A sensitive procedure for determination of morphine in mouse whole blood by high performance liquid chromatography with electrochemical detection.

Morphine is an important drug because of its usefulness in the management of pain as well as its toxicological property of producing addiction. The drug is also important as an experimental tool for defining physiological and pathological mechanisms of pain. Numerous investigators have therefore at tempted to develop procedures for the determination of morphine (1). Radioimmunoassay has been introduced as a method for morphine assay (2-4). Although the procedure is very sensitive, morphine antibody crossreacts with the major metabolite, morphine-3-glucuronide, and with other levorotatory morphinans. A fluorometric procedure has been developed for the quantititation of morphine (5). Liquid chromatography has also been em ployed for the determination of the drug with a fluorescence detector (6). However, both of these methods detect the drug indirectly and are insufficiently sensitive for assays in small animals, especially mice. A sensitive, specific procedure for morphine assay is thus desired. Recently, the possible application of high performance liquid chro matography combined with electrochemical detection (HPLC-ECD) was discussed in relation to morphine determination (7). The present study assesses the application of HPLC-ECD for determining the concentration of morphine in a small amount of mouse blood. Male ICR mice, weighing about 25 g, were injected either intraperitoneally or intra venously with 40 mg/kg of morphine (Merck, Rahway, NJ, USA). The tail vein was incised, and 10 Id of blood was collected at various times after injection from the same animal using a micropipette. The blood was trans ferred to a tube containing internal standard (3,4-dihydroxyphenylpropionic acid) and 90 ,al of 3% trichloroacetic acid. After being vortexed briefly, the tube was centrifuged at 15,000 rpm for 20 min in a refrigerated centrifuge (Sorvall RC-5, Du Pont, Newtown, CT, USA) at 4°C. The supernatant (20 ul) was injected onto an Ultraspliere-ODS column (average particle size, 5 um; 25 cmx 4.6 mm; Altex Scientific, Berkeley, CA, USA) through a six port injector (Rheodyne, Berkeley, CA, USA). An L-2000 chromato graphic system (Yanagimoto, Kyoto, Japan) was employed with an electrochemical detector (VMD-101, Yanagimoto). The work ing electrode was made of glassy carbon. The electrode-applied voltage was set at +725 mV vs. the Ag/AgCI reference electrode. Liquid Send reprint requests to Dr. K. Ishikawa at his present