*2.1. Study Design*

In this experimental, prospective study, the New Zealand White rabbits (NZW) were randomly and equally assigned to three groups, di ffering in the location of patch application: outer surface of the ear (group 1, n = 6), inner surface of the ear (group 2, n = 6) and neck between the scapulas (group 3, n = 6). In all groups, fentanyl patches were covered with a tape (Leukoplast, BSN Medical GmbH, Hamburg, Germany) to ensure a continuous contact throughout the study period.

Venous blood samples withdrawn at di fferent time points (Figure 1) were centrifuged and frozen at −80 ◦C until analysis of fentanyl plasma concentration. Additionally, treatment-related side e ffects were recorded, and practicability of the proposed methods was evaluated to determine the most adequate location for patch application in rabbits. No surgical procedures were performed as only the drug's absorption curve was investigated in this trial.

**Figure 1.** Study design. Fentanyl plasma concentrations higher than 0.5 ng/mL were considered to be analgesic based on previous reports [13,19]. Outcome parameters were time spent above threshold concentration, variability of fentanyl plasma concentrations and practicability of patch application.
