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Trends in Chromatography   Volumes    Volume 3 
Determination of lipoic (thioctic) acid by high-performance affinity chromatography with a trypsin-treated avidin-bound column
Kou Hayakawa, Noriyuki Katsumata, Kazuyuki Yoshikawa, Masahiko Hirano, Tsutomu Ogata, Toshiaki Tanaka, Takeaki Nagamine
Pages: 31 - 42
Number of pages: 12
Trends in Chromatography
Volume 3 

Copyright © 2007 Research Trends. All rights reserved

A method for measuring DL-α-lipoic (thioctic) acid by affinity-chromatography was developed.  Avidin-bound silica gel was treated with trypsin, and a 20 x 4.0 mm I.D. column was made. The eluent was a linearly increasing gradient of 2-propanol in an acidic phosphate buffer system containing sodium chloride (pH 2.4). Lipoic (Thioctic) acid was derivatized with 9-anthryl-diazomethane (ADAM) to the fluorescent lipoic acid-ADAM ester. A linear calibration line was obtained for lipoic acid in amounts from 0 to 29.8 pmol (detection limit, 2.68 pmol: S/N = 2.21). Total lipoic acid was measured after hydrolysis in 4.5 N sulphuric acid solution at 120oC for 1 h. The recovery rate using standard lipoic acid with the total method using acti-vated charcoal was 16.5±0.40%, and a correction factor of 6.06 was used. Free lipoic acid was measured after extraction by the Folch’s method using activated charcoal. The recovery rate using standard lipoic acid with this free method was 69.8±3.70%, and a correction factor of 1.43 was used. Among the foods and fermented foods tested, it was found that egg yolk, spinach, “natto”, rice bran, and dried yeast contained highest amounts of lipoic acid. These foods (except spinach) also contained abundant biotin. Much lipoic acid seemed to be produced by fermentations by Bacillus natto, yeast, and acetic acid bacterium.   Total and free lipoic acids on some fresh serum were also determined.
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