Pharmacokinetics of d-limonene in the rat by GC–MS assay

Abstract

The naturally occurring monoterpene d-limonene has been found to inhibit various stages of tumorigenesis in a number of animal models and is now being evaluated as a chemopreventive agent in humans. To date, there are little or no preclinical pharmacokinetics available nor is there a sensitive assay methodology. In this study, d-limonene and its dideuterium-labeled internal standard, limonene- d 2, in whole rat blood were extracted with n-pentane which was then concentrated on a Kuderna-Danish concentrator. The residue was analyzed by an ion-trap GC–MS under ammonia chemical ionization. The detection limit of d-limonene was 1.0 ng if injected in pure form; however, due to the presence of endogenous d-limonene levels (probably from diet), the routine quantitation limit was set at 1.0 μg ml −1. The monitored assay linearity ranged from 1.0 to 30 μg ml −1 with within-day CV values of 8.0%, 2.4%, and 2.0% at 1.0, 3.0 and 10.0 μg ml −1, respectively (all at n=8), and corresponding accuracy of 100%, 100%, and 101%. The between-day CV values were 12.3, 8.0, and 7.5% at 1, 6, and 20 μg ml −1, respectively (all at n=8). Using this assay, pharmacokinetics of d-limonene were studied in Sprague-Dawley rats following intravenous and oral administration at 200 mg kg −1 each. Blood concentration–time profiles after intravenous administration showed a biphasic decline with a mean initial t 1/2 of 12.4 min and a terminal t 1/2 of 280 min. The plasma:red blood cell partition was found to be 0.84. Plasma protein binding of d-limonene was found to be 55.3% at 20 μg ml −1. The mean total clearance was 49.6 ml min −1 kg −1, the volume of distribution at steady-state 11.7 l kg −1, and median residence time 263 min. The blood concentration–time decline following oral administration also showed a biphasic decline with a mean initial t 1/2 of 34 min and terminal t 1/2 of 337 min. The oral bioavailability of d-limonene was 43.0%.