Analysis of plasma nucleotides in rat by micellar electrokinetic capillary chromatography/electrospray ionization...

The aim of this study was to establish a simultaneous quantitative analysis method of nine endogenous nucleotides in rat plasma using micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry (MEKC/ESI-MS).

To select the optimum conditions for separation of the nucleotides, various pH, concentrations of running buffers and surfactants were tested. Ammonium acetate (20 mM) containing the surfactant dodecyltrimethylammonium bromide (2 mM, pH 3.5) was selected as the micellar running buffer. The plasma samples were prepared by precipitating the proteins with 2 mM EDTA in 60% ethanol. The samples were analyzed using capillary electrophoresis (CE)/MS and selected ion monitoring (SIM) mode with positive ionization. CE was performed using a silica capillary column in reversed polarity mode.

The limits of detection (LODs) and limits of quantification (LOQs) of the nucleotides ranged from 0.05-5 and 2.0-20 μM, respectively. The calibration curves were linear (R(2) >0.99) for all analytes, and the accuracy and precision were within ±15%. The developed method was applied to the analysis of nucleotides in rat plasma that was collected after oral administration of acetaminophen (1000 mg/kg/day) to evaluate the changes in plasma nucleotide levels under hepatotoxic conditions.

Decreased level of GTP and increased level of cytosine nucleotides were found to be associated with liver toxicity, which led to the conclusion that liver toxicity is closely related to changes in nucleotide levels in plasma.