Urinary Benzo[a]pyrene and Its Metabolites as Molecular Biomarkers

Anal. Chem. 2003, 75, 5953-5960

Urinary Benzo[a]pyrene and Its Metabolites as Molecular Biomarkers of Asphalt Fume Exposure Characterized by Microflow LC Coupled to Hybrid Quadrupole Time-of-Flight Mass Spectrometry Jin J. Wang,* David G. Frazer, Samuel Stone, Travis Goldsmith, Brandon Law, Amy Moseley, Janet Simpson, Ali Afshari, and Daniel M. Lewis

National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Morgantown, West Virginia 26505

As a step to study the health effects of asphalt fume exposure, an analytical method was developed to characterize benzo[a]pyrene and its hydroxy metabolites in the urine of asphalt fume-exposed rats. This method is based on microflow liquid chromatography (LC) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). Twenty-four female Sprague-Dawley rats were used in the experiment, with 8 as controls and 16 exposed to asphalt fumes in a whole-body inhalation chamber for 10 days (4 h/day). Generated at 150 °C, the asphalt fume concentration in the animal exposure chamber ranged 76-117 mg/m3. In the urine of the asphalt fume-exposed rats, benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene7,8-dihydrodiol((), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() were identified, and their concentrations were determined at 2.19 ( 0.49, 16.17 ( 0.3, 6.28 ( 0.36, and 29.35 ( 0.26 ng/100 mL, respectively. The metabolite concentrations from the controlled group, however, were either under the detection limits or at a relatively very low level (0.19 ( 0.41 ng/100 mL for benzo[a]pyrene-7,8,9,10-tetrahydrotetrol metabolite). The results clearly indicate that the benzo[a]pyrene and its hydroxy metabolites were significantly elevated (p < 0.001) in the urine of asphalt fume-exposed rats relative to controls. The study also demonstrated that the combination of microflow LC separation and collision-induced dissociation leading to a characteristic fragmentation pattern by hybrid Q-TOFMS offers a distinct advantage for the identifications and characterizations of the benzo[a]pyrene metabolites. A considerable number of workers in several industries are exposed to asphalt fumes. It is estimated that ∼4000 hot-mix asphalt facilities and 7000 paving contractors employ nearly 300 000 workers in the United States.1 Chronic asphalt exposure by inhalation or skin contamination may cause long-term health * To whom correspondence should be addressed. Phone: (304) 285-6329. Fax: (304) 285-6126. E-mail: [email protected]. 10.1021/ac030017a Not subject to U.S. Copyright. Publ. 2003 Am. Chem. Soc.

Published on Web 10/04/2003

effects among road paving and highway maintenance workers.2 Standardized mortality ratios of lung cancer were lower among workers for ground and building constructions than among bitumen workers.3 Many epidemiologic studies also revealed that there were an increase of leukemia in roofer populations and an association between lung, stomach, and nonmelanoma skin cancers and the asphalt fume exposure.4 Studies with animal models also indicated that asphalt fume condensates were genotoxic and could produce skin tumors in mice.5 Hence, occupational exposed to asphalt fume may pose health risks to workers.6 Asphalt fume contains aliphatic, polycyclic aromatic hydrocarbons (PAHs), heterocyclic compounds, and some nitrogen-, oxygen-, and sulfur-containing compounds.7 These persistent organic compounds are very nonpolar and exhibit a high accumulation potential in living systems.8 Chemical characterization of the asphalt fume has confirmed the existence of PAHs including carcinogenic benzo[a]pyrene.9 The amount of 0.1-2 mg/m3 bitumen fume that road pavers could be exposed to may include 10-200 ng/m3 benzo[a]pyrene.10 While the fume condensates displayed genotoxicity, a linear relation exists between 1-hydroxy(1) United States Asphalt Usage Report; Asphalt Institute: College Park, MD, 1989. (2) Binet, S.; Pfohl-Leszkowicz, A.; Brandt, H.; Lafontaine, M.; Castegnaro, M. Sci. Total Environ. 2002, 300, 37-49. (3) Boffetta, P.; Burstyn, I.; Partanen, T.; Kromhout, H.; Svane, O.; Langard, S.; Jarvholm, B.; Frentzel-Beyme, R.; Kauppinen, T.; Stucker, I.; Shaham, J.; Heederik, D.; Ahrens, W.; Bergdahl, I. A.; Cenee, S.; Ferro, G.; Heikkila, P.; Hooiveld, M.; Johansen, C.; Randem, B. G.; Schill, W. Am. J. Ind. Med. 2003, 43 (1), 18-27. (4) Partanen, T.; Boffetta, P. Am. J. Ind. Med. 1994, 26 (6), 721-740. (5) Sivak, A.; Niemeier, R.; Lynch, D.; Beltis, K.; Simon, S.; Salomon, R.; Latta, R.; Belinky, B.; Menzies, K.; Lunsford, A.; Cooper, C.; Ross, A.; Bruner, R. Cancer Lett. 1997, 117 (1), 113-23. (6) Lutes, C. C.; Thomas, R. J.; Burnette, R. Evaluation of emissions from paving asphalts; Final Report to U.S. EPA, Prepared by Acurex Environmental Corp., Research triangle Park, NC, 1994. (7) Machado, M. L.; Beatty, P. W.; Fetzer, J. C.; Glickman, A. H.; McGinnis, E. L. Fund. Appl. Toxicol. 1994, 22 (2), 317. (8) Harvey, R. G. Polycyclic Aromatic Hydrocarbons; Wiley-VCH: New York, 1991. (9) Wang, J.; Lewis, D. M.; Castranova, V.; Frazer, D. G.; Goldsmith, T.; Tomblyn, S.; Simpson, J.; Stone, S.; Afshari, A.; Siegel, P. D. Anal. Chem. 2001, 73, 3691-3700. (10) Burstyn, I.; Kromhout, H.; Kauppinen, T.; Heikkila, P.; Boffetta, P. Ann. Occup. Hyg. 2000, 1, 43-56.

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pyrene excretion and the pyrene content of the fumes.11 Therefore, these compounds are likely the major toxicants that may cause the adverse health effects.12 However, the detailed mechanisms of the specific effects regarding exposure to carcinogenic compounds from asphalt fumes have not been sufficiently understood. The metabolites of these chemicals from a living system may be used as biomarkers for studying the mechanisms of the health effects and the assessment of the exposure.13 Urinary naphthols have been used as biomarkers for assessing the airborne PAH exposure.14 A method for measuring urinary PAH metabolites has been proposed to assess the health risk for each individual at a PAH-burdened workplace.15 It has been reported that PAH exposure to coke plant workers during several consecutive days resulted in fairly constant individual urinary PAH metabolite profiles.16 This study also found that there was a correlation between inhaled PAH and metabolites excreted. A liquid chromatography (LC) method with fluorescence detection was reported for the determination of 3-hydroxybenzo[a]pyrene and 3-hydroxybenz[a]anthracene in the urine of PAH-exposed workers.17 The applications of liquid chromatography-mass spectrometry in analysis of workplace and environmental samples continue to increase.18-20 However, the analytical challenges associated with developing a reliable method for quantifying the PAHs and their metabolites in a living system are still considerable. The collision-induced dissociation with hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (QTOFMS) could lead to a characteristic fragmentation pattern of the benzo[a]pyrene metabolites. The fragment information may help in understanding the structure of the metabolites, identifying new biomarkers for the risk assessment, and studying the mechanisms of asphalt fume-induced health effects. However, such an alternative approach has not been applied to the study of asphalt fume exposure. Therefore, the specific aims of current study are (i) to develop an analytical method based on a microflow LC/Q-TOFMS technology, (ii) to establish the fragmentation patterns to identify the benzo[a]pyrene metabolites, and (iii) to quantify urinary benzo[a]pyrene and its hydroxy metabolites in the urine of asphalt fume-exposed rats. (11) Genevois, C.; Brandt, H. C. A.; Bartsch, H.; Obrecht-Pflumio, S.; Wild, C. P.; Castegnaro, M. Polycyclic Aromat. Compd. 1996, 8, 75-92. (12) World Health Organization. IARC Monogr. Eval. Carcinogenic Risk Chem. Hum. 1985, 35. (13) Butler, M. A.; Burr, G.; Dankovic, D.; Lunsford, A.; Miller, A.; Nguyen, M.; Olsen, L.; Sharpnack, D.; Snawder, J.; Stayner, L.; Sweeney, M. H.; Teass, A.; Wess, J.; Zumwalde, R. A report of CDC/NIOSH, Health effects of Occupational Exposure to Asphalt, 2000. (14) Yang, M.; Koga, M.; Katoh, T.; Kawamoto, T. Arch. Environ. Toxicol. 1999, 36, 99-108. (15) Grimmer, G.; Jacob, J.; Dettbarn, J.; Naujack, K. W. Int. Arch. Occup. Environ. Health 1997, 69, 231-239. (16) Buchet, J. P.; Gennart, J. P.; Mercado-Calderon, F.; Delavignette, J. P.; Cupers, L.; Lauwerys, R. Br. J. Ind. Med. 1992, 49, 761-768. (17) Gundel, J.; Angerer, J. J. Chromatogr., B 2000, 738, 47-55. (18) Wang, J.; Siegel, P. D.; Lewis, D. M.; Ashley, K.; Stettler, L. E.; Wallace, W. E.; Vo, E. In Spectroscopic Techniques in Industrial Hygiene. Encyclopedia of Analytical Chemistry; Meyers, R. A., A., Ed.; John Wiley & Sons: Ltd.: Chicester, U.K., 2000; Vol. 6, (19) Baere, S. D.; Cherlet, M.; Baert, K.; Backer, P. D. Anal. Chem. 2000, 74, 1393-1401. (20) Melikian, A. A.; O’Connor, R.; Prahalad, A. K.; Hu P.; Li, Heyi; Kagan, M.; Thompson, S. Carcinogenesis 1999, 20 (4), 719-726.

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EXPERIMENTAL SECTION Materials and Instruments. Reagent grade dichloromethane (99.9+%), acetonitrile, hexane, and perdeuterated anthracene were purchased from Aldrich (Milwaukee, WI). Reference metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(() and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() were purchased from NCI Chemical Carcinogen Reference Standard Repository (Kansas, MI). 6-Hydroxychrysene (ring 13C6, 98%) was purchased from Cambridge Isotope Laboratories (Andover, MA). QTM PAH Mix reference material was purchased from Supelo (Bellefonte, PA). ES Tuning Mix was purchased from Agilent Technologies (Wilmington, DE). Poly(tetrafluoroethylene) (PTFE) filters (37 mm, 0.45-µm pore size) and XAD-2 traps (treated with 2-(hydroxymethyl)piperidine) were purchased from SKC (Eighty Four, PA). Solid-phase extraction cartridges of EnvirElut PAH (500 ng/ 2.8 mL) were purchased from Varian (Harbor City, CA). PTFE tubes (30 mL) and glass tubes (10 mL) were purchased from Fisher Scientific (Pittsburgh, PA). Target DP vials (1.5 mm with 200-µL inserts) were obtained from Alltech Associates, Inc. (Deerfield, IL). Extraction of asphalt fumes from collection media was performed with dichloromethane by ultrasonic extraction (FS220, Ultrasonic power 320 W, Fisher Scientific, Fairlawn, NJ). A BenchMate II Workstation (solid-phase extractor, Zymark, Hopkinton, MA) and syringe filter (25 mm, 0.2-µm pore size) specially designed for HPLC samples (Gelman Sciences, Ann Arbor, MI) were employed to perform the purification and filtration of the sample solutions. Extracts were reduced under a nitrogen stream using a TurboVap LV evaporator (Zymark). Liquid nitrogen, highpurity helium, and argon were purchased from Butler Gas Products Co. (Mckees Rocks, PA), and used as GC/MS (HewlettPackard, Wilmington, DE) and hybrid Q-TOFMS II (Micromass Inc. Beverly, MA) carrier gases. The GC column was HP-5 MS, 95% dimethylpolysiloxane, nonpolar, 30-m length, 0.53-mm i.d. (thick film 530 id, Hewlett-Packard), and the microflow LC column used was Nucleosil C18 PAH, 5 µm, 1000-µm i.d. (LC Packings, San Francesco, CA). The test asphalt was used by the paving industry (Hot Performance Grade Asphalt PG 64-22). Twenty-four female Sprague-Dawley rats (6-8 weeks) were purchased from Hilltop Lab Animals Inc. (Scottdale, PA). Animal Inhalation Exposure and Asphalt Fume Sample Collection. Twenty-four female Sprague-Dawley rats were certified to be pathogen free and postexamined by a veterinarian. The rats were individually housed in metabolism cage during the twoweek acclimatization period. The treatment of animals for inhalation exposure study followed strictly the regulation of new Centers for Disease Control and Prevention Animal Use protocol. The test rats received the asphalt fume exposure in a whole-body inhalation chamber for 4 h/day over 10 days. The generation of asphalt fume was conducted in the National Institute for Occupational Safety Health inhalation facility. A dynamic asphalt fume generation system (Heritage Research Group, Indianopolis, IN) was modified to provide the asphalt fume.9 A computer control system was incorporated into the system to improve performance and to simplify operation. The test asphalt was representative of the formulation used throughout midwestern United States. For fume generation, the asphalt was initially preheated in an oven to 170 °C and then pumped to a large bitumen kettle that maintained the asphalt temperature. The heated asphalt was then transferred

to the generator where the fume was produced above the asphalt surface as the asphalt flowed over a heated generator plate. Air, heated to 150 °C, passed over the upper surface of the asphalt and transported the volatile fraction to the animal exposure chamber via a short heated transfer line. The test chamber can hold eight rats for the exposure period. The sampling train for the fumes consisted of a PTFE filter collection of particles, followed by a second stage, an XAD-2 tube (treated with 2-(hydroxymethyl)piperidine) that trapped the vapor fraction from the influent (1.0 L/min) vapor. Fume samples were collected immediately post the generator and at the entry into the exposure chamber. The test animals were sacrificed immediately after the last exposure. The urine of exposed rat was collected once per day, and the nonexposed rat urine acted as controls. Urine Sample Collection and Preparation. The urine from exposed rats was collected into a polypropylene tube every 24 h through a rat metabolism cage and was frozen immediately at -20 °C. The control urine was collected from nonexposed rats for 8 days. A few processing steps were performed to manipulate the urine samples into a form ready for analysis. The procedures involved hydrolysis of conjugated benzo[a]pyrene metabolites and purification of them from interferences before determination by microflow LC/Q-TOFMS. The condition of enzymatic hydrolysis conjugates in urine was optimized by testing several levels of enzyme concentrations. Briefly, to a 20-mL volume of urine was added 20 µL of β-glucuronidase/arylsulfatase (stock solution) and the pH was adjusted to 5.0 with 1 M hydrochloric acid. The sample solution was incubated for 16 h at 37 °C. An internal standard, 6-hydroxychrysene (ring 13C6), customized from Cambridge Isotope Laboratories, was added to the sample at a final concentration of 10 ng/µL. The urine purification stage was performed using a solid-phase extraction to remove impurities that could interfere in target analyte detection. In operation, the urine was transferred to a smooth-walled test tube. This tube was loaded onto the sample position of the BenchMate II Workstation. The EnvirElut PAH cartridge (Varian) was conditioned with a solution of 80% water + 20% acetonitrile (6 mL). After the sample was loaded, the cartridge was washed with water (6 mL) and dried for 1 min by air. Then, the target analytes were slowly eluted and collected with diethyl ether (6 mL). Finally, the extract solutions were preconcentrated by reducing the solvent of sample under a nitrogen stream using a TurboVap LV evaporator. Control urine samples were prepared using the entire analytical procedure as well as the same reagents as those used for treating asphalt fume urinary samples. A recovery experiment was carried out using spiked solutions (range of 10-40 ng/µL) of 6-hydroxychrysene, benzo[a]pyrene, 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8dihydrodiol((), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol((). Asphalt Fume in Animal Exposure Chamber. Asphalt fume sample preparation involved desorption, filtration, and preconcentration to determine the asphalt fume concentration in the animal exposure chamber. Samples from either the PTFE filter or the XAD-2 trapping absorbant were transferred to separate PTFE tubes, and dichloromethane/hexane (50:50) was added. Ultrasonic extraction was performed using an FS-220 ultrasonicator (320 W). After desorption of asphalt fumes from the collection medium, the extract was filtered using a BenchMate II Workstation, which was programmed to perform procedures automatically. Precon-

centration was performed under nitrogen using the TurboVap LV evaporator. Sample extracts were reconstituted with dichloromethane. Concentrations of asphalt fumes in animal exposure chamber were determined by GC/MS. The instrument was calibrated by a mixture of 16 reference PAHs, and perdeuterium anthracene was used as internal standard. The total ion chromatograms were acquired with 3-min solvent delay. Separation was performed on a HP-5 MSD capillary column (30-m length, 0.53-mm i.d) with a temperature program from 50 to 310 °C at an increasing rate of 5 °C/min. The calibration curve was developed with five-point measurements, and the asphalt fume was determined by GC/MS. Microflow LC/Q-TOFMS. The coupling of a microflow LC to hybrid a Q-TOFMS system offers high-accuracy mass measurement (∼5 ppm) and resolution (∼10 000) for developing a selective bioanalytical method. Electrospray ionization combined with TOF MS/MS is particularly useful for selected precursor ion monitoring. A Nucleosil C18 PAH column (5 µm, 1000-µm i.d) was used in microflow LC. A gradient elution profile (20 µL/min) was generated with solvents (A) 90% water + 10% acetonitrile + 0.1% formic acid and (B) 90% acetonitrile + 10% water + 0.1% formic acid. The separation was conducted with a mobile phase that contained 95% A at 0-10 min to 85% A at 10-15 min and then to 75% A at 15-18 min, to 60% A at 18-20 min, to 30% and at 20-25 min, and to 5% A at 30 min. After running for 30 min, the mobile phase was back to 95% A at 31-50 min. The source temperature was set at 90 °C. The instrument was calibrated with a multipoint calibration technique using selected fragment ions (m/z: 333, 480, 684, 813, 942, 1056, and 1285) that resulted from the collisioninduced decomposition of [Glu]-fibrinopeptide B (Sigma Chemical Co.). The limits of detection was less than 20 fmol of [Glu]fibrinopeptide B determined by the signal-to-noise ratio greater than 3:1 for TOFMS acquisition. The limit of the measurement (LOQ) was determined at less than 30 fmol for TOF MS/MS acquisition. The linear dynamic range was greater than 3 orders of magnitude (0.1-50 pmol). In all cases, calibration regression coefficients were between 0.95 and 0.99. The precision was determined by repeating the injection 5 times (RSD < 15). Once optimized, operating parameters were maintained constant throughout the experiment. RESULTS LC Separation and Tandem MS Fragmentation Pattern. Benzo[a]pyrene and its hydroxy metabolites were separated by microflow and characterized by Q-TOFMS. A gradient elution from a PAH column as described in the Experimental Section was optimized. The detection limits of reference standard benzo[a]pyrene metabolites were 40-60 pg based on a signal-to-noise ratio greater than 3:1. With the conditions as described above, the greatest ion intensities in the current experiment were observed via proton transfer. During collision-induced dissociation, the metabolite ion under investigation is collided with a collision gas and acquires internal energy, which leads to its decomposition into fragment ions and generation of a specific TOFMS/MS spectrum pattern. For the Q-TOF MS/MS scan, the precursor ions of m/z 321 (C20H16O4 + H)+, m/z 287 (C20H14O2 + H)+, m/z 269 (C20H12O + H)+, and m/z 253 (C20H12 + H)+ were selected to determine benzo[a]pyrene-7,8,9,10-tetrahydrotetrol((), benzo[a]pyrene-7,8-dihyAnalytical Chemistry, Vol. 75, No. 21, November 1, 2003

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Figure 1. Microflow LC/Q-TOFMS/MS acquisition profiles with selected precursor ions (a) benzo[a]pyrene-7,8,9,10-tetrahtdrotetrol((), (b) benzo[a]pyrene-7,8-dihydrodiol((), (c) 3-hydroxybenzo[a]pyrene, (d) 2-hydroxychrysene (ring 13C6), and (e) benzo[a]pyrene.

drodiol((), 3-hydroxybenzo[a]pyrene, and benzo[a]pyrene. Figure 1 presented Q-TOF MS/MS acquisitions with selected precursor ions from a mixture of a reference standards solution. With the optimized microflow LC/Q-TOFMS conditions as described in the Experimental Section, benzo[a]pyrene-7,8,9,10tetrahydrotetrol (MH+, 321) was eluted at retention time (RT) 5-7 min and acquired through channel 1 (Figure 1a). Benzo[a]pyrene7,8-dihydrodiol (MH+, 287) was eluted at RT 22-24 min and acquired through channel 2 (Figure 1b). 3-Hydroxybenzo[a]pyrene (MH+, 269) was eluted at RT 23-25 min and acquired through channel 3 (Figure 1c). 6-Hydroxychrysene (MH+, 251) used as an internal standard was eluted at RT 30-32 min and acquired through channel 4 (Figure 1d). Benzo[a]pyrene (MH+, 253) was eluted at RT 38-41 min and acquired through channel 5 (Figure 1e). A Q-TOF MS/MS fragmentation pattern was produced to further identify the target analyte. Monitoring a metastable decomposition reaction with a TOF MS/MS scan provides a means by which to increase the selectivity of an assay for a given analyte. An optimized TOF MS/MS spectrum of benzo[a]pyrene7,8,9,10-tetrahydrotetrol(() was presented in Figure 2a, and the collision energy was optimized at 10 eV. The singly charged benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() metabolite ion was observed at m/z 321.1298 (C20H16O4 + H)+. The fragmentation was observed to cause dehydration to occur. The major fragment m/z 303.1604 resulted from the precursor ion when it lost one H2O. The fragment m/z 285.2782 was formed from ion m/z 303.1604 when it lost another H2O. The other fragment included ion m/z 257.2604 under the experimental conditions. An optimized TOF MS/MS spectrum of benzo[a]pyrene-7,8-dihydrodiol(() was presented in Figure 3a, and the collision energy was optimized at 12 eV. The singly charged metabolite ion was observed at m/z 287.1064 5956 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

(C20H14O2 + H)+. Three major fragments were observed under the experimental conditions. The fragments included ions m/z 241.0912, 258.0933, and 269.0847 produced from the precursor ion when it lost one H2O. An optimized TOF MS/MS spectrum of 3-hydroxybenzo[a]pyrene was presented in Figure 4a. The singly charged metabolite ion was observed at m/z 269.0948 (C20H12O + H)+. Two major fragments were observed under the experimental conditions. The fragments included ions m/z 241.2085 and 251.1559 produced from the precursor ion when it lost one H2O. An optimized TOF MS/MS spectrum of benzo[a]pyren was presented in Figure 5a. The singly charged molecular ion was observed at m/z 253.1070 (C20H12 + H)+. Two major fragments m/z 226.2949 and 202.2258 were observed under the experimental condition. An optimized TOF MS/MS spectrum of an internal standard molecular ion, 6-hydroxychrysene, was observed at m/z 251.2143 (13C6C12H12O + H)+. Two major fragments m/z 231.2603 and 209.2609 were observed. Determination of Benzo[a]pyrene and Its Metabolites in Urine. The levels of benzo[a]pyrene and its metabolites 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol((), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() were determined by combination of LC separation and a unique tandem MS fragmentation pattern. To achieve maximal selectivity, two criteria, (1) retention time from microflow LC column and (2) a characteristic fragmentation pattern of the TOF MS/MS spectrum, which matched the behavior of its reference standard, were set to quantify benzo[a]pyrene and its hydroxy metabolites. Animal exposure trials were conducted twice (eight rats for each period) with the same exposure conditions (exposure time and temperature of fume generation). To evaluate the recovery of the target analytes, a stable isotope 6-hydroxychrysene (ring 13C6C12H12O) was used as an internal standard. The relative recovery of the

Figure 2. Optimized tandem MS fragmentation pattern of benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() metabolite detected from (a) a reference standard solution and (b) urine of asphalt fume-exposed rats.

Figure 3. Optimized tandem MS fragmentation pattern of benzo[a]pyrene-7,8-dihydrodiol(() metabolite detected from (a) a reference standard solution and (b) urine of asphalt fume-exposed rats.

internal standard can account for losses of the analytes during sample preparation and detection processes. The recoveries of spiked reference standard metabolites ranged 65-75%, and slightly lower recovery was observed for benzo[a]pyrene-7,8dihydrodiol((). The selected precursor metabolite ion was monitored for quantification of each target analyt. The results were obtained based on calculations from a calibration curve that was developed from the total counts (peak area) of TOF MS/MS acquisition of a reference standard. The concentrations of experimentally determined benzo[a]pyrene and its hydroxy metabolites were summarized in Table 1. The urine from each rat was individually analyzed. The mean

concentration (n ) 50) of benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() was determined at 29.35 ( 0.26 ng/100 mL of urine from exposed rats. The data were obtained based on a TOF MS/MS acquisition profile with selected precursor ion m/z 321 (C20H16O4 + H)+. The target metabolite eluted from LC column at retention times of 5-7 min. An optimized TOF MS/MS spectrum from urine of asphalt fume-exposed rats is presented in Figure 2b. The singly charged benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() metabolite ion was observed at m/z 321.1256 (C20H16O4 + H)+. Three major fragments were observed. The fragments included ions m/z 257.3124, 303.1406 produced from molecular ion when it lost one H2O, and 285.2278 produced from ion m/z 303.1406 when it lost Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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Figure 4. Optimized tandem MS fragmentationi pattern of 3-hydroxybenzo[a]pyrene metabolite detected from (a) a reference standard solution and (b) urine of asphalt fume-exposed rats.

Figure 5. An optimized tandem MS fragmentation pattern of benzo[a]pyrene detected from (a) a reference standard solution and (b) urine of asphalt fume-exposed rats.

another H2O. The fragmentation pattern was identical to that observed from reference standard benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() solution (Figure 2a). The concentration of benzo[a]pyrene-7,8-dihydrodiol(() based on a TOF MS/MS acquisition profile (RT 22-24) with selected precursor ion m/z 287 (C20H14O2 + H)+ was determined at 6.28 ( 0.36 ng/100 mL of urine from exposed rats. An optimized TOF MS/MS spectrum from urine of exposed rats was presented in Figure 3b. The singly charged benzo[a]pyrene-7,8-dihydrodiol(() metabolite ion was observed at m/z 287.1164 (C20H14O2 + H)+. Three major fragments were observed. The fragments included ions m/z 241.1075, 258.1098, and 269.1015 produced from molecular ion when it lost one H2O. The fragmentation pattern was identical to that observed from 5958 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

the reference standard solution (Figure 3a). The concentration of 3-hydroxybenzo[a]pyrene based on a TOF MS/MS acquisition profile (RT 23-25) with selected precursor ion m/z 269 (C20H12O + H)+ was determined at 16.17 ( 0.35 ng/100 mL of urine from exposed rats. An optimized TOF MS/MS spectrum from urine of exposed rats is presented in Figure 4b. The singly charged 3-hydroxybenzo[a]pyrene ion was observed at m/z 269.0948 (C20H12O + H)+. Two major fragments m/z 241.2085 and 251.1494 produced from molecular ion when it lost one H2O were observed. The fragmentation pattern was identical to that observed from the reference standard solution (Figure 4a). The concentration of benzo[a]pyren based on a TOF MS/MS acquisition profile (RT 38-41) with selected precursor ion m/z 253 (C20H12 + H)+ was

Table 1. Determined Concentrations of Benzo[a]pyrene and Its Hydroxy Metabolites from Urine of Asphalt Fume Exposed Rats

major m/z found

exposed urine mean ( RSDa (ng/100 mL)

control urine mean ( RSDa (ng/100 mL)

321.1352, 303.1404, 285.2991, 257.2669

29.35 ( 0.26

0.19 ( 0.41

287.1164, 269.1015, 258.1098, 241.1075

6.28 ( 0.36

BDLb

269.0948, 251.1494, 241.2085 253.1134, 226.2010, 202.2374

16.17 ( 0.35 2.19 ( 0.49

BDL BDL

benzo[a]pyrene and its hydroxy metabolite benzo[a]pyrene-7,8,9,10tetrahydrotetrol (() benzo[a]pyrene-7,8dihydrodiol(() 3-hydroxybenzo[a]pyrene benzo[a]pyrene a

Relative standard deviation, n ) 50. b BDL, below detection limit.

Table 2. Concentrations of Asphalt Fumes in Animal Exposure Chamber exposure (day)

asphalt fume filter collection (mg/m3 ( RSDb)

asphalt fume XAD-2 collection (mg/m3 ( RSDb)

concentrations total (mg/m3 ( RSDb)

1-5 6-10

36.66 ( 0.16 48.06 ( 0.21

40.11 ( 0.20 69.74 ( 0.29

76.77 ( 0.18 117.80 ( 0.20

a

Ten exposure days, 4 h/day. b RSD, relative standard deviation.

determined at 2.19 (0.49 ng/100 mL of urine from exposed rats. An optimized TOF MS/MS spectrum is presented in Figure 5b. The singly charged benzo[a]pyrene ion was observed at m/z 253.1134 (C20H12 + H)+. Two major fragments m/z 226.2010 and 202.2374 were observed that were identical to those observed from benzo[a]pyrene reference standard solution (Figure 5a). 6-Hydroxychrysene acted as an internal standard. Molecular ion m/z 251.2143 of 6-hydroxychrysene, and two major fragments (m/z 231.2603 and 209.2609) were observed from the optimized TOF MS/MS spectrum. The relative standard deviation for urine from asphalt fume-exposed rats ranged 26-49% over 10 days. The control urine samples were analyzed under the same experimental conditions. The concentrations of benzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol((), and 3-hydroxybenzo[a]pyrene from the control group rats were under the limits of detection except that benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() was determined at 0.19 ( 0.41 ng/100 mL. Concentrations of Asphalt Fumes in Animal Exposure Chamber. Concentrations of asphalt fumes in the animal exposure chamber were determined by positive electron ionization GC/ MS. Isotope dilution with perdeuterated anthracene as an internal standard was used in the analysis. The dynamic range of GC/ MS exhibited by the PAH mixture was linear over 2 orders of magnitude (10-1000 ng). The coefficients of determination, r2, ranged from 0.98 to 0.99 for the calibration curves. Isotope dilution with perdeuterated anthracene as an internal standard was used in the analysis. The relative recovery of the internal standard can account for losses of the analytes during sample preparation and detection processes. The results of cumulative exposure to asphalt fume over 10 days are summarized in Table 2. The asphalt fume particulates collected on filters ranged 36.66-48.06 mg/m3, and the semivolatile fumes trapped on XAD-2 tubes ranged 40.1169.74 mg/m3. The total asphalt fume concentrations ranged 76.77-117.80 mg/m3, and the relative standard deviation ranged 18-28% over the 10 days of exposure.

DISCUSSION AND CONCLUSION Asphalt fume is a complex mixture that contains a wide variety of toxicants. The determination of the exact correlation between the specific health effects and the specific exposure conditions is a difficult task. To advance understanding of the exposure and its health effects, and to develop more a effective approach for exposure risk assessment, it is required not only to quantify the benzo[a]pyrene but also to identify its metabolites resulting from the exposure. In this study, an analytical method based on microflow LC and Q-TOFMS technologies was developed and used to characterize benzo[a]pyrene and its metabolites in the urine of asphalt fume-exposed rats. Three benzo[a]pyrene metabolites including 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol((), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(() are reliably identified in this study. The results clearly indicate that the concentrations of benzo[a]pyrene and its hydroxy metabolites from urine of asphalt fumeexposed rats are significantly (p < 0.001) higher than those from the control animals. This confirms that the benzo[a]pyrene from the asphalt fumes can be absorbed by a living system and be involved in the metabolic processing. Therefore, benzo[a]pyrene and its hydroxymetabolites from the urine of the exposed living system can be used as biomarkers for exposure assessment. When the LC flow is reduced to nanolitters, it is possible to further reduce the detection limit to the subpicogram level. The accuracy and reliability of the method may thus be further enhanced and used to detect benzo[a]pyrene and its hydroxy metabolites in the samples of urine from workers exposed to asphalt fumes in the field. Other techniques such as HPLC/UV and HPLC/fluorescence detection can also be used to measure metabolites in the living system.18 Such techniques may also be cost-effective. Their identifications of the compounds, however, are only based on their retention time. This approach cannot provide specific information on the chemical identity and the toxic progenitor. With the combined LC and Q-TOFMS techniques, the identifications are based on three criteria: (i) retention time, (ii) selected precursor ion monitoring, and (iii) fragment pattern. The current approach greatly increases the reliability of the identification. Furthermore, it provides some information on the chemical structures of the benzo[a]pyrene and its metabolites. This information may be useful to advance understanding of how the hazards attribute to toxicity and to cause adverse health effects. In summary, while the method developed in this study can be potentially used as an alternative tool for the assessment of asphalt Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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fume exposure, it provides a new opportunity to investigate the mechanisms of the adverse health effects associated with the exposure.

extended to Aaron T. Timperman and Vo Evanly for review of the manuscript.

ACKNOWLEDGMENT The authors gratefully thank Al Munson, Paul Siegel, Vincent Castranova, Nancy Bollinger, Jean Meade, Judy Mull, and Beverly Carter for their project support and coordination. Thanks are also

Received for review January 7, 2003. Accepted July 18, 2003.

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