Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide

Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5–25 μM for homocysteine and glutathione, and in the range of 0.5–200 μM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 μl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35–4.38% and 0.89–4.13%, respectively.