High performance liquid chromatography with fluorescence detection for the determination of phenylpropanolamine in human plasma and rat's blood and brain microdialysates using DIB-Cl as a label.

A high performance liquid chromatographic method for the determination of phenylpropanolamine (PPA) in human plasma and rat's brain and blood microdialysates using fluorescence (FL) detection after precolumn derivatization with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) is described. PPA was extracted from plasma samples by a liquid-liquid extraction method with ethyl acetate followed by derivatization with DIB-Cl, while the blood and brain microdialysates were directly subjected for derivatization. The DIB-derivatives of PPA and the internal standard, ephedrine (EP), were then separated using an isocratic HPLC-FL set at excitation and emission wavelengths of 325 and 430 nm, respectively, on an ODS column. Calibration curves of PPA in spiked human plasma were linear over the concentration range of 5-5000 nM (0.755-755 ng/ml) and those in spiked blood and brain microdialysates were linear over the range of 25-5000 nM (3.775-755 ng/ml) with limits of detection of 17, 48 and 40 fmol on column in plasma and blood and brain microdialysates, respectively. As well, the intra- and the inter-assay precisions were lower than 12% for human plasma and the microdialysates. The method was successfully applied for the monitoring of PPA levels in rat's brain and blood microdialysates administered with a single oral dose of PPA (2.5 mg/kg).