High Throughput Analysis of the Polycyclic Aromatic Hydrocarbon Metabolite Phenanthrene Tetraol in Human Urine by Gas Chromatography-Tandem Mass Spectrometry

摘要

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous carcinogens believed to play a significant role as causes of lung cancer in smokers. PAH require metabolic activation to exert their carcinogenic effects. One major pathway proceeds through formation of bay region diol epoxides, which react with cellular DNA to form adducts that cause mutations in critical growth control genes. Phenanthrene is the simplest PAH with a bay region. It is metabolized via the bay region diol epoxide pathway yielding the urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT). We have proposed the use of PheT as a biomarker of PAH uptake and metabolic activation in smokers. Our goal is to use this and other biomarkers to identify those smokers at high risk for lung cancer. In this study, we developed a high throughput method for analysis of PheT in 100 μl human urine samples. The internal standard [~(13)C)_6]PheT was added and the urine was treated with β-glucuronidase and arylsulfatase in 96-well plates. Solid-phase extraction was carried out using Strata SDB-L plates in a 96-well format. The plate was conditioned with MeOH and equilibrated with H_2O before the incubation mixture and rinse were added. Clean up consisted of four successive washes: 1% NH_4OH:10% MeOH in H_2O; H_2O; 1% HCOOH:10% MeOH in H_2O; and H_2O. PheT was eluted with 0.7 mL 50% MeOH in H_2O, and condensed to dryness. The analyte fraction was derivatized with bis-trimethylsilyltrifluoroacetamide using [D_(10)]PheT as a derivatization and injection standard. The derivatization was carried out under anhydrous conditions to avoid crystallization of the by-products from reaction of BSTFA with H_2O. The 96-well plates were analyzed by GC-MS/MS in the negative ion chemical ionization mode, using a 30 m × 0.25 mm, 15 μm film thickness DB-17MS column, with selected reaction monitoring for at m/z 372.130 → m/z 210.70 for PheT-TMS, m/z 378.016 → m/z 216.070 for [~(13)C_6]PheT-TMS, and m/z 382.160 → m/z 220.070 for [D_(10)]PheT-TMS.