Determination Of Nucleotides By Capillary Electrophoresis And Its Application In Pharmaceutical Analysis

Nucleotides and nucleotide derivatives are very important nitrogen-containing organic molecules for organisms. Nucleotides are the basic structural unit of nucleic acid DNA and RNA, which is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information, nucleotides participate in nearly all biochemical processes. Nucleotides play a variety of key roles in cellular metabolism:ATP and GTP are carriers of chemical energy in the cell; adenine nucleotides are components of three major coenzymes, NAD+, FAD, and Co A, organic molecules that assist in various biochemical reactions by serving as carriers; nucleotides are intermediates in cellular communication and signal transduction(such as cAMP and cGMP); they are also donor substrates for glycobiology.Nowadays, nucleotides are usually quantified using ultraviolet-visible absorption spectroscopy, fluorescence spectrophotometry, high-performance liquid chromatography, capillary electrophoresis and electrochemical methods. Capillary electrophoresis is a separative analytical technique which is widely accepted because of its ability to simultaneously determine different analytes with both high efficiency and resolution, short analysis times and low consumption of samples and electrolytes. It has become one of the most advanced separation techniques in recent years, although quality analysis of nucleotides currently is performed predominantly with high-performance liquid chromatography.In this manuscript, we developed the new method for the determination of nucleotides by capillary electrophoresis. The mainly points are as follows:1. The determination of guanosine3’,5’-bis (diphosphate) by capillary electrophoresis with UV detection. Factors affecting separation and quantification such as buffer concentration, buffer pH and buffer additive have been investigated and optimized. The method was validated with respect to the linearity, sensitivity and precision. The analytical time for the detection of ppGpp by CE-UV has been proved to be less than15min under the optimized conditions(20℃,31cm×50μm capillary,50mmol/L phosphate buffer,5mmoI/L sodium dodecylbenzenesulfonate, pH9.2, applied voltage15kV, UV detection at254nm). Different sample-preparation reagents commonly used to stop cell metabolism were also tested with respect to the yield of nucleotides, the stability of the treated sample and the compatibility with the subsequent capillary electrophoresis analysis. The linearity curve could be expressed as the equation of y=113.6x+21920, with a correlation coefficient of0.9943with the range of0.05-0.60mmol/L for ppGpp. This proposed method was rapid, simple, effective, and so could be used for the determination of ppGpp.2. Quality control of vidarabine monophosphate in pharmaceutical formulations by capillary zone electrophoresis. The quantitative determination results for vidarabine monophosphate by national standard method usually higher than the real level because the samples often contain adenosine, which has similar absorption spectra with vidarabine monophosphate. Thus, we developed a rapid capillary electrophoresis method for the determination of vidarabine monophosphate, which realized successfulseparation of these two compounds. The influence of capillary length, buffer pH, buffer concentration, applied voltage, capillary temperature was systemically investigated and the proposed method was then successfully applied to the quality control of vidarabine monophosphate in its pharmaceutical formulations. This method was suitably validated with respect to the linearity, limit of detection and quantification, accuracy, precision, specificity and stability.