Application of High Performance Liquid Chromatography with Fluorescence Detection to Determination of Ampicillin in Plasma Deproteinized by Phenol Method

Pretreatment methods applicable to routine high performance liquid chromatography determination of ampicillin (ABPC) in many samples at plasma level concentration were examined and the deproteinization by phenol was found to be most suitable. The method consists of deproteinization of plasma by phenol, separation of ABPC (separation column ; Lichrosorb RP-8, mobile phase ; tetrahydrofuran : 0.02M phosphate buffer (pH 7.0)=1 : 30, flow rate ; 1 ml/min), post-column labeling with fluorescamine and measurement of fluorescence intensity at excitation wavelength of 385 nm and emission wavelength of 480 nm. ABPC was spiked in the plasma in the range of 0.05 to 10 μg/ml and the calibration curve showed a linear relationship (r=1.000), the limit of detection being 0.01 μg/ml. By use of this method, variation of ABPC concentration in the human plasma after oral administration of talampicillin, a prodrug of ABPC, was measured. As a result of comparative analysis, good correlation was found between this method and bioassay (r=0.990).