ION-PAIR EXTRACTION AND LIQUID-CHROMATOGRAPHIC ANALYSIS OF MORPHINE IN RAT-BRAIN AND PLASMA

A highly efficient and reproducible two-step liquid-liquid ion-pair ex traction technique for the isolation of morphine from biological sampl es is described. A rapid normal phase high-performance liquid chromato graphic procedure coupled with amperometric electrochemical detection has also been developed for subsequent quantification of morphine. Ext raction involves the disruption of brain tissue or plasma in methanol, centrifugation, evaporation and reconstitution in ethyl acetate conta ining 10 mM di-(2-ethylhexyl) phosphoric acid, a liquid cation-exchang er, and back-extraction into 170 mM orthophosphoric acid. An acidic el uent consisting of acetonitrile-76 mM orthophosphoric acid-ammonia buf fer (pH 3.0) (15:85, v/v) in combination with a strong cation-exchange column allows complete separation of morphine and the internal standa rd, nalbuphine. The limit of detection for morphine is 1.3 ng oncolumn .