High performance liquid chromatography method for the detection of released purinergic and biogenic amine signaling molecules from in vitro ileum tissue

Abstract

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

Mucosal adenosine triphosphate mediates serotonin release from ileal but not colonic guinea pig enterochromaffin cells

“…High performance liquid chromatography was utilized to monitor levels of purines and 5‐HT . A 1 cm 2 segment of either distal ileum or colon was transferred to a well containing warm (∼37 °C) oxygenated Krebs buffer within a modified tissue well plate.…”

Chromatographic analysis of age-related changes in mucosal serotonin transmission in the murine distal ileum

“…The tissue samples were then cut along the mesenteric border and pinned out into a modified tissue culture plate forming a whole mount preparation. Fabrication of this modified well plate has been described previously [29]. Once the tissue section was pinned within the individual well, the tissue was washed twice using Krebs buffer to remove any waste or excised mucosa which may have arisen during tissue transfer.…”

“…However, in contrast, high performance liquid chromatography (HPLC) [18,29,30] is capable of measuring all parameters noted in Scheme 1. Within the field of gastroenterology, HPLC has been predominantly used to measure levels of analytes in tissue homogenates [18,28,30] and hence only intracellular neurochemicals are monitored, meaning the release of 5-HT from EC cells is entirely missed.…”

Environmentally friendly solid‐phase microextraction coupled with gas chromatography and mass spectrometry for the determination of biogenic amines in fish samples

“…There are many classic analytical methods for the determination of BA levels in foods such as HPLC , CE , TLC , and GC . In general, HPLC–MS is an effective technology to determinate BAs contents without derivatization, but it also has some limitations, such as expensive instrument and high test cost.…”