Adenosine triphosphate (ATP) and serotonin (5-HT) are known to play key roles in the function and activity of the gastrointestinal tract; however no methods have been established for monitoring of these signalling molecules within one assay. We have developed a simple chromatographic methodology using UV/visible detection for the analysis of purinergic and biogenic amine signalling molecules. The chromatographic separation was achieved in an isocratic mode, where the mobile phase consisted of 5 % methanol and 95 % ammonium phosphate buffer with 10 mM tetrabutylammonium bisulfate. Column temperature of 45 °C provided the means to separate all analytes within 14.7 minutes. Good resolution and tailing factors were observed for all components within the separation. The limit of detection for ATP and 5-HT was 30 nM and 317 nM respectively with a linear range from 10 -0.02 µM. In vitro measurements were carried out by using aliquots from buffer the tissue was stored in after 30 mins to measure released molecules. In vitro assay of ileum tissue in the presence and absence of endogenous ATP was carried out. Results showed that ATP can elevate 5-HT release. This method can be used study alterations in these key signalling molecules with gastrointestinal disease.2
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