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A sensitive, precise and accurate LC–MS/MS method was validated to quantify to bramycin in plasma and microdialysate samples.A very low injection volume (4 μL) was used and a short retention time (˜6 min) was obtained.Microdialysis probe relative recovery for tobramycin is 30% in vitro and in vivo for a flow rate of 1.0 μL min−1.Tobramycin relative recovery is independent of drug concentration and technique used (dialysis or retrodialysis).Tobramycin has a high penetration in interstitial fluid with similar free lung and plasma concentrations in healthy rats.A bioanalytical LC–MS/MS method was developed and validated to determine tobramycin in plasma and lung microdialysate samples. Tobramycin was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 10 mM of heptafluorobutyric acid, eluted as gradient. Apramycin was used as internal standard (IS) for plasma samples. Drugs were monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transitions 468.2 > 163.3 m/z for tobramycin and 540.3 > 217.2 m/z for IS. The method showed linearity in the concentration range of 0.1–50 μg mL−1 for microdialysate and 0.5–100 μg mL−1 for plasma with coefficients of determination ≥0.991. The inter- and intra-day precision, the accuracy and the stability of the drug in different conditions were in accordance with the limits established by US Food and Drug Administration guideline. The concentrations of tobramycin in plasma and lung microdialysate, determined using CMA/20 probes and a Ringer solution at a flow rate of 1 μL min−1, were evaluated in healthy Wistar rats after a 10 mg kg−1 i.v. bolus dose. Samples were harvested up to 12 h post-dose. Before animal's experiments, probe recovery was determined by dialysis and retrodialysis in vitro and by retrodialysis in vivo. Probes recovery was independent of drug concentration and method of determination. In vivo recovery was 27.74 ± 7.70%. Pharmacokinetic parameters were estimated by non-compartmental analysis using the software Phoenix®. The estimated area under the curve (AUC0-∞) was 128 ± 19 μg h mL−1 and 105 ± 12 μg h mL−1 for plasma and lung, respectively. Tobramycin plasma clearance was 0.07 ± 0.01 L/h/kg and the volume of distribution was 0.49 ± 0.09 L/kg. Elimination half-life in plasma was 4.4 ± 0.7 h and in lung, 4.2 ± 0.56 h. The free tissue/free plasma AUC0-∞ ratio was 0.94. This is the first study showing a validated method to quantify tobramycin in microdialysate samples and to evaluate the lung interstitial concentration of the drug.