Development of a quantitative analytical method for 6PPD, a harmful tire antioxidant, in biological samples for toxicity assessment

This study developed and validated a high-sensitivity analytical method for the quantification of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), a hazardous antioxidant used in tires, to support the effective management of its environmental exposure. Using an HPLC-UV system with acetonitrile and deionized water as the mobile phase, the method did not require salts or acids. The calibration curve exhibited excellent linearity (R 2 = 0.9999) with detection limits of 0.17 pg μL –1 (LOD) and 5.51 pg μL –1 (MDL), underscoring its ultra-trace detection capability. A pretreatment protocol was developed to quantify 6PPD in mouse liver samples. The protocol achieved a relative recovery rate of 71.89 % and high precision across all spiking concentrations (mean RSD = 2.20 ± 0.92 %). The validated methods were applied to analyze 6PPD residues in mouse liver following intratracheal instillation exposure. At 9 h post-exposure, residual 6PPD concentrations increased significantly in a dose-dependent manner (R 2 = 0.9906), with a relative distribution of 0.06 ± 0.01 %. This cost-effective and reliable method provides a practical tool for quantifying 6PPD in environmental and biological matrices. The findings enhance the understanding 6PPD exposure dynamics and support the establishment of regulatory guidelines for managing its ecological and human health risks. • 6PPD is a toxic rubber antioxidant released into the environment through tire wear. • A simple HPLC-UV method was developed to quantify 6PPD without mobile phase additives. • A pretreatment method was developed to quantify 6PPD in mouse liver samples. • The MDL for 6PPD was 5.5 pg/μL, with a relative recovery of 72 % after pretreatment. • The relative distribution of 6PPD in mouse liver was determined to be 0.06 %.