Four female and three male rats that were not involved in the PET imaging study, and kept under a regular 12-h dark/light cycle, were anesthetized using isoflurane and injected intravenously with [
11C]carfentanil (39.5 ± 17.5 MBq) to assess the in vivo stability of the tracer in plasma and tissues (Fig.
1B; Supplementary Fig.
S1). Blood samples (0.2–0.6 mL) were drawn using cut-tail method 5 and 10 min after injection. Twenty minutes after injection, blood was collected by cardiac puncture, and the rats were euthanized by cervical dislocation. Blood samples were collected into heparinized gel tubes (Microtainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and centrifuged at 14,400 ×
g for 90 s. The separated plasma was mixed with ice-cold acetonitrile (plasma:acetonitrile 2:3 (
v/
v)) to precipitate the plasma proteins and then centrifuged at 14,400 ×
g for 90 s. The supernatants were analyzed using a high-performance liquid chromatograph coupled to a radiodetector (radio-HPLC: Merck Hitachi L-7100 gradient pump system with Radiomatic 150 TR, Packard, Palo Alto, CA, USA). The brain, BAT, liver, stomach, muscle, and duodenum were dissected and homogenized in 1:1 (
v/
v) methanol:water using an electric homogenizer (Ultra-Turrax T8, IKA, Staufen, Germany). The homogenates were filtered (Amicon Ultra-4, 50 k, Merck KGaA, Darmstadt, Germany) to remove particulate matter and proteins, and then injected into the radio-HPLC. In the radio-HPLC, a µBondapak C18 column (7.8 × 300 mm, 10 µm; Waters, Milford, MA, USA) was used, along with a gradient of 50 mM phosphoric acid and acetonitrile, and the results were analyzed as previously described [
19].