Development and validation of an analytical method for quantitation of alpha-pinene oxide in rodent blood and mammary tissue by 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 lumber industry. Despite widespread exposure, toxicity data for AP are limited. Alpha pinene oxide (APO) is a potential metabolite of AP in rodents and humans. Given its potential reactivity in tissues, establishing the extent of its formation and concentration in blood and tissues is an important component of the evaluation of the toxicity of AP. The objective of this work was to validate a method to quantitate APO in rat and mouse blood and mammary tissue, a potential target, in support of the toxicokinetic and toxicology studies. Standards were prepared by adding 5 μL of APO spiking solution to 100 μL rat blood followed by 300 μL ethyl acetate containing internal standard (IS, (+) limonene oxide). Sample was vortexed for 3 min and centrifuged for 3 min. The supernatant was analyzed by GC-MS in EI mode. The ions monitored were m/z 109 (APO) and 94 (IS). The method was successfully validated in male Sprague Dawley rat blood over the concentration range of 5.00 to 250 ng/mL. Matrix standard curves were linear (r ≥ 0.99), and accuracy measured as the percent relative error (%RE) values were ≤ ±15% for standards at all levels. Intra- and inter-day precision (% relative standard deviation, RSD) and accuracy (%RE) were ≤ 6.3% and ≤ ±5.4% respectively. The limit of detection determined from the SD of the lower limit of quantitation (5 ng/mL), was 1.07 ng/mL. Absolute recoveries were 100-114% across all standard levels. Standards as high as 25000 ng/mL could be analyzed with 1000x dilution with %RE ≤ ±7.1% and %RSD ≤4.5%. APO was stable in rat blood for at least 70 days in frozen storage (-80 °C). The method was evaluated in male and female Harlan Sprague Dawley rat blood and B6C3F1 mouse blood: %RE values were ≤ ±5.3% and %RSD ≤7.8%. The method was also evaluated in female B6C3F1 and SD rat mammary tissue. Approximately 50 mg mammary tissue was homogenized with a 950 μL of water. APO was added to give concentration in the range 25-500 ng/g mammary, 100 μL of homogenate was extracted with ethyl acetate and analyzed as described above for blood. Mean %RE values were ≤ ±14.6% and %RSD ≤8.1%. These results demonstrate that the method is suitable for the analysis of APO in rodent blood and mammary tissues generated from toxicokinetic and toxicology studies.