Anal. Chem. 2001, 73, 2819-2827
Accelerated Articles
Quantification of the Heterocyclic Aromatic Amine DNA Adduct N-(Deoxyguanosin-8-yl)-2-amino3-methylimidazo[4,5-f]quinoline in Livers of Rats Using Capillary Liquid Chromatography/ Microelectrospray Mass Spectrometry: A Dose-Response Study John R. Soglia,† Robert J. Turesky,‡,§ Axel Paehler,§ and Paul Vouros*,†
Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, Division of Chemistry, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Nestle´ Research Centre, Nestec Ltd., CH-1000, Vers-chez-les Blanc, Lausanne 26, Switzerland
Capillary liquid chromatography/microelectrospray-mass spectrometry (capillary LC/µESI-MS) was used to quantify DNA adducts of the heterocyclic aromatic amine 2-amino3-methylimidazo[4,5-f]quinoline (IQ) in livers of male Fischer-344 rats. Animals received a single oral dose of either 0.05, 0.50, 1.0, or 10 mg/kg IQ and were sacrificed 24 h following treatment. The major lesion identified at all doses was N-(deoxyguanosine-8-yl)-2amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ). The capillary LC/µESI-MS method provided the means for quantifying 17.5 fmol of dG-C8-IQ (2.0 adducts in 108 nucleosides) (S/N 10) in 300 µg of liver DNA with an intra- and interday precision of 3.5 and 6.6% (RSD), respectively. dG-C8-IQ was quantified with a mean intraand interday accuracy of 105 ( 26 and 106 ( 28 (SD) based on back-calculated adduct masses from five standard curves analyzed over a four-week period. This is the first report on development of a capillary LC/µESI-MS method to quantify dG-C8-IQ adducts in liver DNA of rats following dosing with IQ at different levels. Furthermore, the ability to accurately and precisely quantify dG-C8IQ at a level of 2.0 adducts in 108 nucleosides in vivo 10.1021/ac010218j CCC: $20.00 Published on Web 05/18/2001
© 2001 American Chemical Society
makes this method well suited for use in future studies relating carcinogen exposure to risk in humans.
Diet is thought to account for about one-third of human cancer in the United States.1,2 Several factors, including high caloric and fat intake enhance cancer development.3 Some foods also contain mutagenic and/or carcinogenic substances as very minor components. During the past twenty years, a series of mutageniccarcinogenic compounds known as heterocyclic aromatic amines (HAAs) have been found in cooked meats and fish.4-7 These compounds are carcinogenic to rodents at multiple sites and one HAA, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; Figure 1), is a powerful liver carcinogen in nonhuman primates.8-14 Since HAAs * To whom correspondence should be addressed: Phone: (617) 373-2840. Fax: (617) 373-8478. † Northeastern University. ‡ Current address: National Center for Toxicological Research. § Nestec Ltd. (1) Doll, R.; Peto, R. J. Natl. Cancer Inst. 1981, 66, 1191-1308. (2) Ames, B. N.; Gold, L. S.; Willet, W. C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5258-5265. (3) Wakabayashi, K.; Nagao, M.; Esumi, H.; Sugimura, T. Cancer Res. 1992, 52 (Suppl.), 2092s-2098s. (4) Sugimura, T. Cancer (Philadelphia) 1982, 49, 1970-1984. (5) Sugimura, T. Science (Washington, DC) 1986, 233, 312-318.
Analytical Chemistry, Vol. 73, No. 13, July 1, 2001 2819
Figure 1. Structures of IQ and of the IQ-DNA adducts N-(deoxyguanosine-8-yl)-2-amino-3-methyl[4,5-f]quinoline (dG-C8-IQ) and (deoxyguanosine-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ). Asterisk (*) marks site of deuteriation for internal standard compounds.
are present in a variety of foods, there is concern that these compounds may contribute to nutritionally linked human cancers.15,16 The covalent modification of DNA by genotoxic carcinogens such as HAAs is regarded as a critical step in the development of cancer. Therefore, the measurement of HAADNA adducts may provide a means for assessing human cancer risk and proposing acceptable limits of exposure.17,18 IQ is found in cooked meats and fish and is also present in cigarette smoke condensate.19-21 In order for IQ to bind to DNA, it must be metabolically activated, which may occur by cytochrome (6) Sugimura, T.; Sato, S.; Wakabayashi, K. Mutagens/carcinogens in pyrolysates of amino acids and proteins and in cooked foods: heterocyclic aromatic amines. In Chemical Induction of Cancer, Structural Bases and Biological Mechanisms; Woo, Y.-T., Lai, J. C., Argus, M. F., Eds.; Acedemic Press: San Diego, 1988; Vol. 3C, pp 681-710. (7) Sugimura, T. Trends Pharmacol. Sci. 1988, 9, 205-209. (8) Sugimura, T. Mutat. Res. 1988, 205, 33-39. (9) Adamson, R. H.; Takayama, S.; Sugimura, T.; Thorgeirsson, U. P. Environ. Health. Perspect. 1994, 102, 190-193. (10) Adamson, R. H. Cancer Detect. Prev. 1989, 14, 215-219. (11) Adamson, R. H.; Thorgeirsson, U. P.; Snyderwine, E. G.; Reeves, J.; Dalgard, D. W.; Takayama, S.; Sugimura, T. Jpn. J. Cancer Res. 1990, 81, 10-14. (12) Thorgeirsson, U. P.; Dalgard, D. W.; Reeves, J.; Adamson, R. H. Regul. Toxicol. Pharmacol. 1994, 19, 130-151. (13) Ohgaki, H.; Takayama, S.; Sugimura, T. Mutat. Res. 1991, 259, 399-410. (14) Tanaka, T.; Barnes, W.; Williams, G. M.; Weisberger, J. H. Jpn. J. Cancer Res. 1985, 76, 570-576. (15) Nagao, M.; Sugimura, T. Mutat. Res. 1993, 290, 43-51. (16) Weisburger, J. H. ISI Atlas Sci.: Pharmacol. 1987, 1, 162-167. (17) Groopman, J. D.; Kensler, T. W. Chem. Res. Toxicol. 1993, 6, 764-770. (18) Poirier, M. C.; Beland, F. A. Chem. Res. Toxicol. 1992, 5, 749-755. (19) Felton, J. S.; Knize, M. G.; Shen, N. H.; Andresen, B. D.; Bjeldanes, L. F.; Hatch, F. T. Environ. Health Perspect. 1986, 67, 17-24. (20) Felton, J. S.; Knize, M. G. Hetercyclic aromatic. mutagens/carcinogens in foods. In Handbook of Experimental Pharmacology; Cooper, C. S., Grover, P. L., Eds.; Springer-Verlag: Berlin and Heidelberg, 1990; Vol. 94/I, pp 471-502. (21) Manabek, S.; Tohyama, K.; Wada, O.; Aramaki, T. Carcinogenesis 1991, 12, 1945-1947.
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P450 1A2-mediated N-oxidation to produce 2-(hydroxyamino)-3methylimidazo[4,5-f]quinoline (HONH-IQ).22,23 The HONH-IQ metabolite is believed to undergo either O-acetylation or Osulfonation thereby further enhancing its reactivity and rate of DNA adduction.24,25 IQ adduction in rodents and nonhuman primates occurs primarily at the C8 position of guanine bases in the DNA backbone, although adduction at the N2 position also occurs to a lesser extent.26-30 To date, a number of investigations attempting to correlate carcinogen exposure of HAAs to the onset of cancer have used IQ as a model compound due to its structural representation of this class of compounds, its presence in a number of foods, and its potent mutagenicity.31-34 These studies measured the level of IQ-DNA adducts in animal models having close interspecies relationships with humans so that the resultant tumor incidence data could be used for human risk assessment. The technique used to detect and quantify the IQ-DNA adducts (22) Battula, N.; Schut, H. A. J.; Thorgeirsson, S. S. Mol. Carcinog. 1991, 4, 407-414. (23) Guengerich, F. P.; Shimada, T. Chem. Res. Toxicol. 1991, 4, 391-407. (24) Kato, R. CRC: Crit. Rev. Toxicol. 1986, 16, 307-348. (25) Snyderwine, E. G.; Wirth, P. J.; Roller, P. P.; Adamson, R. H.; Sato, S.; Thorgeirsson, S. S. Carcinogenesis 1988, 9, 411-418. (26) Hashimoto, Y.; Shudo, K.; Okamoto, T. Biochem. Biophys. Res. Commun. 1980, 96, 355-362. (27) Hashimoto, Y.; Shudo, K.; Okamoto, T. ′′ J. Am. Chem. Soc. 1982, 104, 7635-7640. (28) Snyderwine, E. G.; Roller, P. P.; Adamson, R. H.; Sato, S.; Thorgeirsson, S. S. Carcinogenesis 1988, 9, 1061-1065. (29) Lin, D.; Turesky, R. J.; Miller, D. W.; Lay, J. O. Jr.; Kadlubar, F. F. Chem. Res. Toxicol. 1992, 5, 691-697. (30) Schut, H. A. J.; Putnam, K.; Randerath, K. Cancer Lett. 1988, 41, 345352. (31) Turesky, R. J.; Markovic, J. Chem. Res. Toxicol. 1994, 7, 752-761. (32) Schut, H. A. Environ. Health Perspect. 1994, 102 (Suppl.), 57-60. (33) Turesky, R. J.; Markovic, J. Carcinogenesis 1995, 16, 2275-2279. (34) Turesky, R. J.; Gremaud, E.; Markovic, J.; Snyderwine, E. G. Chem. Res. Toxicol. 1996, 9, 403-408.
in these studies was 32P-postlabeling.35,36 32P-Postlabeling is very useful for detecting the DNA adducts resulting from exposure to many diverse classes of chemical carcinogens, enabling the detection of 1 adduct in 1010 nucleotides.37 However, quantification by this procedure can lead to an underestimation of the number of DNA adducts, due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides.38-41 The use of liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) to quantify drug compounds in vivo is well documented.42,43 The accuracy and precision of this analytical technique has been proven to be acceptable due to its widespread use in federally regulated pharmaceutical laboratories. However, the use of this technique in the area of DNA adduct research has been limited due the level of DNA adduction in vivo being far below the limits of detection attainable by standard LC/ ESI-MS. The recent emergence of micro- and nanoflow electrospray ionization (µESI and nESI) sources has resulted in profound decreases in the limits of detection attainable using mass spectrometry.44 Furthermore, operating at microliter and nanoliter flow rates has enabled the coupling of packed capillary liquid chromatography systems to mass spectrometry thereby allowing the detection of analyte compounds in biological samples.44-48 Specifically, a capillary LC/µESI-MS method was recently developed to detect IQ-DNA adducts in the kidney of nonhuman primates undergoing carcinogen bioassay at levels approaching 1 adduct in 107 bases.49 However, with current studies aimed at exposing animals to carcinogen levels close to what would be expected in humans, it is essential to develop a capillary LC/µESIMS method capable of detecting less than 1 adduct in 108 bases. It is also imperative that the method should be able to quantify adduction at those levels with high accuracy and precision. This paper reports on the use of a capillary LC/µESI-MS method to quantify the level of liver IQ-DNA adducts in rats dosed with IQ at 0.05, 0.50, 1.0, and 10 mg/kg of body weight. The accuracy and precision of the method based on statistical (35) Randerath, K.; Reddy, M. V.; Gupta, R. C. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6126-6129. (36) Gupta, R. C.; Reddy, M. V.; Randerath, K. Carcinogenesis 1982, 3, 10811092. (37) Randerath, K.; Randerath, E.; Agrawal, H. P.; Gupta, R. C.; Schurdak, M. E.; Reddy, M. V. Environ. Health Perspect. 1985, 62, 57-65. (38) Beach, A. C.; Gupta, R. C. Carcinogenesis 1992, 13, 1053-1074. (39) Randerath, E.; Agrawal, H. P.; Weaver, J. A.; Bordelon, C. B.; Randerath, K. 3 Carcinogenesis 1985, 6, 1117-1126. (40) Shields, P. G.; Harris, C. C.; Petruzzeli, S.; Bowman, E. D.; Weston, A. Mutagenesis 1993, 8, 121-126. (41) Beland, F. A.; Doerge, D. R.; Churchwell, M. I.; Poirier, M. C.; Schoket, B.; Marques, M. M. Chem. Res. Toxicol. 1999, 12, 68-77. (42) Jeanville, P. M.; Woods, J. H.; Baird, T. J., 3rd; Estape, E. S. J. Pharm. Biomed. Anal. 2000, 5, 897-907. (43) Zhao, J. J.; Xie, I. H.; Yang, A. Y.; Roadcap, B. A.; Rogers, J. D. J. Pharm. Biomed. Anal. 2000, 9, 1133-1143. (44) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aeserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802-3805. (45) de Wit, J. S.; Parker, C. E.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1987, 59, 2400-2404. (46) Parker, C. E.; Perkins, J. R.; Tomer, K. B.; Shida, Y.; O’Hara, K. J. Chromatogr., B. 1993, 616, 45-57. (47) Vanhoutte, K.; Van Dongen, W.; Hoes, I.; Lemiere, F.; Esmans, E. L.; Van Onckelen, H.; Van den Eeckhout, E.; van Soest, R. E. J.; Hudson, A. J. Anal. Chem. 1997, 69, 3161-3168. (48) Robins, R. H.; Guido, J. E. . Rapid Commun. Mass. Spectrom. 1997, 11, 1661-1666. (49) Gangl, E. T.; Turesky, R. J.; Vouros, P. Chem Res. Toxicol. 1999, 12, 10191027.
evaluation of the data generated from five standard curves over a four-week period is also presented. Finally, verification of the capillary LC/µESI-MS method was accomplished by comparing the rat liver dose-response adduction results acquired during this study to those obtained during a similar dose-response study utilizing the 32P-postlabeling technique. MATERIALS AND METHODS Caution: IQ and several of its derivatives are carcinogenic to rodents and should be handled carefully. Chemicals. The following chemicals were obtained from Sigma Chemical Co. (St. Louis, MO): calf thymus DNA, 2′deoxyguanosine (2′-dG), deoxyribonuclease 1 (type 2 from bovine pancreas), alkaline phosphatase (type 3s), ammonium acetate (Sigma ultra 98%), potassium phosphate, monobasic, ethylenediaminetetraacetic acid (EDTA), triethylamine, tris(hydroxymethyl)aminomethane (TRIZMA), magnesium chloride. Phosphodiesterase 1 (Crotalus adamanteous venom) was obtained from Amersham Pharmacia Biotech. Hydrochloric acid was obtained from Fisher Scientific (Pittsburgh, PA). All solvents used were HPLC grade unless otherwise stated, and prior to use, water was purified using a Millipore Milli-Q plus system (resistance 18 MΩ minimum). Chemical Synthesis. The nitro derivative of IQ was synthesized by the method of Grivas with minor modifications.50,51 N3IQ and N3-IQ-d3 were synthesized and purified by the method of Turesky and Markovic.33 Electrospray ionization mass spectrometry confirmed the structures of both the deuterated and undeuterated azides. The parent ions ([M + H]+) of the undeuterated and deuterated compounds occurred at 224.8 and 227.9, respectively, with the major fragment ion occurring at [M + H - 28]+ corresponding to the loss of N2. dG-C8-IQ, dG-C8-IQ-d3, and dG-N2-IQ standards were synthesized by photoactivating either the N3-IQ or N3-IQ-d3 in the presence of 2′-deoxyguanosine, where the yield of the C8 or N2 adduct depended on the reaction solvent pH and ionic strength.31 The standards were purified using HPLC with each adduct standard peak identified on the basis of its characteristic UV spectra and further examined for purity by capillary LC/MS.31,52 Following purification, the HPLC fraction containing the adduct standard was lyophilized to dryness, reconstituted with 0.5 mL of 50:50 (v/v%) methanol/water and stored at - 20 °C in 2-mL siliconized screw cap tubes. Analytical Solutions. The concentration of adduct in each stock solution was quantified spectrophotometrically (Spectronic Genesis 5, model 336008) based on molar extinction coefficients in CH3OH of 250 ) 50 000 (dG-C8-IQ, dG-C8-IQ-d3) and 274 ) 41 000 (dG-N2-IQ). The final stock solution concentrations were 5.88, 2.37, and 11.2 µg/mL for dG-C8-IQ, dG-C8-IQ-d3, and dGN2-IQ, respectively. dG-C8-IQ and dG-N2-IQ analyte working solutions were prepared by diluting the respective stock solution to the appropriate concentration in 20:80 (v/v%) methanol/water. The dG-C8-IQ-d3 internal standard working solution was prepared by diluting the stock solution to the appropriate concentration in 10:90 (v/v%) methanol-5 mM TRIS/10 mM MgCl2, pH 7.6. (50) Grivas, S. J. Chem. Res. (S) 1988, 84. (51) Turesky, R. J.; Bracco-Hammer, I.; Markovic, J.; Richli, U.; Kappeler, A. M.; Welti, D. H. Chem. Res. Toxicol. 1990, 3, 524-535. (52) Turesky, R. J.; Rossi, S. C.; Welti, D. H.; Lay, J. O.; Kadlubar, F. F. Chem. Res. Toxicol. 1992, 5, 479-490.
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Animals and Treatments. Male Fischer-344 rats at ∼200 g of body weight and eight weeks of age were used for this study. Animals were purchased from Iffa Credo L’Abresle, France. The protocol was reviewed and approved by the Swiss Federal Review Board. Animals were acclimated for one week with three animals per cage and consumed Nafag 890 rat chow and water ad libitum. IQ was prepared as a suspension in 10 mM phosphate-buffered saline (pH 7.4) in a final volume of 1 mL per rat and administered by gavage at a dose of 0.05, 0.50, 1.0, and 10 mg/kg of body weight. In all cases, IQ was administered 24 h prior to autopsy. Animals were sacrificed with an overdose of sodium pentobarbital (60 mg/kg ip). Liver tissue was obtained immediately following sacrifice, quickly frozen in liquid nitrogen, and stored at -80 °C (52). Approximately 1.0-1.7 mg of nuclear DNA was isolated from each liver tissue, diluted to 1 mL with water, and stored at -80 °C.53,54 32P-Postlabeling. Livers of male Fischer-344 rats were used in this study.57 Nuclear DNA was isolated from tissues by Qiagen chromatography.31,33 The enzymatic digestion of DNA (30 µg) and adduct enrichment by solid-phase extraction were performed as previously described.33 The 32P-postlabeling analyses were performed as described by Randerath et al. with modifications of the TLC solvents used for adduct resolution.31,37 Recovery of synthetic 3′-phospho-dG-C8-IQ (100 fmol), which had been added to 30 µg of rat liver DNA from untreated animals (1 adduct per 106 bases) prior to MNSPD hydrolysis and solid-phase extraction, was 44 ( 8.0% (N ) 3) and was not taken into account for adduction calculations.33 Adduct visualization and quantification were determined as previously described, and adduct identity was corroborated by HPLC using a protocol previously described.31,33 Preparation of IQ-Dosed Fischer-344 Rat Liver DNA for Capillary LC/µESI-MS Analysis. The rat liver tissue DNA was digested to nucleosides according to a procedure described previously49 with the following modifications. DNA (300 µg) was transferred to an incubation vial, spiked with 85 fmol of C8-dGIQ-d3 and 5 mM TRIS/10 mM MgCl2 (pH 7.6) was added to achieve a final volume of 300 µL. DNASE 1 (dissolved at 10 mg/ mL in 5 mM TRIS/10 mM MgCl2, pH 7.6) was added at 770 units/ mL and incubated at 37 °C for 5 h. Then, alkaline phosphatase (straight) and phosphodiesterase (dissolved at 1.0 mg/mL in 5 mM TRIS/10 mM MgCl2, pH 7.6) were added at 4.0 and 0.30 units/mL respectively, and the digest incubated at 37 °C for 18 h. Enzymatic hydrolysis was terminated by the addition of 900 µL of ice cold ethanol (0 °C) followed by rapid vortex mixing. Insoluble precipitates were pelleted by centrifugation for 15 min (1100 rpm) (Savant Instruments, model SC110A, Farmingdale, NY). The supernatant was then isolated and evaporated under vacuum to ∼100 µL. Preparation of dG-C8-IQ Standard Curves. Standard curves were prepared by adding 300 µg of calf thymus DNA to (53) Turesky, R. J.; Markovic, J.; Aeschlimann, J.-M. Chem. Res. Toxicol. 1996, 9, 397-402. (54) Heflich, R. H.; Morris, S. M.; Beranek, D. T.; McGarrity, L. J.; Chen, J. J.; Beland, F. A. ′ Mutagenesis 1986, 1, 201-206. (55) Turesky, R. J.; Constable, A.; Richoz, J.; Varga, N.; Markovic, J.; Martin, M. V.; Guengerich, F. P. Chem. Res. Toxicol. 1998, 11, 925-936. (56) Gangl, E. T.; Turesky, R. J.; Vouros, P. Anal. Chem. 2001, 73, 23972404. (57) Turesky, R. J.; Box, R. M.; Markovic, J.; Gremaud, E.; Snyderwine, E. G. Mutat. Res. 1997, 376, 235-241.
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five independent vials containing 260 µL of 5 mM TRIS/10 mM MgCl2, pH 7.6. Then, 85 fmol of C8-dG-IQ-d3 (10 µL total volume) and 30 µL of a preprepared serial dilution solution containing a known amount of adduct were added to each respective vial to achieve a final volume of 300 µL. Each sample was then digested and processed in the same manner as stated for the IQ-dosed rat liver DNA samples. To ensure accurate rat liver IQ adduct quantification, duplicate standard curves were prepared in the same manner as stated for the calf thymus standard curve samples except that 300 µg of liver tissue DNA from rats dosed with phosphate-buffered saline (control) was used as a matrix. Adduct Enrichment and Sample Cleanup prior to Capillary LC/µESI-MS Analysis. Solid-phase sample extraction was facilitated by using a vacuum manifold fitted with disposable, valved, Teflon needles (Alltech Associates Inc., Deerfield, IL) to prevent sample cross-contamination. Isolute C18 (100 mg) SPE cartridges were conditioned with 3 mL of methanol followed by 3 mL of 5 mM triethylamine in water, pH 3.5-3.7 (pH adjusted with 1 M HCL). The addition of triethylamine to both the conditioning and wash solutions was found to elute a large majority of polar and nonpolar endogenous digest components while concurrently retaining the adduct of interest. Reduction in digest background helped reduce matrix-related analyte signal suppression during analysis. Following evaporation to 100 µL, each DNA digestion mixture was diluted with 1.5 mL of 5 mM triethylamine in water, pH 3.5-3.7, and then applied to the preequilibrated SPE cartridges. Each cartridge was then washed with 3 mL of 5 mM triethylamine in 10:90 (v/v%) methanol/water, pH 3.5-3.7 (pH adjusted with 1 M HCl), followed by 3 mL of water, and then the adducts were eluted with 1 mL of methanol. Each methanol eluent was filtered through 13 mm of PTFE 0.45-µm Minispike filters (Waters Corp., Milford, MA) and then evaporated to dryness under vacuum centrifugation at 0.02 mbar. Prior to injection into the capillary LC/µESI-MS system, the lyophilized sample was reconstituted with 20 µL of 10:90 (v/v%) methanol/5 mM ammonium acetate, pH 4.0. The SPE recovery for dG-C8-IQ was ∼60%. Chromatography. Capillary LC Column Packing. All chromatographic analyses were conducted on a 75 µm i.d. × 360 µm o.d. × 16.5 cm bare fused-silica column packed to a length of 8 cm with Nucleosil, ODS (end capped), 5-µm reversed-phase particles. Columns were manufactured in-house following a protocol based on previously described procedures.45,47 The frit slurry solution was prepared by combining 20 µL of formamide with 80 µL of potassium silicate (Kasil-1, PQ Corp., Valley Forge, PA), vortex mixing to ensure complete homogeneity, and then centrifuging for 5 min (1100 rpm). Bare fused-silica capillary (Polymicro Technologies, Pheonix, AZ), 75 µm i.d. × 360 µm o.d., was cut to a length of 30 cm and the end inserted into the visibly clear supernatant of the frit slurry solution until capillary action loaded 3 cm of slurry into the capillary. The end containing the slurry was cut so that 1 cm of solution remained, and then the entire capillary was baked at 90 °C for 2 h. Once fixed, the frit was cut to 2 mm and conditioned with water, methanol, and dichloromethane prior to packing. Columns were packed to a length of 8 cm, cut to a total length of 16.5 cm (8.5 cm unfilled), and then conditioned with methanol for 2 h. Each column was
Figure 2. Schematic of the capillary LC/µESI-MS system
used without introducing a forefrit at the head of the packing material. Capillary Liquid Chromatography. Figure 2 shows a schematic of the capillary LC system used for this study.49 An HP1090 series 2 liquid chromatograph was used to generate solvent flow at a rate of 0.4 mL/min (Agilent Technologies, Palo Alto, CA). To reduce the solvent flow rate into the µLC interface, an LC Packings IC-400-VAR (LC Packings, San Fransisco, CA) splitter equipped with a fused-silica calibrator (5 µm i.d. × 100 µm o.d. × 13 cm) was placed in-line. The solvent flow rate, postsplitter, measured at ∼200 nL/min., resulted in a flow rate split ratio of ∼2000:1. The flow from the splitter was then directed to the integrated capillary LC/µESI system. A four-port microbore (0.15 mm) valve equipped with a stainless steel stator and 0.5-µL PAEK internal sample loop was used for sample introduction (VICI, Valco, Houston, TX). To eliminate the analyte carryover issues associated with use of valves equipped with external injector ports, a negative pressure sample introduction system was developed. Specifically, a bare fused-silica capillary (75 µm i.d. × 360 µm o.d. × 10 cm) was fitted to the injection port using a PAEK sleeve and ferrule so that the dead volume between the capillary end and the through-port was minimized. A second bare fused-silica capillary of the same dimension was fitted to the waste port, again using a PAEK sleeve and ferrule. Sample introduction was achieved by placing the open end of the injection port capillary into a sample and applying a negative pressure to the end of the waste port capillary using a 500-µL gastight syringe (Hamilton Co., Reno, NV). The total volume injected per sample using this system, including capillary and sample loop, is ∼1.0 µL. Following sample injection, the fluidics were flushed with DMSO, methanol, and 10:90:0.05% (v/v/v%) methanol/water/acetic acid to ensure no analyte carryover from injection to injection. Isolation of both dG-C8-IQ and dG-C8-IQ-d3 from endogenous in vivo sample components was achieved using a binary mobile phase consisting of 5 mM ammonium acetate, pH 4.0/methanol/0.05% acetic acid. For the first 10 min of each run, the mobile phase was held isocratic at 10% methanol/0.05% acetic acid/90% 5 mM ammonium acetate, pH 4.0, to ensure the sample loop was flushed completely and the analyte was focused at the head of the capillary column.
Then, the methanol/0.05% acetic acid was increased linearly from 10 to 90% over 5 min and held isocratic at 90% for 5 min, to ensure stable electrospray as the analyte eluted as well as to purge the capillary column of late eluting species. The capillary column was then allowed to reequilibrate by stepping the mobile phase from 90 to 10% methanol/0.05% acetic acid and holding for 5 min. Simultaneous analysis for dG-N2-IQ and dG-C8-IQ in vivo was achieved using a binary mobile phase consisting of water/0.05% acetic acid and methanol/0.05% acetic acid. Following a 10-min isocratic period at 10% methanol/0.05% acetic acid, the methanol/ 0.05% acetic acid was stepped to 90% in 0.01 min and held isocratic for 7 min. The methanol/0.05% acetic acid was then stepped to 10% in 0.01 min and the system allowed to reequilibrate for 5 min prior to subsequent analyses. µESI and Mass Spectrometry. Microelectrospraying of the capillary column effluent was achieved using a liquid junction interface platform similar in design to an interface shown previously.56 All mass spectral analyses were conducted using a TSQ 700 triple quadrupole mass spectrometer (Thermoquest, San Jose, CA). On a weekly basis, the mass spectrometer was tuned on [M + H]+ m/z 464 using a 1:10 (v/v) dilution of a dG-C8-IQ stock solution (0.588 µg/mL final) in 10:90:0.05% (v/v%) methanol/ water/0.05% acetic acid. Prior to daily analysis, the heated capillary temperature was set to 175 °C, the collision cell pressure set to 0.9 mTorr, second quadrupole offset voltage set to -30 V, and conversion dynode set to -15 kV. Electrospraying of the capillary column effluent was initiated by positioning the µESI tip ∼3 mm from the entrance hole of the TSQ700 heated capillary and applying a potential of 1.5 kV to the stainless steel liquid junction housing. Following in vivo sample injection, an instrument control language (ICL) procedure was initiated that set the instrument to acquire positive ions in a multiple reaction monitoring mode. Specifically, the first quadrupole was set to transmit [M + H]+ ions of m/z 464 (C8-dG-IQ) and 467 (C8-dG-IQ-d3) (dwell time 0.4 and 0.2 s, respectively, default interchannel delay and scan width of 0.4 Da each) while the third quadrupole monitored the AH2+ fragment ions (-116 Da) m/z 348 and 351, respectively. At 16.5 min, the electron multiplier voltage was increased to 1600 V, while the same scanning parameters were maintained, until the Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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Table 1. Capillary LC/µESI-MS Method Inter- and Intraday Accuracy and Precisiona
standard curve ID 082500-1 083000-1 090600-1a 090600-1b 091900-1
090600-1a 090600-1b
analysis date
mass of dG-C8-IQ (fmol) spiked into 300 µg of DNA (predigestion) 148 119 73.8 43.4 17.5
(A) Interday Accuracy and Precision Data 8/25/00 153 120 60.1 42.9 8/30/00 162 105 68.7 38.3 9/06/00 159 125 58.9 37.1 9/06/00 142 121 62.2 41.3 9/19/00 151 130 57.6 35.8
25.4 27.7 26.8 25.5 29.7
mean SD % RSD
153 7.7 5.1
120 9.4 7.8
61.5 4.4 7.1
39.1 2.9 7.5
27.0 1.8 6.6
% accuracy
103
101
83.3
90.0
154
(B) Intraday Accuracy and Precision Data 9/06/00 159 125 58.9 37.1 9/06/00 142 121 62.2 41.3
26.8 25.5
mean SD % RSD
151 12 8.0
123 2.8 2.3
60.6 2.3 3.9
39.2 3.0 7.6
26.2 0.90 3.5
% accuracy
102
103
82.0
90.3
149
a The data for each standard curve represent the back-calculated mass dg-C8-IQ in each digest. Standard curves ID 090600-1a and -b were prepared using liver DNA from a rat dosed with phosphatebuffered saline. The remaining standard curves were prepared using calf thymus DNA.
analyte and internal standard were detected after which the electron multiplier was returned to its prerun value. For simultaneous analysis of the dG-C8-IQ and dG-N2-IQ adducts, the instrument was set to acquire positive ions in a selected reaction monitoring mode where the first and third quadrupoles were set to scan for m/z 464 and 348, respectively (scan time of 0.4 s, scan width 0.4 Da), and the electron multiplier voltage increased to 1600 V at 10 min. Quantification. The control rat liver DNA standard curve was constructed by plotting the mean analyte to internal standard peak height ratio resulting from duplicate sample digest analysis at each mass level versus mass of analyte in 300 µg of DNA digest. The resulting linear regression data was used to quantify the mass of dG-C8-IQ in liver DNA from each rat involved in the study. Three individual 300-µg DNA aliquots were analyzed from rats 1-9 to allow statistical evaluation of the resultant quantification data. Because of the high dG-C8-IQ adduct content in samples 1012, only 100 µg of DNA was taken for analysis. The data were then normalized to reflect the adduct content in 300 µg of DNA. Accuracy and Precision. Accuracy and precision of the assay were evaluated by analyzing the back-calculated dG-C8-IQ mass from a total of five standard curves analyzed over a four-week period (Table 1). The RSD of the mean predicted mass for the independently assayed standard curve digests provided a measure of precision. The accuracy of the assay was assessed by expressing the mean predicted dG-C8-IQ mass as a percentage of the known mass added to each digest. RESULTS Mass Spectrometric Analysis of IQ-DNA Adduct Standards and Internal Standard. The structures of dG-C8-IQ and dG-N2-IQ are shown in Figure 1. To ensure the presence of both 2824
Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
standard and internal standard compounds, each stock solution was analyzed using flow injection analysis µESI-mass spectrometry. The product ion spectra of the [M + H]+ ions of the two isomeric dG-C8-IQ and dG-N2-IQ adducts show very characteristic fragmentation patterns when subjected to collision-induced dissociation (CID) (Figure 3). The [M + H]+ ions appear at m/z 464 while the AH2+ ions ([MH - deoxyribose + H]+) appear at m/z 348. Further fragmentation of the AH2+ ion of both species results in specific cross ring cleavage products, m/z 303 and 331 for C8 and N2 adducts (Figure 3D and F), and are typical for adducts of this type.49 Panels B and E of Figure 3 show the product ion spectrum for C8-dG-IQ-d3, where the mass offset of +3 Da for each parent and product ion is due to the presence of the three deuterium atoms at the N-methyl group of the IQ adduct. The facile loss of the deoxyribose group from the parent ion was exploited during subsequent analyses to enable the use of selected reaction monitoring (SRM). By optimizing the argon gas pressure in the TSQ700 mass spectrometer collision chamber as well as the energy at which the parent ion enters the chamber, the m/z 464 to 348 transition can be induced to occur with greater yield of product ions. Thus, by monitoring this transition (SRM) during sample analyses, a large majority of the background noise is filtered out, leading to an increased signal-to-noise level for the analyte peak. The absence of standard and internal standard stock solution cross-contamination was established using the capillary LC/µESIMS system operating in SRM mode. All standard solutions were determined to be pure and isotopic purity exceeded 99%. Determination of the Ratio of dG-N2-IQ versus dG-C8IQ in the Rat Liver DNA. To determine the approximate level of dG-N2-IQ in the rat liver DNA samples, a 100-µg DNA aliquot from rat 12 was processed and analyzed following the procedure outlined in Materials and Methods for the simultaneous determination of dG-C8-IQ and dG-N2-IQ. As shown in Figure 4, the dG-N2-IQ eluted at 15.4 min while the dG-C8-IQ eluted at 16.2 min. Based on their relative peak heights, the ratio of the N2 to the C8 adduct is ∼1:6. This ratio is in agreement with published results where the ratio of N2 to C8 adduction in animals receiving a single oral dose of IQ and sacrificed after 24 h was ∼1:5.34 Intra- and Interday Assay Accuracy and Precision. The dG-C8-IQ to dG-C8-IQ-d3 peak height ratio increased in proportion with the added mass of dG-C8-IQ in 300 µg of blank rat liver DNA from 17.5 to 148 fmol. Linear regression performed without a weighting factor yielded the best fit of the standard curves. The limits of quantification and detection for the control rat liver DNA standard curve were 17.5 and 6.00 fmol based on respective analyte peak signal-to-noise ratios of 10:1 and 3:1. Interand intraday accuracy and precision for determinations of each adduct mass comprising the standard curve are presented in Table 1. The adduct was quantified with a mean intraday accuracy of 105 ( 26 (SD) and the lowest mass included in the standard curve, 17.5 fmol, was determined with a RSD of 3.5% (Table 1B). Backcalculated adduct masses from five standard curves analyzed over a four-week period were used to assess interday accuracy and precision of the capillary LC/µESI-MS method (Table 1A). Mean values (( SD) of the regression parameters for these standard curves were as follows: slope, 8.36 × 10-3 ( 0.0010; y-intercept, -0.167 ( 0.038; correlation coefficient, 0.984 ( 0.0050. The
Figure 3. Flow injection analysis (FIA) of dG-C8-IQ (A), dG-C8-IQ-d3 (B), and dG-N2-IQ (C) standards. (A-C) represent product ion spectra of the [M + H]+ ion of each respective standard utilizing a collision offset voltage of -20 V. (D-F) represent product ion spectra of the [M + H]+ ion of each standard, only the collision offset voltage was increased to -50 V. Prior to FIA, the collision cell pressure was adjusted to 0.9 mTorr.
Analysis of IQ-Dosed Fischer-344 Rat Liver DNA. Representative mass chromatograms showing the presence of dGC8-IQ adduct at all dose levels as well as the absence of adduct in the control animal are shown in Figure 5. The mass of dGC8-IQ present in each 300-µg rat liver DNA sample aliquot was quantified based on regression results obtained following the analysis of a standard curve prepared using blank rat liver DNA. The mean mass of dG-C8-IQ in each rat sample and the corresponding number of adducts in 108 bases are shown in Table 2. Figure 6 shows the mean pooled dG-C8-IQ mass in 300 µg of rat liver DNA as a function of IQ dose at each dose level.
Figure 4. Selected ion chromatogram showing the ratio of dG-N2IQ versus dG-C8-IQ in Fischer-344 rat 12 dosed with IQ at 10 mg/ kg. (A) Blank 10:90 (v/v%) methanol/water reconstitution solution. (B) dG-N2-IQ and dG-C8-IQ content in 100 µg od liver DNA from rat 12. (C) System test solution containing dG-N2-IQ and dG-C8-IQ at 10 and 1 ng/mL, respectively.
accuracy of adduct quantification over a four-week period was 106 ( 28 (SD). The interday precision of the assay showed no trends with RSD values ranging from 5.1 to 7.8%. These results demonstrate that the assay is accurate and reproducible.
DISCUSSION The capillary LC/µESI-MS method developed for this study was used to successfully quantify the level of dG-C8-IQ adducts in the liver of rats involved in a dose-response study. The measures taken to ensure the validity of the study and the implications of our findings are summarized in this section. Chromatography. In an effort to achieve optimal chromatographic efficiency and response for dG-C8-IQ and dG-N2-IQ, several different mobile-phase combinations were evaluated. Use of a binary system consisting of water/0.05% acetic acid and methanol/0.05% acetic acid produced excellent peak sharpness for both adducts. In addition, when this combination was used Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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Figure 5. Selected ion chromatograms of Fischer-344 rat liver DNA dosed with IQ at 0.05, 0.5, 1.0, and 10.0 mg/kg of body weight. The top trace in each group corresponds to the level of dG-C8-IQ adduct in a 300-µg liver DNA aliquot, 24 h posttreatment, while the bottom trace corresponds to the internal standard dG-C8-IQ-d3. (A) Rat 1, 0.05 mg/kg IQ dose. (B) Rat 4, 0.5 mg/kg IQ dose. (C) Rat 7, 1.0 mg/kg IQ dose. (D) Rat 11, 10.0 mg/kg IQ dose. (E) Rat 14, PBS dose (blank). Table 2. Comparison of the DG-C8-IQ Adduct Levels in Rat Liver DNA as a Function of IQ Dose Using Different Detection Methodologiesa (B) 32P-postlabeling analysisb
(A) capillary LC/µESI-MS analysisa IQ dose (mg/kg)
animal code
mass of dG-C8-IQ (fmol)
adducts/108 basesc
0.05
rat 1 rat 3 rat 4 rat 5 rat 6 rat 7 rat 8 rat 9 rat 10 rat 11 rat 12
31.8 ( 6.7 28.4 ( 1.3 75.7 ( 18 67.9 ( 22 62.5 ( 9.8 122 ( 30 90.4 ( 33 95.2 ( 7.6 351 ( 32 349 ( 0.60 340 ( 52
3.5 ( 0.70 3.1 ( 0.10 8.4 ( 2.0 7.6 ( 2.4 6.9 ( 1.1 14 ( 3.3 10 ( 3.7 10 ( 0.80 39 ( 3.4 38 ( 0.10 37 ( 5.6
0.50 1.0 10
IQ dose (mg/kg)
adducts/108 basesd
0.05
0.40 ( 0.20
0.30
1.0 ( 0.20
1.0
2.0 ( 0.40
10
33 ( 12
a(A) DG-C8-IQ adduction level dose-response data determined using capillary LC/µESI-MS. b(B) DG-C8-IQ adduction level dose-response data generated during a separate study where the C8-IQ adduct content in each rat liver dna was determined by 32P-postlabeling.57 32P-Postlabeling values do not take into account 50% labeling efficiency. c Mean ( SD of three independent analyses for rats 1-9 and two independent analyses for rats 10-12. d Values obtained from three independent analyses ( SD.
and equal masses of C8 and N2 adduct were injected, the MS responses for both adducts were approximately equal. As shown in Figure 4, the use of the aforementioned mobile-phase components in conjunction with the optimized capillary LC method allowed both the C8 and N2 adducts to be observed in the liver DNA of rat 12 dosed at 10 mg/kg IQ. However, following peak integration, the signal-to-noise ratio of the dG-C8-IQ peak was ∼30. On the basis of this calculation, the limits of detection were insufficient to quantify the dG-IQ adducts at lower dose treat2826 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
ments. Therefore, alternative solvent conditions were investigated to enhance the signal for the dG-C8-IQ adduct. As stated in Materials and Methods, a mobile-phase combination of 5 mM ammonium acetate, pH 4.0/methanol/0.05% acetic acid was used in conjunction with the optimized capillary LC method to successfully quantify dG-C8-IQ in each rat liver DNA at all dose levels. As shown in Figure 5D, the dG-C8-IQ peak response resulted in a signal-to-noise ratio of 305 for rat 10 dosed at the same level of rat 12. This is a dramatic improvement over
formation does not increase as a linear function of IQ dose. This nonlinear relationship occurs because IQ must first undergo metabolic N-oxidation by cytochrome P450 1A2 in the liver before it can bind to DNA. The rate at which IQ is metabolized to HNOHIQ is dictated by the level of cytochrome P450 1A2 expressed in rat liver.55 At high IQ concentrations, the cytochrome P450 1A2 is saturated by the substrate and some of the dose will be eliminated as unmetabolized IQ. Direct phase two pathways will also be implicated in metabolism of IQ at high doses.51 Therefore, the percent of the dose that goes through bioactivation to form the HNOH-IQ metabolite decreases as a function of increasing dose, resulting in a lower percentage of the IQ that adducts to DNA.
Figure 6. Log-log relationship between IQ dose and dG-C8-IQ adduct formation in the liver tissue of Fischer-344 rats; Pooled results.
the dG-C8-IQ peak signal-to-noise ratio observed when the water/ 0.05% acetic acid/methanol/0.05% acetic acid mobile-phase components were used. However, when the buffered mobile-phase system was used to analyze equal masses of dG-C8-IQ and dGN2-IQ, the response for the N2 adduct was only 1/10 the response of the C8 adduct, hence the reason the N2 adduct peak is not observed in any of the chromatograms shown in Figure 5. Since it was determined that the major lesion resulting from this doseresponse study would be dG-C8-IQ based on 32P-postlabeling results, all efforts were directed toward optimizing its peak response and not that of dG-N2-IQ.31,33 Thus, future studies aimed at quantifying both adducts following IQ dosing at levels similar to those described here will require further chromatographic optimization. Comparison of Capillary LC/µESI-MS Dose-Response Results to 32P-Postlabeling Data. The method used during this study to quantify rat liver IQ-DNA adducts was shown to be accurate and precise. However, to verify our method, it was necessary to compare our dose-response data with doseresponse data generated using an established technique. In a previous dose-response study using Fischer-344 rats, the level of dG-C8-IQ present in the liver DNA of each animal was measured by 32P-postlabeling and the results are shown in Table 2.57 There exists a good correlation between the adduction data acquired using our capillary LC/µESI-MS system to those acquired using 32P-postlabeling, which did not utilize an internal standard to account for losses and efficiency of the postlabeling procedure. Not taking into account labeling efficiencies of the 32Ppostlabeling procedure, which was ∼50%, the adduct levels are within a factor of 2-3-fold for these respective methods, thereby reinforcing the validity of the dose-response data presented here. The lower estimates of adduct levels obtained by the 32Ppostlabeling procedure as compared to capillary LC/µESI-MS suggest that the percent recovery and 32P-postlabeling labeling efficiency of adducts at low levels of IQ-modified DNA in vivo (
