Development of a Method for Separation of Adenine Nucleotides
Abstract: Adenosine tetraphosphate, or Ap4A, is a nucleotide with very interesting proposed biological effects. Previous studies have indicated that blood glucose levels in mammals might increase in the presence of Ap4A, and that Ap4A might antagonize pancreatic ATP channels. In order to study these effects further, a reliable method of separating Ap4A from other nucleotides in biological samples is required. This is made difficult by the high polarity and the high structural resemblance of different nucleotides. The aim of this study was to develop a method for separation of Ap4A from ATP and AMP on the Agilent Poroshell 120 HILIC-Z column, using conditions compatible with mass spectrometry. A method previously employed to separate simple nucleotides, using 10 mM ammonium acetate buffer at pH 9, a column temperature of 25℃, a flow rate of 300 µL/min, and a re-equilibration time of 33.3 minutes was used as a starting point.  In order to investigate the retention mechanism of the separation, an array of parameters (column temperature, mobile phase flow rate, re-equilibration time, buffer concentration, and mobile phase pH) were varied one by one while all the other parameters were held constant. Out of all evaluated parameters, increasing the buffer concentration to 20 mM was the only adjustment that had a positive effect on the separation. In this method two peaks were observed; one corresponding to ATP + AMP and the other corresponding to Ap4A, with a resolution of about 1.3. Furthermore, it was found that reproducible results could be achieved with a re-equilibration time as short as 8.3 minutes.
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