Development and Validation of an Analytical Method for Quantitation of Alpha-Pinene Oxide in Rodent Blood and Mammary Gland by GC-MS.

Alpha-pinene is a monoterpene found in the oil of coniferous trees and has a wide variety of applications. Alpha pinene oxide is a potential reactive metabolite of alpha-pinene in rodents. The objective of this work is to validate a gas chromatography-mass spectrometry method to quantitate alpha pinene oxide in rat and mouse blood and mammary gland in support of studies investigating the toxicity and toxicokinetic behavior of alpha-pinene. The method was validated in male Sprague Dawley rat blood over the concentration range 5 to 250 ng/mL. Matrix standard curves were linear (r ≥ 0.99), and accuracy (percent relative error %RE) was ≤ ±15% for standards at all levels. Intra- and inter-day precision (% relative standard deviation, RSD) and accuracy (%RE) were evaluated at three concentration levels (10, 50, and 200 ng/mL) and were ≤ 6.3% and ≤ ±5.4%, respectively. The limit of detection, determined from the SD of the limit of quantitation (5 ng/mL), was 1.06 ng/mL. Standards as high as 25,000 ng/mL could be accurately quantified after diluting into the validated range (%RE ≤ ±7.1%; RSD ≤5.8%). Alpha pinene oxide was stable in rat blood for at least 70 days in frozen storage (-80 °C). Alpha pinene oxide could accurately be quantified in male and female Hsd:Sprague Dawley®SD® rat and B6C3F1 mouse blood (mean %RE ≤ ±5.3%; %RSD ≤7.8%) and female B6C3F1 and SD rat mammary gland (mean %RE ≤ ±14.6%; %RSD ≤8.1%) using primary matrix standard curve. These results demonstrate that the method is suitable for the analysis of alpha pinene oxide in rodent blood and mammary gland generated from toxicokinetic and toxicology studies.