LC-MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behaviour of its main bio-active spilanthol

Abstract

N-Alkylamides are a promising group of naturally occurring bio-actives, with evidence for immune stimulating properties, which find applications i.a. in buccal preparations. In Spilanthes extracts, these properties are mainly ascribed to the most abundant N-isobutylamide, spilanthol. Yet, other N-alkylamides present in these extracts may contribute to this effect, as well as to its potential toxicity and physiologic interactions. Therefore, N-alkylamide profiling of an ethanolic Spilanthes extract was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS(1) and MS(2) fragmentation data were used for identification purposes. Moreover, the transmucosal behaviour of spilanthol, formulated in this ethanolic extract and in two commercially available oral gels, was evaluated using porcine buccal mucosa in a Franz diffusion cell experimental set-up. A high-throughput HPLC-UV method was used for the quantification of spilanthol in the receptor phase. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were obtained using different propylene glycol/water ratios. Eight N-isobutylamides, two 2-methylbutylamides and one 2-phenylethylamide were detected, with spilanthol as most abundant N-alkylamide (88.8%). Up till now, two of these N-isobutylamides were not yet described in Spilanthes extracts. We demonstrated for the first time that spilanthol permeates the buccal mucosa. Depending on the formulation, a more pronounced local or systemic effect is achieved, which is important for the regulatory product classification. The purely aqueous permeation coefficient K(p,aq) (+/-SEM) was found to be 11.3 (+/-0.403)10(-3)cm/h.