Determination of Acyclovir in Blood Serum and Plasma by Micellar Liquid Chromatography with Fluorimetric Detection

Abstract

A method is described for determination of acyclovir (ACV) in blood serum and plasma by micellar liquid chromatography (MLC) on silica. A novel micellar mobile phase containing mixed micelles of sodium dodecylsulfate (SDS) and hexadecyltrimethylammonium bromide (HTMABr) was introduced to MLC and enhanced fluorimetric detection of ACV in acidic media was applied. The method was optimized with respect to detection sensitivity and separation. Addition of SDS to mobile phase caused an increase in the sensitivity of the fluorimetric detection by a factor of five. Additions of isopropanol, tetraethylammonium chloride, triethylammine, Brij 35, and HTMABr to the mobile phase containing SDS were studied. Ail of them diminished the positive effect of SDS on the detection sensitivity but the mixture of SDS and HTMABr still yielded in the increase of detection sensitivity by a factor of three compared with the mobile phase without surfactants. Octadecylsilica, nitril, and silica stationary phases were tried out, the last being optimal. The retention of ACV on silica which was too low in a mobile phase containing SDS was shown to increase with additions of HTMABr to the mobile phase. Influences of SDS and HTMABr concentrations, ionic strength, and pH on the relative intensity of fluorescence and the retention of ACV were studied. Under the final conditions (0.050 mol/1 SDS, 0.010 mol/1 HTMABr, 0.050 mol/1 phosphate, pH=2.05) the recovery from serum samples after protein precipitation with perchloric acid was (97.3±3.1)%, the relative standard deviation 3% and the limit of detection 0.8 ng ACV that corresponds to 0.08 μg ACV in 1 ml sample when 10 μl injected.