Dispersive liquid–liquid microextraction combined with microwave-assisted derivatization for determining lipoic acid and its metabolites in human urine

•A DLLME method is proposed for clean-up and concentration of LA and its metabolites in human urine.•MAD was performed to render LA chromophores for UV detection and also high ionization efficiency in MALDI.•LA and its major metabolites were identified by MALDI-TOF MS.•LA and its metabolites were identified by LTQ Orbitrap in human urine after oral medication.

This study explored dispersive liquid–liquid microextraction for extraction and concentration of lipoic acid in human urine. To improve the detection of lipoic acid by both capillary liquid chromatography (CapLC) with UV detection and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin was performed to render lipoic acid chromophores for UV detection and also high ionization efficiency in MALDI. All parameters that affected lipoic acid extraction and derivatization from urine were investigated and optimized. In the analyses of human urine samples, the two methods had a linear range of 0.1–20 μM with a correlation coefficient of 0.999. The detection limits of CapLC–UV and MALDI-TOF MS were 0.03 and 0.02 μM (S/N ≧ 3), respectively. The major metabolites of lipoic acid, including 6,8-bismethylthio-octanoic acid, 4,6-bismethylthio-hexanoic acid, and 2,4-bismethylthio-butanoic acid were also extracted by dispersive liquid–liquid microextraction and detected by MALDI-TOF MS. The minor metabolites (undetectable by MALDI-TOF MS), bisnorlipoic acid and tetranorlipoic acid were also extracted by dispersive liquid–liquid microextraction and identified with an LTQ Orbitrap mass spectrometer. After dispersive liquid–liquid microextraction and microwave-assisted derivatization, all lipoic acid derivatizations and metabolites were structurally confirmed by LTQ Orbitrap.