Monitoring Polycyclic Aromatic Hydrocarbon Metabolites in Human

Institute of Hydrochemistry, Technical University Munich, Marchioninistrasse 17, 81377 Munich, Germany, and GSF-National Research Center for Environme...
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Anal. Chem. 2001, 73, 5669-5676

Monitoring Polycyclic Aromatic Hydrocarbon Metabolites in Human Urine: Extraction and Purification with a Sol-Gel Glass Immunosorbent Matthias Schedl,† Gottfried Wilharm,† Stefan Achatz,‡ Antonius Kettrup,‡ Reinhard Niessner,† and Dietmar Knopp*,†

Institute of Hydrochemistry, Technical University Munich, Marchioninistrasse 17, 81377 Munich, Germany, and GSF-National Research Center for Environment and Health, Institute of Ecological Chemistry, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany

A new, rapid method for selective extraction of hydroxylated polycyclic aromatic hydrocarbons metabolites (OHPAHs) in human urine was developed using an immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix. Resulting chromatograms after immunoextraction of urine samples and HPLC analysis of the extracts were free from matrix interferences. The LODs for the determination of OHPAHs in these difficult samples were in the low-ppt range (1-16 ng/L). In addition to its high selectivity, the immunosorbent proved to be robust and reusable. Obtained recoveries in spiked urine samples ranged from 83 to 107% for the hydroxyphenanthrene and hydroxypyrene compounds under investigation, while recovery for 3-hydroxybenzo[a]pyrene was only 45-62%. In a biomonitoring study, the SGG immunosorbent was successfully used for trace-level analysis of OH-PAHs in 20 human urine samples. Results were compared to data obtained by an independent reference analysis method and revealed good correlation between both methods. In risk assessment as in occupational and environmental health, one of the most critical points is the characterization of exposure of the population to the pollutants under consideration. Occupational hygienists have spent much effort selecting and validating several types of biomarkers. They can be divided into several different categories, one of them is biomarkers of internal dose, that is, indicators of the occurrence and extent of exposure of a human being.1 The presence of the exogenous substance itself or its metabolites confirms that the compound has entered the body by some route of penetration and takes into account individual differences in absorption and bioaccumulation. Analytical methods with sufficient accuracy, specificity, and sensitivity are required to detect very low levels of chemicals in biological specimens. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous substances to which man is exposed in the environment and in * Corresponding author: (e-mail) [email protected]; (fax) +49-897095-7999. † Technical University Munich. ‡ Institute of Ecological Chemistry. (1) Vainio, H. Toxicol. Lett. 2000, 102-103, 581-589. 10.1021/ac010868n CCC: $20.00 Published on Web 11/01/2001

© 2001 American Chemical Society

the work atmosphere of several industries. Their physicochemical characteristics determine whether they are found as gases (the two- and three-ringed compounds) or adsorbed on particles. In contrast to most of the environmental pollutants discussed presently, the hazardous potential of PAHs is supposed to be rather underestimated, first of all with regard to environmentally caused cancer disease.2 The International Agency for Research on Cancer (IARC) and the United States Environmental Protection Agency (U.S. EPA) have classified several of these compounds as probable or possible human carcinogens.3,4 These compounds are metabolized by mixed-function cytochrome-dependent oxygenases (phase 1 enzymes) that may form metabolites that are either excreted in urine or act as ultimate carcinogens with DNA or other macromolecules. Due to its low dose and extensive metabolism, the determination of PAH metabolites in human urine is a challenging analytical task, especially when the internal exposure level of the population in general urbanized regions should be estimated. The lack of availability of chemical standards is another limitation for performing quantitative and qualitative determinations. Therefore, in most biomonitoring studies that are focused on the occupational near-term PAH exposure, only 1-hydroxypyrene has been analyzed and used as a biomarker.5 Mainly employed after hydrolysis of glucuronate and sulfate bioconjugates is HPLC with fluorescence detection, using off-line sample cleanup by C18-SPE. However, because of strong matrix interferences, the isolation of more polar metabolites (e.g., hydroxyphenanthrenes) with conventional SPE techniques is difficult and error prone. Therefore, numerous variants have been proposed to improve the sensitivity, specificity, and speed of the method which was originally described by Jongeneelen et al. several years ago.6 Moreover, the simultaneous (2) Grimmer, G.; Jacob, J.; Dettbarn, G.; Naujack, K.-W.; Heinrich, U. Exp. Toxic. Pathol. 1995, 47, 421-427. (3) International Agency for Research on Cancer. Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. Polynuclear Aromatic Compounds, part 1; International Agency for Research on Cancer: Lyon, France; 1983; Vol. 32. (4) Patnaik, P. In Handbook of Environmental Analysis; Patnaik, P., Ed.; CRC Press: Boca Raton, FL, 1997; pp 165-169. (5) Dor, F.; Dab, W.; Empereur-Bissonet, P.; Zmirou, D. Crit. Rev. Toxicol. 1999, 29, 129-168. (6) Jongeneelen, F. J.; Anzion, R. B. M.; Leijdekkers, C. M.; Bos, R. P.; Henderson, P. T. Int. Arch. Occup. Environ. Health 1985, 57, 47.

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detection of a variety of PAH metabolitessa profile analysissgives more detailed information on both the internal exposure rate and interindividual variations of biological concentrations as caused by the genetic polymorphism of involved enzymes, possibly. Coupled column chromatography using on-line sample cleanup with a “tailor-made” copper phthalocyanine-modified silica precolumn is a well-established technique for HPLC analysis of PAH metabolites in urine.7-10 Capillary gas chromatography with massselective detection (GC/MS) generally gives better separation performance, but sample preparation is more time-consuming and requires considerable amounts of solvent. Corresponding methods use either headspace silylation of PAH metabolites with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or derivatization to the corresponding methyl ethers by treatment with diazomethane.11,12 Traditional alkyl-bonded silica or copolymer sorbents, which are used to achieve preconcentration of analytes, are of limited selectivity. Many matrix constituents are also enriched and can disturb the chromatographic separation and detection. Therefore, methods that use immunoaffinity-based solid-phase extraction (immunoextraction) with antibodies immobilized on a stationary phase (immunoaffinity support) for specific removal of compounds from a sample are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds, including PAHs in sediment samples.13-15 Only a few papers were published on the application of immunochemical methods16-19 including immunoextraction for the determination of PAH metabolites in human urine samples.20-22 In the studies that applied immunoextraction, the immunosorbent was prepared by immobilization of an anti-benzo[a]pyrene-tetrol-DNA-IgG (8E11) on CNBractivated Sepharose.23 Most interesting, however, are supports that exhibit higher rigidity and porosity and, therefore, higher mechanical stability at high flow rates and pressures in order to be useful in on-line immunoextraction-HPLC systems also known as (7) Boos, K.-S., Lintelmann, J.; C.; Kettrup, A. J. Chromatogr. 1992, 600, 189194. (8) Angerer, J.; Mannschreck, C.; Gu ¨ ndel, J. Int. Arch. Occup. Environ. Health 1997, 70, 365-377. (9) Lintelmann, J.; Kettrup, A.; Boos, K.-S. J. Chromatogr. 1992, 600, 189194. (10) Lintelmann, J.; Hellemann, C.; Kettrup, A. J. Chromatogr., B 1994, 600, 67-73. (11) Gmeiner, G.; Krassnig, C.; Schmid, E.; Tausch, H. J. Chromatogr., B 1998, 705, 132-138. (12) Jacob, J.; Grimmer, G.; Dettbarn, G. Biomarkers 1999, 4, 319-327. (13) Stevenson, B. J. Chromatogr., B 2000, 745, 39-48. (14) Delaunay, N.; Pichon, V.; Hennion, M.-C. J. Chromatogr., B 2000, 745, 1537. (15) Perez, S.; Ferrer, I.; Hennion, M. C., Barcelo´; D. Anal. Chem. 1998, 70, 4996-5001. (16) Gomes, M.; Santella, R. M. Chem. Res. Toxicol. 1990, 3, 307-310. (17) Herikstad, B. V.; Ovrebo, S.; Haugen, A.; Hagen, I. Carcinogenesis 1993, 14, 307-309. (18) Scheepers, P. T. J.; Fijneman, P. H. S.; Beenakkers, M. F. M.; de Lepper, A. J. G. M.; Thuis, H. J. T. M.; Stevens, D.; Van Rooij, J. G. M.; Noordhoek, J.; Bos, R. P. Fresenius J. Anal. Chem. 1995, 351, 660-669. (19) Knopp, D.; Schedl, M.; Achatz, S.; Kettrup, A.; Niessner, R. Anal. Chim. Acta 1999, 399, 115-126. (20) Bentsen-Farmen, R. K.; Botnen, I. V.; Note, H.; Jacob, J.; Ovrebo, S. Int. Arch. Occup. Environ. Health 1999, 72, 161-168. (21) Strickland, P. T.; Kang, D. H.; Bowman, E. D.; Fitzwilliam, A.; Downing, T. E.; Rothman, N.; Groopman, J. D.; Weston, A. Carcinogenesis 1994, 15, 483-487. (22) Weston, A.; Bowman, E. D.; Carr, P.; Rothman, N.; Strickland, P. T. Carcinogenesis 1993, 14, 1053-1055. (23) Santella, R. M.; Lin, C. D.; Cleveland, W. L.; Weinstein, I. N. Carcinogenesis 1988, 5, 373-377.

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high-performance immunoaffinity chromatography (HPIAC).24 Since its inception about 10 years ago, sol-gel encapsulation has opened up a new promising way to immobilize biomolecules.25,26 Corresponding bioceramic supports, beside other advantages, exhibit mechanical robustness and long-term stability of the embedded biologicals. To date, only a few studies on encapsulated antibodies have been published,27-33 even less combined the solgel glass (SGG) immunosorbent with conventional chromatographic techniques.34-37 The objective in the present study was to determine urinary PAH metabolites by immunoextraction-HPLC (IE-HPLC) using for the first time sol-gel glass-entrapped antibodies with proven affinity to several hydroxylated PAHs, including 1-hydroxypyrene and 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrenes, and to apply this method in a biomonitoring study of 20 individuals of the general population. For validation and comparison of this novel method, metabolites were determined by a coupled-column HPLC analysis, using a copper phthalocyaninemodified silica precolumn. EXPERIMENTAL SECTION Chemicals. All reagents were of analytical reagent grade, the solvents were of HPLC quality. All water used was ultrapure and obtained by reversed osmosis including UV treatment (Milli-RO 5 Plus, Milli Q185 Plus; Millipore, Eschborn, Germany). β-Glucuronidase/arylsulfatase solution was obtained from Recipe (Mu¨nchen, Germany) for coupled-column LC and from Boehringer (Mannheim, Germany) for IE/LC. 1-Hydroxypyrene (1-OH-PYR), 2-, 3-, 4-, and 9-hydroxyphenanthrenes (2-, 3-, 4-, and 9-OH-PHE), and 3-hydroxybenzo[a]pyrene (3-OH-BaP) were purchased from Dr. W. Schmidt (Institute of PAH Research, Greifenberg, Germany). 1-Hydroxyphenanthrene (1-OH-PHE) was generously provided by Dr. A. Seidel (Institute of Toxicology, University of Mainz, Germany). Tetramethoxysilane (TMOS) was obtained from Fluka (Buchs, Switzerland). HighTrap protein A columns were from Pharmacia (Uppsala, Sweden). BSTFA, sodium ascorbate, and uric acid was obtained from Sigma (Deisenhofen, Germany). Preparation of the Sol-Gel Glass Immunoaffinity Cartridge. The immunoglobulin (IgG) fraction was isolated from (24) Hage, D. S. J. Chromatogr., B 1998, 715, 3-28. (25) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M.; Chem. Mater. 1994, 6, 16051614. (26) Ellerby, L. M., Nishida, C. R.; Nishida, F.; Yamanaka, S. A., Dunn, B.; Valentine, J. S.; Zink, J. L. Science 1992, 255, 1113-1115. (27) Wang, R.; Narang, U.; Prassad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671-2675. (28) Turniansky, A.; Avnir, D.; Bronshtein, A.; Aharonson, N. Altstein, M. J. SolGel Sci. Technol. 1996, 7, 135-143. (29) Jordan, J. D.; Dunbar, R. A.; Bright, F. V. Anal. Chim. Acta 1996, 332, 83-91. (30) Shabat, D.; Grynszpan, F.; Saphier, S.; Turniansky, A.; Avnir, D.; Keinan, E. Chem. Mater. 1997, 9, 2258-2260. (31) Bronshtein, A.; Aharonson, N.; Avnir, D.; Turniansky, A.; Altstein, M. Chem. Mater. 1997, 9, 2632-2639. (32) Altstein, M.; Bronshtein A.; Glattstein, B.; Zeichner, A.; Tamiri, T.; Almog, J.; Anal. Chem. 2001, 73, 2461-2467. (33) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 11711175. (34) Cichna, M.; Markl, P.; Knopp, D.; Niessner, R. Chem. Mater. 1997, 9, 26402646. (35) Spitzer, B.; Cichna, M.; Markl, P.; Sontag, G.; Knopp, D.; Niessner, R. J. Chromatogr., A 2000, 880, 113-120. (36) Doody, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Chem. Mater. 2000, 12, 1142-1147. (37) Zuehlke, J.; Knopp, D.; Niessner R. Fresenius J. Anal. Chem. 1995, 352, 654-659.

polyclonal rabbit anti-PAH antiserum using a protein A column according to the instructions given by the supplier. The sol-gel glass was prepared by a two-step procedure, in which hydrolysis was followed by polymerization of TMOS. An acidic silica sol was obtained by mixing 2.5 mL of TMOS with 0.5 mL of MeOH and 50 µL of 0.05 M HCl and throughly mixed for 20 min under ice cooling. The purified antibody fraction was dissolved at a concentration of 1.5 mg/mL in phosphate-buffered saline (PBS), which is 0.08 M sodium phosphate (pH 7.6) containing 0.15 M sodium chloride, cooled on ice, mixed with an equivolume amount of the silica sol, and immediately poured into a Petri dish. The sol-gel was allowed to age at 4 °C until a loss of weight of ∼70% occurred (after ∼36 h). Afterward the glass was ground in a mortar and packed into 3-mL glass columns (Merck, Darmstadt, Germany). PTFE frits (porosity 10 µm, Merck) were placed above and below the sorbent bed. The columns were washed with 10 mL PBS and stored at 4 °C. Off-Line Immunoextraction-Liquid Chromatography Analysis (IE-LC). For enzymatic hydrolysis, an aliquot of urine (10 mL) was diluted 1:1 in 0.1 M sodium acetate buffer (pH 5.0; v/v). The solution was incubated overnight (16 h) with 10 µL (1000 Fishman units) of β-glucuronidase/sulfatase solution at 37 °C in an oven and 5% (v/v) of ACN was added. Sample enrichment and purification was performed with the immunoextraction cartridge containing 0.6 g of the antibody-doped sol-gel glass and using a flow rate of ∼2 mL/min and a negative pressure (MSP peristaltic pump, Ismatec, Wertheim-Mondfeld, Germany). The column was conditioned, prior to sample application, with 3 mL of ACN/water (1:1), followed by 6 mL of 0.05 M sodium acetate buffer (pH 5.0). A 20-mL aliquot of the hydrolyzed sample was applied onto the column. To remove unretained and weakly bound impurities, the immunosorbent was flushed successively with 3 mL of acetate buffer (0.1 M with 5% ACN) and 3 mL of water (with 5% ACN); both washing solutions contained 10 mg/L uric acid to inhibit oxidation of the OH-PAHs. The retained compounds were desorbed from the immunoaffinity column with 3 × 1-mL fractions of ACN/water (1:1). The eluate was collected in an amber glass vial, which contained 15 µL of an aqueous ascorbate solution (1 g/L), and after filtration with an 0.45-µm PP syringe filter (Whatman, Springfield Mill, U.K.) could be directly analyzed by the LC method or further concentrated by solid-phase extraction (C18-SPE) (see section Combined IE-C18-SPE). After elution, the immunoextraction column was regenerated for the next sample by successive washing with 10 mL of ACN/water (1:1), 6 mL of acetate buffer (0.1 M with 5% ACN), and 6 mL of water (with 5% ACN) to remove any residual components from the previous sample. After six cycles of loading, elution, and regeneration, the column back pressure increased significantly due to the clogging of the Teflon frits. Therefore, the sol-gel glass was removed and a new column was packed by replacing the Teflon frits. For storage, the immunosorbent not in use was kept at 4 °C in PBS. Combined IE-C18-SPE. For RP-SPE, the volume of the eluate obtained by immunoextraction was brought to a final volume of 10 mL with water in order to reduce the concentration of ACN to 15% (v/v), and additional ascorbate (50 µL, 1 g/L) was added. The sample was loaded into a SPE cartridge filled with 500 mg of Lichrolut RP18e (Merck) which had been preconditioned with 6 mL of MeOH, followed by 6 mL of water. The sample

was drawn through the cartridge at a flow rate of 3 mL/min. After another wash with 6 mL of ascorbate solution, the cartridge was dried in a gentle stream of nitrogen. The analytes were eluted with 3 mL of acetone/CH2Cl2 (3:2) into an amber glass bottle which contained 15 µL of ascorbate solution. The extract was then dried using a nitrogen stream. The residue was reconstituted with 500 µL of MeOH/water (6:4). HPLC. Analysis was performed on a Shimadzu LC system consisting of a SCL-6A controller, two LC-6A pumps, photodiode array UV-visible detector SPD-M6A, CTO-10A column oven (Shimadzu, Duisburg, Germany), and S3400 fluorometric detector (Sykam, Gilching, Germany). Chromatographic separations were carried out on a LiChrocart column (Merck) 250 × 4 mm i.d. packed with Superspher 100 RP 18 (end capped, 5-µm particle size) material. The column was equipped with a 4 × 4 mm i.d. guard column (Merck) of the same stationary phase. Injection was performed with a model 7125 injector (Rheodyne, Cotati, CA) equipped with a 100-µL sample loop. Analysis was carried out using a gradient solvent program. The initial composition of the mobile phase was 60% MeOH and 40% water, both acidified with 1 mg/L ascorbate. After keeping the MeOH content constant for 20 min, the gradient was programmed to linearly increase the amount of MeOH until 95% in 20 min. To clean the column, the amount of MeOH was kept constant at 95% for 15 min. The flow rate was set at 0.8 mL/min, the analytical column temperature was kept at 40 °C. Fluorescence detection wavelengths were set to λex/em ) 244/370 nm (hydroxyphenanthrenes), λ ex/em ) 242/ 388 nm (1-OH-PYR) and λex/em ) 265/430 nm (3-OH-BaP). Hydroxyphenanthrenes eluted at 18-23 min, hydroxypyrene at 33 min, and 3-OH-BaP at 42 min. Data acquisition and evaluation were performed with the CSW v.1.7 package (Data Apex, Prague, Czech Republic). Peak areas were used for quantification. Calibration was performed by multiple injections of standard solutions (from 0.02 to 5 ng/mL) of the OH-PAHs. Calibration curves were calculated by linear regression analysis. Correlation coefficients (squared) ranged from 0.990 to 0.999. 2- and 3-OH-PHE cannot be separated with this method; therefore, data treatment was performed as described in the section “Coupled-column Chromatography”. Gas Chromatography/Mass Spectrometry. The eluate of a smoker’s urine sample, which was obtained after immunoextraction with subsequent C18-SPE, was dried under a stream of nitrogen and then redissolved in 200 µL of BSTFA. After heating at 70 °C for 1 h, a 1-µL aliquot of sample was injected into a HP 5980 Series II gas chromatograph combined with a high-resolution mass selective detector (VG AutoSpec). Separation was performed on a DB5-MS fused-silica capillary column, 60 m × 0.25 mm i.d., 0.25-µm film thickness (J&W Scientific, Folsom, CA). Carrier gas was helium (Messer-Griesheim, Krefeld, Germany). The following GC temperature program was used: 100 °C for 1 min, 15 °C/min to 160 °C, 8 °C/min to 305 °C, and held until 1 h. The mass spectrometer was operated in electronic impact (EI) mode using an ionization energy of 70 eV and a trap current of 600 µA. The analysis was performed in the selected ion monitoring (SIM) mode detecting masses 266.113, 290.113, and 340.113, corresponding to the TMS-ethers of monohydroxyphenanthrenes, monohydroxypyrene, and monohydroxybenzo[a]pyrene, respectively. Coupled-Column Chromatography. The LC system consisted of an autosampler (Triathlon, Spark Holland, Emmen, The Analytical Chemistry, Vol. 73, No. 23, December 1, 2001

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Netherlands), a gradient pump (L-6200, Merck-Hitachi, Darmstadt, Germany), an automatic six-port switching valve (AW 700, Walfort & Partner, Reinhardshagen, Germany), a second LC pump (l-655A11LC with L-5000 controller, Merck-Hitachi), a column thermostat (Mistral, Spark Holland), and a fluorescence detector (9070, Varian, Darmstadt, Germany) connected with a PE Nelson 900 Series Interface (Perkin-Elmer, U ¨ berlingen, Germany). Data acquisition was done with Turbochrom Navigator 4.0 software Perkin-Elmer). Sample enrichment was achieved by use of a copper phthalocyanine-modified silica precolumn (5 × 4 mm i.d., 30 µm); for analytical separation, a LiChrocart column was used (Superspher 100 RP 18, 250 × 4 mm i.d., 4 µm, Merck) with guard column (4 × 4 mm i.d.). Sample centrifugation was done with a Labofuge Ae (Heraeus, Osterode, Germany). Stock solutions of 1-OH-PYR and 9-OH-PHE in MeOH were prepared by weighing in. The other phenanthrene metabolites were purchased as stock solutions in ACN. Standard solutions of different concentrations were obtained by diluting the stock solutions with water/MeOH (1:1). Sample preparation and the coupled-column HPLC method with system-integrated sample processing were performed according to Lintelmann et al. with small modifications.9,10 A 2.5mL aliquot of urine was adjusted to pH 5.0 with 1 M hydrochloric acid and made to a final volume of 5.0 mL with sodium acetate buffer (0.1 M, pH 5.0). To hydrolyze the conjugates of the metabolites, 7 µL of the enzyme solution was added and the solutions were incubated under continuous shaking for 3 h at 37 °C in a thermostated water bath. After that the samples were centrifuged in glass tubes at 2000g for 5 min, transferred into autosampler vials, and injected into the HPLC system without mixing. To enhance sensitivity, the injection volume of samples and standard solutions was enlarged to 500 µL. Compared to chromatograms of 100-µL injections, no additional matrix interference was observed. Sample processing and transfer were carried out at room temperature, while the analytical column was thermostated to 40 °C. Pump 1 constantly delivered solvent A (MeOH/water (10:90)) at 1.0 mL/min; pump 2 operated in the gradient mode with solvents B (MeOH/water (60:40) and C (MeOH) at a flow rate of 0.8 mL/min. Fluorescence detection wavelengths were set to λex/em ) 242/388 nm. Calibration was performed by multiple injection of OH-PAH standard solutions in the coupled-column mode; concentrations were used in the range of 0.02-4 ng/mL. Calibration curves were obtained by linear regression with a weighting by 1/x; curves were forced through the origin. Correlation coefficients (squared) for the calibration curves were higher than 0.990. Since 2- and 3-OH-PHE were not separated by the applied method, an exact quantification could not be performed for these analytes. Calibration curves of both substances were determined yielding a response factor that was 1.8 times lower for 2-OH PHE compared to the 3-OH isomer. To get a rough estimation of the content of both analytes, the calibration curve of 3-OH-PHE was applied and the amount of 2+3OH-PHE was calculated by a mean response factor, assuming a distribution of 1:1. Samples were analyzed twice in subsequent injections. In the following, the mean value of both analyses was used. All substances were quantified by peak area. Study Population. All individuals participating in this study were recruited on a voluntary basis and could be considered as 5672

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occupationally nonexposed. They were classified into smokers (mean age 40.5 ( 13; mean body weight 83.5 ( 10.1 kg) and nonsmokers (mean age 30.8 ( 6.4; mean body weight 77 ( 12.8 kg), each group with equal numbers (n ) 10) of individuals. A short self-explanatory questionnaire was delivered to each participant on the day before urine collection together with distribution of urine vessels (glass containers). The questionnaire contained items on actual and recent (last 4 months) smoking habits, passive smoking, and the estimated number of meals within the last 4 months that contained grilled or smoked food. The spot urine samples were encoded, and 10-mL aliquots were stored frozen at -20 °C for no more than 3 weeks prior to analysis. PAH metabolite determination was performed with two independent methods and without knowledge of sample source. Creatinine in Urine. To account for differences in dilution of the urine due to diuresis, urinary PAH metabolite concentrations found in the biomonitoring study were corrected for creatinine (nmol/mol creatinine). The creatinine in fresh urine samples was reacted with alkaline picrate, and the creatininepicrate complex was measured by spectrophotometry.38 Statistical Analysis. Statistical data evaluation was performed with the Mann-Whitney U-test distinguishing between smokers and nonsmokers. Statistical significance was assumed for p-levels lower than 0.05. Statistica for Windows 5.1 B software (StatSoft, Tulsa, OK) was used. RESULTS AND DISCUSSION Capacity of the Immunosorbent. The pyrene antiserum was chosen for this study because the cross-reactivity is well documented for parent PAHs and selected PAH metabolites and proved to be useful for environmental screening and human biomonitoring.19 No leaching of the immobilized antibodies from the solgel glass matrix was observed in PBS, which was used for washing of new prepared immunoextraction columns. This was confirmed by two different methods (data not shown). At first, proteins were determined using the BCA protein assay (Pierce, Rockford, IL). The result was negative; i.e., proteins could not be detected. In a second experiment, undiluted aliquots of the PBS wash were used as antibody dilution in the indirect PAH-ELISA, which was performed on microtiter plates as described earlier.19 Also, the ELISA was negative; i.e., there was no signal higher than the background signal obtained., To estimate the capacity of the prepared immunosorbents, 10 mL of PBS was spiked with 0.5 and 2 µg/L 1-OH-PYR and applied on the immunoextraction columns (1.5 mg of IgG/column). After a washing step with 5 mL of water with 5% ACN, the retained compounds were eluted with 3 mL of elution solution (ACN/water (1:1). The effluent, washing, and eluted fractions were analyzed by HPLC. For loading with 500 ng (2000 ng) of 1-OH-PYR, 420 ng (640 ng) of analyte was found in the eluate, 30 ng (520 ng) in the effluent, and 10 ng (640 ng) in the washing solution (n ) 2). These capacities exceed the expected sum of OH-PAH metabolites in human urine by a factor of 5-20 (using data from previous studies).19 Therefore, the capacity of the immunosorbent with 1.5 mg of immobilized antibody was sufficient for the analysis of real samples. (38) Foster-Swanson, A.; Swartzentruber, M.; Roberts, P.; Feld, R.; Johnson, M.; Wong, S.; Bartsch, J.; Dees, K.; Toner, M.; Cloyed, D.; Pacquette, J.; Wu, A.; Bradley, H.; Trundle, D. Clin. Chem. 1994, 40, 1057.

Table 1. Recoveries after Fortifying 10 mL of a Nonsmoker’s Urine Sample (with Proven Low PAH Metabolite Concentration) Prior to Enzymatic Hydrolysis with (A) High OH-PAH Levela or (B) Low OH-PAH Levelb and Subsequent IE-LC Analysis (n ) 3)c recovery ( SDd (%)

Figure 1. Elution profile of adsorbed 1-OH-PYR from the immunosorbent. The immunosorbent was loaded with 50 ng of 1-OH-PYR in 10 mL of PBS (with 5% ACN), washed with 5 mL of water/ACN (95: 5), and eluted with 6 × 1 mL of water/ACN (1:1) (open bars) or MeOH/ H2O (1:1) (shaded bars). 1-OH-PYR concentration in the fractions was analyzed by HPLC. For normalization, the sum of recovered 1-OH-PYR was set to 100%. Error bars indicate relative standard deviation (1s).

Based on the found maximum capacity of the sol-gel support and the assumptions that all encapsulated antibodies were specific and both binding sites of an IgG molecule should be accessible and, therefore, could bind two molecules of 1-OH-PYR, the calculated amount of antibodies that retained their activity after entrapment was 14.7%. However, one has to take into account that the used antibody was polyclonal, and therefore, only a distinct fraction was specific for the PAHs. According to the literature, the amount of specific antibodies in polyclonal sera is ∼10%.39 Elution of the Analytes. Efficient desorption of the retained analyte from the immunosorbent requires that the total volume of eluent needed should be kept small, to avoid extensive dilution of the analyte in the final extract. Elution is in most cases achieved by increasing the content of organic modifier in the solution (usually MeOH/water or ACN/water mixtures) or by changing the pH value.13,14 As could be demonstrated with our sorbent, 3 mL of ACN/H2O (1:1) was sufficient to remove the trapped analyte completely from the immunoextraction column, whereas 5 mL was needed when MeOH/water (1:1) was used (Figure 1). Compared to 1-OH-PYR, other PAH metabolites generally exhibit lower affinity to the used anti-pyrene-antibodies;19 therefore, these data represent the maximum volume needed for complete desorption of the OH-PAHs. Organic solvent concentration in the elution solution was not increased further, to keep the final extract compatible with subsequent HPLC analysis. During the experiments, we witnessed no negative effect of the desorption solution on the immunosorbent; i.e., capacity and recoveries were not affected adversely (Table 5). Specificity of the Immunosorbent. For determination of the percentage of the analyte that is exclusively retained by the encapsulated antibodies and not by the sorbent matrix or by possible nonspecific protein-analyte interaction, two blank sorbents were prepared containing either no IgG (glass blank) or 1.5 mg of anti-metsulfuron-methyl-IgG (IgG blank).40 Blank sor(39) Rule, GS.; Mordehai, AV.; Henion, J. Anal. Chem. 1994, 66, 230-239.

compound

low OH-PAH concn

high OH-PAH concn

2/3-OH-PHEs 9-OH-PHE 1-OH-PHE 4-OH-PHE 1-OH-PYR 3-OH-BaP

91 ( 12 107 ( 6 83 ( 9 90 ( 11 83 ( 6 45 ( 5

101 ( 11 90 ( 10 92 ( 13 84 ( 7 95 ( 14 62 ( 6

a Added: 30 ng of 1-OH-PYR, 10 ng of 1-OH-PHE, 20 ng of 2-OHPHE, 10 ng of 4-OH-PHE, 10 ng of 9-OH-PHE, and 3 ng of 3-OH-BaP. b Added: 2 ng of 1-OH-PYR, 6 ng of 1-OH-PHE, 9 ng of 2-OH-PHE, 6 ng of 4-OH-PHE, 2 ng of 9-OH-PHE, and 3 ng of 3-OH-BaP. c Spike levels were calculated using data from a previous study.19 d Recovery data were corrected by subtraction of background (blank) OH-PAH level as determined by analysis of the nonspiked sample.

bents and a column with specific IgG were loaded with 10 ng of analyte. After elution of the sorbents, the eluates were analyzed by HPLC. For normalization, recoveries obtained with the specific immunosorbent were set to 100%. Little nonspecific adsorption of 1-OH-PYR on both types of blank sorbents was observed, elution of the glass blank gave 9% relative recovery (compared to specific sorbent) of the analyte, and with the IgG blank, this value was increased to 16%. More polar OH-PHE metabolites were retained only by interaction with the specific IgG; no analyte was found after elution of both blank sorbents. However, due to its hydrophobicity, 3-OH-BaP was strongly adsorbed by the glass matrix (78% relative recovery for glass blank, 72% relative recovery for IgG blank), even if 5% ACN was added to the sample solution. One must consider that these values for nonspecific adsorption represent a “worst-case scenario” for a sorbent with specific IgG, where nonspecific interaction is preferred to the affinity of the antibody toward the analyte. This simplistic approach will result in overestimation of the nonspecific binding forces compared to antibody-analyte interaction. Therefore, retention of OH-PHE and OH-PYR metabolites on the immunosorbent can be attributed to specific antigen-antibody interaction. Nonspecific adsorption of the analytes (except for 3-OH-BaP) by the glass matrix or by nonspecific proteins is negligible in the extraction process. Recoveries. To validate the developed method, urine from a nonsmoker was pooled and spiked with different relevant levels of OH-PAH metabolites. As could be demonstrated, despite the use of a relatively high concentration of organic solvent (5% ACN) during the sample-loading step, recoveries were still excellent for both spike levels, owing to the high-capacity reserve provided by the immunosorbent (Table 1). Recoveries for 3-OH-BaP were below average, because of the poor affinity of the antibodies to the analyte.19 Furthermore, combination of immunoextraction with C18-SPE, despite the additional extraction step, did not significantly decrease recoveries, but provided increased sensitivity. In addition, no influence of salt concentration (0-200 mmol/L NaCl) or pH value (between 5 and 9) of the sample solution on the (40) Simon, E.; Bou Carrasco P.; Knopp, D.; Niessner, R. Food Agric. Immunol. 1998, 10, 105-120.

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Table 2. Limits of Detection (LOD) for the Different Analytes after Injection of Standard Solution or Enrichment of a 10-mL Urine Sample LODa (ng/L)

compound

std solnb

after SGG-IE, final vol 3 mLb

after SGG-IE combined with C18-SPE, final vol 0.5 mLb

1-OH-PHE 2-/3-OH-PHEs 4-OH-PHE 9-OH-PHE 1-OH-PYR 3-OH-BaP

31 33 33 52 10 10

11 11 11 16 4 6

2 2 2 3 1 1

a LODs were calculated by using a signal-to-noise ratio of 3. b A 100µL injection volume.

Figure 2. Chromatograms obtained after (a) C18-SPE (upper plot) and (b) immunoextraction (lower plot). Ten milliliters of urine from the same subject was analyzed, final volume 3 mL, 100-µL injection volume. Peaks (1) 2-/-3-OH-PHEs, (2) 9-OH-PHE, (3) 1-OHPHE, (4) 4-OH-PHE, and (5) 1-OH-PYR.

recoveries was observed during our studies (data not shown). These factors did not inhibit or promote binding of the analytes to the antibodies. Therefore, the developed extraction procedure might be applied to other aqueous matrixes as well (e.g., sea/ river water, diluted organic extracts of marine organisms, etc.). With a previously described immunoextraction procedure, recoveries ranging from 47 to 76% for hydroxyphenanthrenes and 1-OHPYR were obtained, which, in contrast to the presented methodology, only allowed semiquantitative determination of the PAH metabolites.20 Comparison C18-SPE versus Immunoextraction. Figure 2 shows the difference between chromatograms obtained after conventional extraction of human urine with C18-SPE material and immunoextraction with the SGG immunosorbent. Although fluorescence detection is more selective compared to UV detection, still a multitude of peaks is visible after C18-SPE. High background signal levels originate from matrix components that are also enriched with the rather nonselective C18 material. Therefore, detection of the more polar OH-PHEs in complex matrixes such as urine becomes impossible using conventional extraction methods and sorbents. We were not able to remove these matrix interferences by using modified washing procedures during sample enrichment. The high amount of nonspecific absorption of matrix compounds is further confirmed by the fact that, after enrichment of the urine sample, the C18 sorbent became dark brown and the color of the extract was yellow to amber. In contrast to these findings, the SGG immunosorbent remained almost totally white, even after several extraction cycles, with the eluate being completely colorless. In the chromatogram after immunoextraction, matrix interferences are almost completely absent; only sharp peaks are visible. Consequently, OHPHE metabolites can easily be detected and quantified, and even more polar compounds are detectable. This efficient removal of matrix components is achieved by the superior selectivity of the immunosorbent. Only analytes that share common structural features are recognized by the antibodies and subsequently retained on the sorbent. Matrix compounds are not adsorbed, and therefore, the resulting chromatograms are extremely “clean”. This advantageous quality of the immunosorbent also allows 5674 Analytical Chemistry, Vol. 73, No. 23, December 1, 2001

Figure 3. Comparison of data obtained after analysis of 1-OH-PHE by coupled-column HPLC and by off-line IE/HPLC in 20 urine samples. One datum point represents the mean value from two independent measurements for each method, Values were corrected for individual creatinine levels. Fit was performed by least-squares regression. Table 3. Correlation between OH-PAH Metabolite Values from Real Urine Samples Measured with Two Different Methodsa compound

correlation equationb

SD slope

SD intercept

R2

1-OH-PYR 1-OH-PHE 2/3-OH-PHEs 9-OH-PHE 4-OH-PHE

Y ) 5.5x + 12.1 Y ) 0.76x + 16.1 Y ) 0.75x + 40.9 Y ) 0.66x + 10.4 Y ) 0.72x + 14.1

0.25 0.08 0.05 0.09 0.27

5.5 12.2 13.1 5.8 5.1

0.963 0.863 0.937 0.734 0.271

aY represents data obtained with off-line immunoextraction (nmol/ mol creatinine); x represents data obtained with coupled-column HPLC; one datum point is calculated as mean value from two independent measurements. Data were not corrected by recovery factor). b Leastsquares regression equation.

higher throughput in routine analysis of urine samples as impurities of the sample are completely removed and consequently cannot impair the operation of sensitive analytical equipment, e.g., HPLC columns and LC/MS detectors. In addition to the peaks

Figure 4. Chromatograms obtained after (a) coupled-column HPLC (upper plot) and (b) Immunoextraction (lower plot). A 500-µL (on-line HPLC) or a 10-mL (immunoextraction) aliquot of urine from the same individual was analyzed, Peaks: (1) 4-OH-PHE, (2) 9-OH-PHE, (3) 3-OHPHE, (4) 1-OH-PHE, (5) 2-OH-PHE, and (6) 1-OH-PYR. Table 4. Concentrations of OH-PAH Metabolites Found in Human Urine Using Sol-Gel Glass Immunosorbent mean/median ( SD (nmol/mol creatinine)

a

subgroup

n

2-/3-OH-PHEs

9-OH-PHE

1-OH-PHE

4-OH-PHE

1-OH-PYR

all subjects nonsmokers smokers (p-level)a

20 10 10

208/186 ( 142 143/131 ( 67 273/216 ( 160 (0.0191)

40/32 ( 34 14/14 ( 10 66/46 ( 27 (0.0072)

124/104 ( 81 84/86 ( 22 165/129 ( 94 (0.0013)

25/21 ( 16 14/13 ( 6.5 36/24 ( 13 (0.0002)

95/58 ( 87 41/45 ( 12 149/120 ( 92 (0.0008)

p-Level was determined by Mann-Whitney U-test distinguishing between smokers and nonsmokers.

originating from PAH metabolites that were used for this study, we observed several times signals that were caused by more polar compounds with a retention time of less than 15 min. As these molecules obviously have also been enriched by the immunosorbent, they must be structurally related to PAHs and should contain a fused aromatic system of at least three benzenoid rings. Future investigations will employ LC/MS analysis to identify these unknown compounds. LODs. As interfering matrix components are effectively removed during the immunoextraction procedure, a chromatogram after injection of a standard solution is basically identical to the chromatogram of a urine sample extract after immunoextraction. Subsequently, as illustrated in Table 2, LODs are rather low and do largely depend on recoveries and the chosen enrichment factor (sample volume compared to final volume of the extract). Other methods that have been described previously offer comparable LODs ranging from 5 to 16 ng/L for OH-PHE metabolites.7-10 Therefore, obtained detection limits are sufficient for reliable detection of OH-PAH compounds even in urine of occupationally nonexposed individuals.19 Human Biomonitoring. Twenty urine samples of smokers and nonsmokers were analyzed both by off-line immunoextraction and by a previously described procedure using a coupled-column HPLC method with a copper phthalocyanine-modified silica precolumn.9,10 As illustrated in Figure 3 for 1-OH-PHE, obtained data show a good correlation between the on-line HPLC and off-line IE-LC method. Except for 4-OH-PHE, where concentration levels

were very close to the LOQ, and for 1-OH-PYR, where the on-line HPLC procedure led to a systematic 5-fold underestimation, concentrations of the other metabolites were quite comparable (Table 3). However, after (a) comparing the results with data from our previous biomonitoring study,19 (b) repeatedly checking recoveries of the immunoextraction method, and (c) analysis of 1-OH-PYR in selected samples by off-line C18-SPE, we maintain the correctness of the 1-OH-PYR values found with the SGG-IE method. The reason for the considerable underestimation provided by the coupled-column technique is still under investigation. 3-OHBaP was not detectable in any of the samples. Figure 4 shows the chromatograms obtained from the same sample by the two different methods. Once again, very few matrix interferences were visible after extraction with the immunosorbent, whereas an elevated background noise level, particularly in the retention time window of more polar PAH metabolites, was observed using the coupled-column method. In addition, the results revealed significant higher excretion levels of PAH metabolites in urine of smokers compared to nonsmokers (Table 4). This confirms findings made in previous studies,8,19,41 although other publications did not show a significant influence of smoking habit on the excretion of PAH metabolites.12 GC/HRMS Analysis. Confirmation of identity of the PAH metabolites was performed by GC/MS analysis after derivatization of an extract of selected urine samples.11 The mass traces of the (41) Van Delft, J.; Steenwinkel, M. Ann. Occup. Hyg. 2001, 45, 395-408.

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Table 5. Comparison of Data Obtained after Immunoextraction of 10-mL Urine Sample with Used and New Immunosorbent Columns (n ) 3) ng/mL analyte found compound

with new immunosorbent

with used immunosorbent

2-/3-OH-PHEsa 1-OH-PHE 1-OH-PYR

1.85 ( 0.02 0.854 ( 0.044 0.577 ( 0.014

2.11 ( 0.31 0.852 ( 0.058 0.603 ( 0.063

a 9- and 4-OH-PHE levels were lower than the LOQ and were therefore not compared.

Figure 5. GC/HRMS chromatogram after immunoextraction combined with C18-SPE of a 10-mL urine sample followed by derivatization with BSTFA. (a) Mass trace m/z 266.113, TMS-ether of OHPHE; (b) Mass trace m/z 290.113, TMS-ether of 1-OH-PYR. Upper plots, standard solution; lower plots, sample. Peaks: (1) 4-OH-PHE, (2) 9-OH-PHE, (3) 3-OH-PHE, (4) 1-OH-PHE, (5) 2-OH-PHE, and (6) 1-OH-PYR.

TMS-ethers of the monohydroxylated phenanthrene and pyrene compounds were recorded. Five OH-PHEs and 1-OH-PYR metabolites (Figure 5) were successfully identified, while the relative intensities of the peaks were comparable to those measured by HPLC with fluorescence detection. In contrast to the described HPLC method, all OH-PHEs were separated by GC. Also, there was some indication for the existence of other mono- or polyhydroxylated PAHs, but due to low concentrations and lack of suitable standard reference material, this could not be confirmed yet. Reusability. The immunosorbents were designed and prepared for multiple use, as there is a limited supply of wellcharacterized polyclonal antibodies. To investigate the influence of repeated cycles of sample loading, elution, and regeneration, the capacity of one immunoextraction column was checked before and after the biomonitoring study. The capacity was found to be 640 ( 20 ng of 1-OH-PYR prior to the experiments and 600 ( 52 ng after processing 20 samples of 10 mL of human urine each. In

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addition, recoveries for spiked urine samples remained unchanged for used columns (data not shown). To confirm this result, three freshly prepared and three “used” immunosorbents (used for ∼20 urine extractions) were chosen for the analysis of identical samples. Results are summarized in Table 5. Within-run and betweenrun imprecisions were between 5 and 14% (analysis of four different urine samples on (a) the same day (n ) 3) or (b) on three different days). It could be demonstrated that different, independently prepared IS columns yielded the same results even after extensive usage. Therefore, the immunosorbent is ideally suited for repeated use, making it a very cost-effective analytical tool. Overall, the newly developed sol-gel glass immunosorbent proved to be a valuable tool for the selective enrichment of hydroxylated PAH metabolites from a complex matrix. Its uncomplicated handling and robustness allow reliable and rapid trace-level analysis of the analytes in human urine and may aid in the identification of additional metabolites in the future. In addition, due to its versatility, the extraction of the analytes in several other aqueous matrixes is possible. Further studies will focus on the integration of an IS precolumn into an automated on-line LC/MS extraction system, which should lead to enhanced reproducibility, increased sample throughput, and lower detection limits. ACKNOWLEDGMENT Harald Beck is thanked for performing the GC/HRMS measurements. Prof. K.-S. Boos and Dr. D. Mu¨hlbayer from the Institute of Clinical Chemistry of the University Hospital “Mu ¨nchenGrosshadern” at the Ludwig-Maximilians-University Munich are acknowledged for creatinine determination. AC010868N