Separation and Determination of (I)-Ephedrine and (d)-Pseudoephedrine in Plasma by High-Performance Liquid Chromatography with Fluorescence Detection

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Abstract
The separation and determination of (l)-ephedrine (l-Ep) and (d)-pseudoephedrine (d-Ps) in Plasma by high-performance liquid chromatography (HPLC) is described. The newly developed method is based on precolumn derivatization with 5-dimethylaminonaphthalene-l-sulfonyl chloride (DNSCl) in 0.03% triethylamine in acetonitrile at 50 C°. The diastereomers formed were separated on a reversed phase column by HPLC with fluorescence detection employing 0.6 % phosphate buffer (pH 6.5)-methanol (3:8, v/v) as mobile phase. Clean-up of the ephedrine stereoisomers in plasma was efficiently attained by the combined use of a Sep-pak C18 cartridge and an ion-exchange gel, Carboxymethyl Sephadex LH-20 (CM-LH-20). The detection limit of each ephedrine stereoisomer was 100 pg at a signal-to-noise ratio of 3:1. The plasma-level profile of l-Ep and d-Ps in a guinea pig was investigated by the newly developed method. The determination of l-Ep and d-Ps in a volunteer's plasma after oral administration of Xiao Qing-long Heji [an oriental pharmaceutical preparation clinically provided now in China] was also performed.