HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF LIPOIC ACID IN THE FORM OF 2-S-PYRIDINIUM DERIVATIVE

Lipoic acid (LA) is a naturally occurring compound found in plants, animals and various microorganisms that exists in oxidized and reduced form. Both forms together constitute the universal antioxidant redox couple of many biological systems [1,2].There is a rapid growth in the number of publications confirming beneficial effect of LA in therapy of many diseases, including diabetes, atherosclerosis, degenerative processes in neurons, diseases of joints, or acquired immune deficiency syndrome.Therefore, the analysis of LA in biological samples is important in biochemistry, nutritional and clinical chemistry.We have introduced a simple method for determination of LA in biological samples. The method is based on conversion of lipoic acid to its thiol counterpart – dihydrolipoic acid – by reductive cleavage with tris(2-carboxyethyl)phosphine 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 [3]. Bathochromic shift from the reagent absorption maximum at 275 nm to that of derivative maximum at 321 nm, as a result of the reaction, is analytically advantageous. The method was validated for urine and plasma samples. The calibration graphs, obtained with the use of human plasma and urine spiked with growing amounts of LA, were linear in the tested range 0.2 - 20 μmol/L and 0.2 – 50 μmol/L, respectively. The imprecision, expressed as relative standard deviation value, were within range 0.8-7.7% and 0.9-6.2% for plasma and urine samples, respectively. The analytical recovery were from 97.3 to 117.0% for plasma and from 96.5% to 111.5% for urine samples. The lower limit of detection and quantification was 0.1 and 0.2 μmol/L, respectively. This method can be used for routine clinical monitoring of LA in human plasma and urine.