Mass Spectrometry Analysis of Endocannabinoid Secretion

Example 6

The identification of AMEND was further narrowed down by mass spectrometry. Enriched AMEND preparation prepared from epithelial cells treated with or without verapamil were subjected to reversed-phase HPLC, revealing a specific peak in the absence of verapamil that contained potential AMEND compounds (FIG. 2D). Enriched AMEND preparations from control and P-gp deficient mice were subjected to electrospray mass spectrometry run in the positive ion mode (LC/MS), and it was found that levels of several endocannabinoids of both NAE and MAG classes were decreased in the absence of P-gp (Table 2). Qualitative analysis of relative abundance revealed a striking difference in peak profiles for H⁺ and Na⁺ adduct masses consistent with anandamide (AEA) (FIG. 2E). In order to determine which of these P-gp transported ECs exhibited actual AMEND inhibitory activity, purified compounds were tested in the cell-free migration assay. Only AEA, oleoyl ethanolamide (OEA), and alpha-linolenoyl ethanolamide (α-LEA) exhibited significant inhibitory activity, identifying these NAEs as putative AMEND components (FIG. 2F).

With respect to Table 2, semi-quantitative MS analysis was performed to compare enriched AMEND preparations from control T84 cells to those with shRNA-mediated P-gp knockdown (B4-mdr1a and B5-mdr1a). Relative abundance of each compound was calculated by comparison with measured intensity of anandamide-d8 standard; while this is only accurately quantitative for anandamide itself, it allowed for comparison of relative units between samples for the remaining compounds.