Quantitative determination of hydroxy polycylic aromatic hydrocarbons as a biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons

• A new HRGC–HRMS method was developed for determination of 4 OH-PAHs.
• The causes of OH-PAHs instability were identified as photo induced oxidation.
• The current method controlled OH-PAH analyte degradation during analysis.
• Conjugated OH-PAHs are more stable than non-conjugated OH-PAHs.
• Accuracy and precision were 77.4–117% (<15% RSD) in synthetic and human urine samples.

A high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) method was developed for quantitative analysis of hydroxy polycyclic aromatic hydrocarbons (OH-PAHs). Four hydroxy metabolites of known and suspected carcinogenic PAHs (benzo[a]pyrene (B[a]P), benz[a]anthracene (B[a]A), and chrysene (CRY)) were selected as suitable biomarkers of PAH exposure and associated risks to human health. The analytical method included enzymatic deconjugation, liquid − liquid extraction, followed by derivatization with methyl-N-(trimethylsilyl) trifluoroacetamide and instrumental analysis. Photo-induced oxidation of target analytes − which has plagued previously published methods − was controlled by a combination of minimizing exposure to light, employing an antioxidant (2-mercaptoethanol) and utilizing a nitrogen atmosphere. Stability investigations also indicated that conjugated forms of the analytes are more stable than the non-conjugated forms. Accuracy and precision of the method were 77.4–101% (<4.9% RSD) in synthetic urine and 92.3–117% (<15% RSD) in human urine, respectively. Method detection limits, determined using eight replicates of low-level spiked human urine, ranged from 13 to 24 pg/mL. The method was successfully applied for analysis of a pooled human urine sample and 78 mouse urine samples collected from mice fed with PAH-contaminated diets. In mouse urine, greater than 94% of each analyte was present in its conjugated form.