
Multi-element standards were added to 0.3 mL plasma to make the final concentration of internal standard solutions 0.1 μg/mL and 2 μg/mL, respectively. Both the samples were diluted with methanol to 1.5 mL, vortex mixed for 30 s, protein precipitated for 30 min at 4℃ and then centrifuged 5000 r/min for 5 min. The supernatant was filtered through a 0.22 μm filter for HPLC analysis.
The separation was conducted on Agilent 1260 series system, a chitosan derivatized calix[4]arene column with a mobile phase consisting of methanol–water (5:95, v/v) at a flow rate of 1mL/min. The wavelength is 260 nm, and the injection volume is 10 μL. Both pf them were baseline separated within 4 min.
The calibration curves showed good linearity (r2 > 0.999) within the concentration range of 0.05-10 ug/mL. The detection limits of cytosine and uracil are 0.2 ug/mL and 0.1 μg/mL, respectively. The intra-day and inter-day precision values were below 0.195%, and the spiked recoveries of cytosine and uracil were within 80.7%-107.2%.

The authors acknowledge the support from Key Laboratory of Chemical Biology of Guangdong Province (KF2012-01)