Stability study of an anticonvulsant enaminone (E139) using HPLC

A simple high performance liquid chromatography (HPLC) method has been deve loped for the stability study of an anticonvulsant enaminone (E139). Using a Chiral HSA column and a mobile phase of n-octanoic acid (5 mM) and isopro pyl alcohol and disodium hydrogen phosphate solution 1:9 v/v at a flow rate of 1 mL/min., the chromatograms exhibited well resolved peaks at retention times of < 5 min. for the predominant diastereo-isomer. The stability study for E139 was carried out in acid, alkaline, neutral sol ution, and in a phosphate buffer solution of physiological pH. The results confirmed that the hydrolysis of E139 was fastest in acid medium, indicatin g,that protonation of the enaminone system enhanced hydrolysis at a degrada tion rate constant (K-deg) of 0.044 min.(-1) and a degradation half-life (t (1/2)) of 15.75 min. at room temperature (25<degrees>C). Deprotonation of E 139 in alkaline solution also resulted in hydrolysis, but at a slower rate (K-deg of 0.017 min.(-1)) and longer degradation half-life (t(1/2): 40.76 m in.) at 25 degreesC. The enaminone E139 was very stable in the buffer solut ion of physiological pH (K-deg of 0.001 hr (-1), and t(1/2) of 24 days at 2 5 degreesC). Analysis of the acid hydrolysis of E139 by liquid chromatography-mass spect rometry (LC-MS) revealed that the decarboxylated product of E139 was formed . This study offers a great potential for the application of HPLC and LC-MS methods in the bioassay of enaminone compounds.