Quantitation of phenanthrene dihydrodiols in the urine of smokers and non-smokers by gas chromatography-negative ion chemical ionization-tandem mass spectrometry

Polycyclic aromatic hydrocarbons (PAH) are well-established environmental carcinogens likely to be causative agents for some human cancers. Bay-region diol epoxides are ultimate carcinogenic metabolites of multiple PAH. Dihydrodiols are the important intermediate products of this pathway and can be further oxidized to form diol epoxides. We quantified two dihydrodiol metabolites of phenanthrene (Phe), the simplest PAH with a bay-region, in the 6 h urine of smokers (N = 25) and non-smokers (N = 25) using a newly developed and validated analytical method. After hydrolysis by ß-glucuronidase and sulfatase, and solid phase extraction, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). Levels (nmol/6h urine) of Phe-1,2-dihydrodiol (Phe-1,2-D) and Phe-3,4-dihydrodiol (Phe-3,4-D) were 2.04 ± 1.52 and 0.51 ± 0.35, respectively, in smokers, significantly higher than those in non-smokers (1.35 ± 1.11 of Phe-1,2-D, p < 0.05; 0.27 ± 0.25 of Phe-3,4-D, p < 0.005). Cigarette smoking also influenced the regioselective metabolism of Phe, presenting as a significant difference in the urinary distribution pattern of Phe-1,2-D and Phe-3,4-D between smokers and non-smokers: the ratio Phe-3,4-D: Phe-1,2-D increased from 0.20 in non-smokers to 0.28 in smokers (p < 0.01), which can be explained by the induction of the phenanthrene metabolizing enzymes CYP1A2 and CYP1B1 by cigarette smoke. The method described here is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings and will be applicable in clinical and molecular epidemiology studies of PAH metabolism and cancer susceptibility.