Simultaneous determination of loperamide and its desmethylated metabolitesin plasma and urine by high-performance liquid chromatography - Atmospheric pressure ionization mass spectrometry

A selective LC-MS-MS method, with methadone as internal standard, has been developed for the determination of loperamide and its two desmethylated met abolites, desmethylloperamide and didesmethylloperamide, in human plasma an d urine. The body fluids were made alkaline and the analytes were extracted with acetic acid ethyl ester and separated on a Zorbax Eclipse XDB-C8 narr ow-bore column; the mobile phase was a mixture of acetonitrile, methanol, a nd 4 mM ammonium acetate (pH 4.6). A turbo ion-spray was used for ionizatio n. All compounds were characterized by their molecular ions and several fra gments. Multiple reaction monitoring (MRM) was chosen for identification an d quantification. The limits of quantification in plasma were 0.08 mug L-1 for loperamide, 0. 09 mug L-1 for desmethylloperamide, and 0.10 mug L-1 for didesmethylloperam ide. The limits of determination were 0.02 mug L-1 for both loperamide and desmethylloperamide, and 0.03 mug L-1 for didesmethylloperamide. The succes sful determination of loperamide and its metabolites in the plasma and urin e of a volunteer who ingested a single dose of 16 mg loperamide confirms th at the method has satisfactory sensitivity and reliability and can be routi nely used for plasma and urine analysis.