Selective and sensitive determination of lipoyllysine (protein-bound alpha-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing α-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 °C for 1 h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N) = 3) and 0.44 pmol (S/N = 10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1 g wet tissues were 3.67 μg (kidney), 1.97 μg (liver), 2.09 μg (heart), 0.59 μg (brain), 0.30 μg (lung), 0.38 μg (pancreas), and 0.20 μg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.