Sensitive determination of ephedrine and norephedrine in human plasma samples using derivatization with 9-fluorenylmethyl chloroformate and liquid chromatography

A high-performance liquid chromatography procedure for the determination of ephedrine and norephedrine using fluorimetric detection in plasma samples is described. A double liquid–liquid extraction was performed, followed by derivatization with 9-fluorenylmethyl chloroformate. The extracts were chromatographed with a 5-μm C18 (150×4.6 mm I.D.) column using a mobile phase composed of acetonitrile and water (52:48; v/v). The excitation and emission wavelengths were respectively 264 nm and 313 nm. Calibration curves were linear over the range 0 to 300 ng/ml for each analyte. The specificity of the method was demonstrated with several FMOC-reacting drugs. The limits of quantification are similar to those obtained with the reference method: 2 ng/ml for ephedrine and 5 ng/ml for norephedrine. This method has been successfully applied to the determination of ephedrine and norephedrine plasma levels after administration of low doses of ephedrine to healthy subjects.