Synthesis, HPLC measurement and bioavailability of the phenolic amide amkamide

Amkamide is a phenolic amide whose analogues were recently reported to have potent mitochondria protective activity, although their bioavailability is still unknown. Therefore, in this study, amkamide was synthesized and confirmed by nuclear magnetic resonance, and a high-performance liquid chromatography (HPLC) method was developed for analyzing the amides in biological samples. HPLC separation was performed on a Nova-Pak C18 column using a gradient condition and a coulometric electrochemical detector. The HPLC method was able to produce excellent and reproducible separations of amkamide from other amides (oretamide, becatamide, enferamide and veskamide). All five amides yielded outstanding peak resolutions with detection limits as low as 100 fmol. Therefore, the bioavailability of amkamide together with becatamide was determined in a mouse model. Their plasma concentrations were measured following two oral administrations (2 and 4 mg per 30 g body weight) using the HPLC method. As expected, relatively high amounts of amkamide and becatamide (51-58 µM) were detected at the time after administration when the maximum concentration was reached (approximately 20 and 25 min) at the high dose. The data indicate that HPLC methods with amide standards may be utilized to measure amkamide and its analogues in biological samples with excellent resolution and detection limits.