HPLC Determination of Carnitine and Acylcarnitines in Human Plasma by Means of Fluorescence Labeling Using 2-(4-Hydrazinocarbonylphenyl)-4, 5-diphenylimidazole

Abstract

Carnitine and acylcarnitines are important substances involved in the oxidation and metabolism of fatty acids. An HPLC method is presented for the quantitative analysis of these compounds. The method is based on the detection of fluorescent derivatives of carnitine and short- and medium-chain acylcarnitines labeled with 2-(4-hydrazinocarbonylphenyl)-4, 5-diphenylimidazole (HCPI). The labeling of carnitine and acylcarnitines with HCPI was performed at room temperature for 1h using 1-ethyl-3-(3-dimethylaminopropyl)carbodimide as a condensing reagent. The analytes prepared using cation-exchange cartridges were separated on an octadecylsilyl silica gel (ODS) column by a gradient elution system of acetonitrile/Tris-HCl buffer and determined using a synthetic internal standard. Fluorescence detection was performed at 475nm with excitation at 340nm. The detection limits for carnitine, acetyl-, propionyl-, hexanoyl- and octanoylcarnitine ranged from 0.24 to 1.97nmol per ml human plasma, at a signal-to-noise ratio of 3. The within-day and between-day precision of the assay for carnitine and acylcarnitines in plasma samples had relative standard deviations (RSDs) lower than 10.3%. The concentration of free carnitine, acylcarnitines and total carnitine in human plasma could be successfully determined by the proposed method.