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

Abstract

Alpha-pinene (AP), produced by pine trees and other plants, is the main component of turpentine and is used as a fragrance and flavor ingredient. Exposure occurs via use of personal care and household cleaning products and in the lumber industry. Despite widespread exposure, toxicity data for AP are limited. The objective of this work was to develop and validate a method to quantitate AP in rodent blood and mammary glands, in support of toxicokinetic and toxicology studies of AP. The method uses 100 µL of blood or ~100 mg of mammary gland with analysis by headspace gas chromatography-mass spectrometry. The samples are diluted with internal standard (2H3-AP, IS) and sealed in headspace vials; mammary glands are homogenized within the vial. The vials are equilibrated briefly at 60°C before a headspace sample is analyzed. The method was validated in Sprague Dawley rat blood over the range 5-500 ng/mL and mammary gland over the range 100-5000 ng/g. The method was linear (r ≥0.99), accurate (mean relative error (RE) ≤±13.4%) and precise (relative standard deviation (RSD) ≤7.1%) in both matrices. Recoveries incorporating IS were ≥88.7% at all concentrations in both tissues. Standards as high as 1500 ng/mL in blood and 20,000 ng/g in mammary gland could be analyzed using lower injection volume or extrapolating the calibration curve beyond the upper limit of quantitation (mean %RE ≤±18.7; %RSD ≤2.2). Loss of AP occurred during overnight autosampler storage as well as frozen storage in as few as 15 days, but incorporation of IS prior to storage corrected for the loss such that calculated concentrations were within 84.7-117% of day 0 concentrations following frozen storage up to ≥32 days in both matrices. Matrix evaluation was performed in Hsd:Sprague Dawley®SD® rat and B6C3F1 mouse blood and mammary glands (mean %RE ≤±9.2; %RSD ≤4.3). These data demonstrate that the method is suitable for determination of AP in rodent blood and mammary glands.