Enantioselective separation of racemic juvenile hormone III by normal-phase high-performance liquid chromatography and preparation of [2H3]juvenile hormone III as an internal standard for liquid chromatography–mass spectrometry quantification

Juvenile hormone III (JH III) racemate was prepared from methyl (2E,6E)-farnesoate via epoxidation with 3-chloroperbenzoic acid (mCPBA). Enantioselective separation of JH III was conducted using normal-phase high-performance liquid chromatography (HPLC) on a chiral stationary phase. [2H3]Methyl (2E,6E)-farnesoate was also prepared from (2E,6E)-farnesoic acid and [2H4]methanol (methanol-d4) using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP); the conjugated double bond underwent isomerization to some degree. Epoxidation of [2H3]methyl (2E,6E)-farnesoate with mCPBA gave a novel deuterium-substituted internal standard [2H3]JH III (JH III-d3). The standard curve was produced by linear regression using the peak area ratios of JH III and JH III-d3 in liquid chromatography–mass spectrometry (LC–MS).