Methodological aspects of the use of a calibrator in in vivo microdialysis-further development of the retrodialysis method.

To investigate the performance of two alternative retrodialysis recovery methods and to describe the influence of different recoveries on the reliability in estimating unbound extracellular concentrations of morphine. Unbound concentrations of morphine in striatum and in blood were determined by microdialysis after a 10 min i.v. infusion in freely moving rats. In vivo recovery of morphine was determined by morphine itself, retrodialysis by drug, and by the calibrator nalorphine, retrodialysis by calibrator. The low calibrator recovery in striatum (8.6%) resulted in large variability in the estimation of unbound extracellular concentrations when retrodialysis by calibrator was used. In blood, where the recovery was higher (36%), the variability was smaller. Also, when retrodialysis by drug was used, the variability remained low. This difference is caused by the propagation of errors in the way retrodialysis recovery is determined. Therefore, calibrator recovery values > or =20% are preferable in concentration estimations using retrodialysis by calibrator. When no time-dependent change in recovery is observed, retrodialysis by drug determined before the systemic administration is the best method. The calibrator is valuable as a quality control during the experiment.