Determination of r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in human urine by gas chromatography/negative ion chemical ionization/mass spectrometry

r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-B aP-tetraol) is the major hydrolysis product of r-7,t-8-dihydroxy-t-9,10-epo xy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the principal ultimate ca rcinogen of the environmental pollutant benzo[a]pyrene (BaP). As part of a program to establish activation/detoxification profiles of urinary metaboli tes of BaP in humans, we developed a method for quantifying trans-anti-BaP- tetraol. Urine was collected from three groups of individuals exposed to Ba P: psoriasis patients treated with a coal tar-containing ointment, steel wo rkers, and smokers. [H-2(12)]-trans-anti-BaP-tetraol was added to the urine as an internal standard. The urine was treated with beta-glucuronidase and sulfatase, and then the BaP-tetraols were enriched by reverse-phase and ph enylboronic acid solid-phase extraction. The resulting fraction was treated with sodium hydride and methylmethane sulfonate to convert BaP-tetraols to the corresponding tetramethyl ethers (BaP-TME). The mixture was purified b y normal-phase HPLC and analyzed by gas chromatography/negative ion chemica l ionization/mass spectrometry with selected ion monitoring. [(CH3)-C-13](4 )-trans-anti-BaP-TME was used as an external standard. Ions at mit 376, 380 , and 388 were monitored for quantitation of trans-anti-BaP-TME, [(CH3)-C-1 3](4)-trans-anti-BaP-TME, and [H-2(12)]-trans-anti-BaP-TME, respectively. T he instrumental detection limit was approximately 1 fmol of trans-anti-BaP- TME. trans-anti-BaP-tetraol las trans-anti-BaP-TME) was detected in 20 of 2 0 individuals receiving coal tar therapy (mean, 16 fmol/mL of urine), 13 of 13 exposed steel workers (mean, 4.1 fmol/mL of urine), and nine of 21 ciga rette smokers (mean, 0.5 fmol/mL of urine). The means in these groups were significantly different (P < 0.0001). The urine of steel workers was also a nalyzed for cis-anti-BaP-tetraol and cys-syn-BaP-tetraol, but neither was f ound. The results of this study provide a quantitative method for determina tion of parts per trillion levels of trans-anti-BaP-tetraol in human urine. Ultimately, this method can be employed as part of a phenotyping approach for assessing BaP metabolites in human urine.