Development and validation of an analytical method for quantitation of Alpha-Pinene in rodent mammary tissue 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, 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 mammary tissue, a potential target tissue, in support of the National Toxicology Program toxicokinetic and toxicology studies. Standards were prepared by spiking a ~100 mg aliquot of mammary tissue with 100 μL of spiking solution containing AP and internal standard (IS; AP-d3) in 50/50 ethanol/saline in a 2-mL headspace vial containing 18 stainless steel beads. The vial was sealed and homogenized for 30 sec for 2 cycles at 1000 rpm. Each vial was equilibrated for 10 min at 60°C and a 200 μL headspace sample was analyzed by GC-MS using single ion monitoring [m/z 136 (AP); 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 female Sprague Dawley rat mammary tissue over the concentration range 100-5000 ng/g. Matrix standard curves were linear (r ≥ 0.99), and the percent relative error (%RE) values were ≤ ±12% for standards at all levels. Small background peaks were detected in the matrix and method blanks, but the response was low and did not interfere with method performance. Absolute recovery was low (2%) likely due to high lipophilicity of AP. However, the limit of detection, determined from the standard deviation at the lower limit of quantitation (100 ng/g), was 17.7 ng/g, demonstrating adequate sensitivity. Recoveries incorporating the IS were ≥90% at all concentrations. Intra- and inter-day precision (% relative standard deviation, RSD) and accuracy (%RE) were ≤6% and ≤±7%, respectively, for quality control standards prepared at 250 and 2500 ng/g. Standards as high as 20,000 ng/g could be analyzed using a lower injection volume (20 μL) or by extrapolating the calibration curve beyond 5000 ng/g, with mean %RE ≤ ±14% and %RSD ≤3%. Smaller sample sizes (~50 mg) could also be analyzed, with mean %RE ≤ ±2% and %RSD ≤2%. The method was evaluated for female Harlan Sprague Dawley rat and B6C3F1 mouse mammary tissues; %RE values were ≤ ±5% and %RSD ≤2%. These data demonstrate that the method is suitable for the analysis of AP in rodent mammary tissues generated from toxicokinetic and toxicology studies.