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Determination of lipoic acid by precolumn derivatization with monobromobimane and reversed-phase high-performance
1Department of Neonatology, Hospital for Pediatrics, University Hospital Charité, Humboldt University at Berlin, Germany.
Abstract:
A new method for the quantitation of lipoic acid in plasma and tissues based on the selective precolumn derivatization of thiols with the fluorescent label monobromobimane is described. After extraction with diethyl ether, the dithiolane ring of lipoic acid is opened by reduction with NaBH4 before the free thiols can react with the label. Separation and quantitation was achieved by reversed-phase HPLC and fluorescence detection. The concentration-response curve was linear from 20 to 3000 nM in plasma. The recovery as determined with [3H]lipoic acid was 60.9% from plasma and 61.4% from rat heart tissue. The pretreatment of samples with N-ethylmaleimide makes it possible to differentiate between reduced and oxidized forms of lipoic acid.