*2.4. Serum Analysis*

The analyses were performed at Metabolomics Australia (University of Western Australia, Nedlands, Australia). Medetomidine concentrations of the serum samples were analysed using a liquid chromatography-tandem mass spectroscopy (LC-MS/MS) technique. The internal standard during analysis was medetomidine-13C,d3 hydrochloride (Sapphire Bioscience, Redfern, Australia).

To process the serum for analysis, 20 μL of serum were added to 50 μL of working internal standard (50 ng/mL labelled medetomidine-13C,d3 in 50:50 methanol:water plus 0.1% formic acid) and vortexed for 10 s. The mixture was then vortexed with 1 mL ethyl acetate for 120 s, after which they were centrifuged at 3000 rpm for 5 min. Then, 900 μL solvent were evaporated to dryness for 30 min at 40 ◦C before being reconstituted in 70 μL of 50:50 methanol:water.

Processed serum extracts of 2 μL were run on an Agilent 6460 LC-MS/MS in 2D mode using isotope dilution to adjust for instrument response. Solvent A was LC-MS/MS grade water (Thermo Optima) with 0.1% formic acid (Merck). Solvent B was LC-MS/MS grade methanol (B & J) and 0.1% formic acid (Merck). Column one was an Agilent 2.1 × 50 mm 2.6 μm C18 Poroschell and column two was a Phenomenex Kinetex 3 × 150 mm 2.6 μm Biphenyl phase. The flow rate was set at 0.5 mL/min and a gradient was run from 50% B to 80% B in 10 min. The column was washed with 98% B and then returned to 50% B by 7 min. Compounds were heart cut from column one to column two between 0.4–0.9 min. Medetomidine and medetomidine-13C,d3 were monitored with transitions 201 > 95 and 204.1 > 98, respectively, with a collision energy of 15. Assay calibration was achieved by spiking drug free matrix matched rat plasma to create a calibration curve, with the r2 typically >0.9999. Assay precision was assessed during the project by extracting 4 samples in triplicate and the intra-assay CV ranged from 2.1–5.7%. The limit of quantitation for the assay was 0.1 ng/mL.

### *2.5. Pharmacokinetic and Pharmacodynamic Calculations*

The maximum serum concentration ( *C*max) of medetomidine following SC administration was the highest measured concentration for each animal. The time at *C*max (*t*max) was also determined. The elimination rate constant (λ*z*) was calculated as the negative slope of the semilogarithmic plot of each animal created from the terminal three time points (t = 120, 180 and 240 min). The elimination half-life (*t1*/*2*β) was calculated as ln(2)/λ*<sup>z</sup>*. The area under the serum concentration time curve (*AUC*0→∞) was estimated by the trapezoidal rule extrapolated to infinite time. Standard formulae were used to calculate the total body clearance (*Cl*=dose/*AUC*) [53] and volume of distribution at pseudo-equilibrium (*Vdarea* = *Cl*/λ*z*) [54].

The target serum concentration of medetomidine ( *Ctarget*) was obtained from the rats in Group T and was taken as the mean serum concentration of MED at t = 60 and 90 min. The loading dose (LD) was estimated from the product of *Vdarea* and *Ctarget*. The maintenance dose rate (MD) was calculated from the product of *Cl* and *Ctarget*.

### *2.6. Trial of Results*

To trial the calculated drug administration regime for SC administration of medetomidine an additional two rats were administered medetomidine with isoflurane to ensure the conditions for anaesthesia were stable and uneventful. The dose of medetomidine in these two trials was an initial SC dose of 0.12 mg/kg medetomidine delivered over 5 s followed by a SC infusion of 0.08 mg/kg/h with 0.5% (vapouriser setting) isoflurane.
