Determination of reboxetine in rat brain microdialysates and plasma samples using liquid chromatography coupled to fluorescence detection

A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C18 column using gradient elution. The retention time for RBX was found to be 8.8 min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200 ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2 ± 8.2% (mean ± SD, n = 40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700 ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5 ± 7.3% (mean ± SD, n = 40). We demonstrated the applicability of the method to determine the concentration–time profiles of RBX in brain and plasma following systemic administration.

► Reboxetine is a selective noradrenalin reuptake inhibitor.
► We developed a liquid chromatographic method with fluorescence detection.
► Validation was performed according to the existing guidelines.
► Reboxetine was quantified in rat brain microdialysis and plasma samples.
► The method was applicable for pharmacokinetic profiling.