Extraction and measurement of morphine: Correlation of brain level and s.c. dose in drug-naive and morphine-tolerant rats

A method was recently developed by Raffa et al (1) for rapid analysis of brain levels of morphine in rats given the drug subcutaneously. The technique combines the extraction procedure of Spraque and Takemori (2) and the HPLC methodology of Peterson et al (3). The purpose of the present work was verification of the accuracy of this technique and its application to an accompanying study in which the dissociation constants of morphine determined in drug-naive and morphine-tolerant rats were compared. Male, Sprague-Dawley rats (180-220 g) were given morphine sulfate s.c. 60 min prior to testing. Each rat in the "tolerant" group received two 75 mg morphine pellets subcutaneously which were removed 96 h later. Brain levels of morphine were measured a further 24 h later. Morphine levels in rat brain ranged from 52 to 1800 ng, corresponding to subcutaneous doses of 2.5 to 320 mg/kg. This range of brain levels agrees well with those obtained using different methods (4,5). We found no significant difference in brain levels of morphine in naive and tolerant rats given the same doses of morphine (10, 20, 40, and 80 mg/kg, s.c.). Thus, in the determination of dissociation constants for morphine, the same relation can be used for both naive and morphine-tolerant rats when converting administered dose to brain level.