Measurement of Ropivacaine Concentration in the Maxillary Tissue and Serum

The injection of ropivacaine was performed according to our previous report.⁵ Under general anesthesia with an intraperitoneal injection of 50 mg/kg sodium pentobarbital, 20 μL of 0.5% ³H-ropivacaine (Moravek Biochemicals, Brea, Calif; specific activity 0.321 GBq/mmol, 0.093 GBq/mL) or 0.5% ³H-ropivacaine with 10 μg/mL epinephrine was injected into the right palatal mucosa proximal to the first molar of the rat. The maxillary tissue was removed from rats 2, 5, 10, 20, 30, 40, 50, and 60 minutes after the injection (n = 7). The right and left palatal mucosa were removed from the maxilla. Then, the maxilla from the front end of the zygomatic arch to the end of the third molar was divided into right and left maxilla parts (Figure 1). After measurement of wet weight of each extracted tissue sample, the sample (150–200 mg) was put into a glass vial and solubilized with 1 mL of solubilizing agent (Solvable, PerkinElmer, Waltham, Mass) at 60°C for 2 hours. The chemical luminescence of the solubilized sample disappeared upon the addition of 50 μL of acetic acid. An aliquot 0.5 mL of blood was collected from the right femoral vein 2–90 minutes and 2–12 hours after the injection of each anesthetic (n = 7). The blood sample was centrifuged at 15,000g for 20 minutes at 4°C, and then 0.1 mL of serum was treated with the solubilizer and acetic acid.

Divisions of the maxilla. The palatal mucosa was removed from the areas of A and B. The maxilla part from the front end of the zygomatic arch to the end of the third molar was divided into 2 parts (C, D) along the solid line (—). The asterisk indicates the site of injection. The broken line (—) shows the position of a frontal section for the ³H-autoradiogram. (A) Right palatal mucosa. (B) Left palatal mucosa. (C) Right maxilla part (including right molars; the zygomatic arch was removed). (D) Left maxilla part (including left molars; the zygomatic arch was removed).

The radioactivity (dpm) in the tissue or serum sample was measured using a liquid scintillation counter (LSC-6100, Aloka Co., Ltd., Tokyo, Japan) after adding 10 mL of liquid scintillation cocktail (AQUASOL-2R, PerkinElmer). The amount of ropivacaine per wet weight of the tissue (ng/mg) was calculated from the measured value and the specific radioactivity. The concentration of radioactivity derived from ³H-ropivacaine in the serum was expressed as dpm/mL.