Visible diode laser induced fluorescence detection for capillary electrophoretic analysis of amantadine in human plasma following precolumn derivatization with Cy5.29.OSu

Abstract

Visible diode laser induced fluorescence (VDLIF) detection (620–700 nm) has become important in bioanalysis due to the increased sensitivity and selectivity that can be achieved in biological matrices. A selective and sensitive capillary electrophoretic method employing VDLIF detection has been developed for the analysis of amantadine in plasma. Amantadine was extracted from plasma into toluene under alkaline conditions and the residue was derivatized with the far-red label Cy5.29.OSu. The reaction mixture was dried under nitrogen, reconstituted and then injected onto a laboratory constructed capillary electrophoresis system equipped with a laboratory constructed visible diode laser detector temperature tuned to oscillate at 647.8 nm. The selectivity of the technique was evaluated by demonstrating a lack of interfering peaks in extracts of blank plasma. A calibration curve ranging from 1.8 to 461.1 ng ml −1 was shown to be linear. The precision and accuracy of the assay ( n=6) were determined to be within 17% R.S.D. and 15% difference from the nominal concentration respectively. The limits of detection for unextracted amantadine and for amantadine from the extracted concentrate from plasma were determined to be 9.5 fmol and 115 amol respectively.