Determination of Lipoic Acid in the form of 2-S-pyridinium Derivative by High-performance Liquid Chromatography with Ultraviolet Detection
Gra zyna Chwatko, Pawe l Kubalczyk and Edward BaldAffiliation:
Department of Environmental Chemistry, University of Lodz, 163 Pomorska Str., 90-236 Lodz, Poland.
AbstractWe have developed a sensitive, simple and cheap method for determination of lipoic acid in urine and dietary supplement tablets. The method is based on conversion of lipoic acid to its thiol counterpart – dihydrolipoic acid – by reductive cleavage with tris(2-carboxyethyl)phosphine hydrochloride prior to precolumn derivatization with 1-benzyl-2- chloropyridinium bromide, followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet detection of its 2-S-pyridinium derivative at 321 nm. Bathochromic shift from the reagent absorption maximum at 275 nm to that of derivative maximum at 321 nm, taking place during reaction, is analytically advantageous. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. Calibration plot, understood as the relationship between instrument response and known concentration of the analyte, demonstrated linearity of results when applied to normal urine spiked with growing amounts of lipoic acid within the tested range 0.2 – 50 µmol/L. The method was validated for urine samples received from healthy donors and urine concentration of lipoic acid was monitored after oral administration of 600 mg of lipoic acid.
Derivatization, determination, disulfide, HPLC, lipoic acid, thiol, urine.
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