Development and validation of an analytical method for quantitation of Alpha-Pinene Oxide in rodent blood 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 to AP occurs via use of personal care and household cleaning products and in the lumber industry. Despite widespread exposure, the toxicity data for AP are limited. The objective of this work was to develop and validate a method to quantitate AP in rat and mouse blood in support of the National Toxicology Program toxicokinetic and toxicology studies. Standards were prepared by spiking a 100-μL aliquot of blood with 50 μL of spiking solution containing AP and internal standard (IS; AP-d3) in 50/50 ethanol/saline in a 2-mL headspace vial. The vial was sealed with a metal crimp-top cap, equilibrated for 10 min at 60°C, and a 500 μL headspace sample was analyzed by GC-MS using single ion monitoring [m/z 136 (AP) and 139 (IS)]. A DB-5MS column was used with oven temperature ramped from 40°C to 150°C in 9 min. The method was successfully validated in male Sprague Dawley rat blood over the concentration range 5-500 ng/mL. Matrix standard curves were linear (r

≥ 0.99), and the percent relative error (%RE) values were ≤ ±15% for standards at all levels. Small background peaks were detected in the matrix and method blanks, but the response was <30% of the response for the lowest standard and did not interfere with method performance. The limit of detection, determined from the standard deviation at the lower limit of quantitation (5 ng/mL), was 0.5 ng/mL. Absolute recovery was ≥70% at all concentrations. Intra- and inter-day precision (% relative standard deviation, RSD) and accuracy (% relative error, RE) were ≤7% and ≤±13% respectively, for quality control (QC) standards prepared at 10, 100, and 250 ng/mL. Standards as high as 1500 ng/mL could be analyzed using a lower injection volume (150 μL), with %RE values ≤ ±19% and %RSD ≤1%. Loss of AP from blood occurred during overnight autosampler storage as well as frozen (-80°C) storage for 32 days, but incorporation of the IS prior to storage corrected for the loss such that determined concentrations were ≤ ±17% of fresh (Day 0) samples, with %RSD’s ≤5%. The method was evaluated for male and female Harlan Sprague Dawley (HSD) rat blood and B6C3F1 mouse blood; %RE values were ≤ ±9% and %RSD ≤4%. These data demonstrate that the method is suitable for the analysis of AP in rodent blood generated from toxicokinetic and toxicology studies.