A Fast Capillary Electrophoresis Method for Separation and Quantification of Modified Nucleosides in Urinary Samples

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Abstract
Modified nucleosides are formed at the post-transcriptional stage by chemical modification of normal nucleosides within the ribonucleic acid (RNA). These modified nucleosides cannot be reutilized or further degraded, but they are excreted in the urine as intact molecules. The elevated levels of modified nucleosides in the urine samples have served as potential cancer biomarkers in many studies. Although different analytical techniques have been reported for determining nucleosides levels, they are practically difficult to use as a routine tool for early cancer screening. In this paper, a novel method was developed to separate and quantify 10 nucleosides—adenosine, cytidine, guanosine, uridine, inosine, xanthosine, pseudouridine, N2-methylguanosine, 1-methyladenosine, and N2,N2-dimethylguanosine—in urine samples using capillary electrophoresis with an ultraviolet (UV) detector (abbreviated as CE-UV) at a wavelength of 254 nm. A 50 μm (i.d.) × 38 cm (effective length) fused silica capillary was used for the separation, and a borate−phosphate buffer containing 25 mM cetyltrimethylammonium bromide (CTAB) at pH 9.50 was used as a background electrolyte. The separation was performed at 15 kV under reverse polarity and completed within 10 min. The linear range of the analytes was 5.0−500 μmol/L, and the limit of detection was <2.0 μmol/L. The effects of pH, buffer concentrations, CTAB concentration, and the operation voltages on the separation and quantification of the modified nucleosides were also investigated. The technique developed in this study is much simpler and faster, compared to previous studies, and can be used to quantify modified nucleosides in urine samples.

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