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Detection and Quantification of N-(Deoxyguanosin-8-yl)-4-aminobiphenyl Adducts in Human Pancreas Tissue Using Capillary Liquid Chromatography-Microelectrospray Mass Spectrometry Elaine M. Ricicki,† John R. Soglia,† Candee Teitel,‡ Robert Kane,§ Fred Kadlubar,‡ and Paul Vouros*,† The Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, Division of Molecular Epidemiology, National Center for Toxicological Research (HFT-100), Jefferson, Arkansas 72079, and Thermo Electron Corporation, San Jose, California 95134 Received November 5, 2004
Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (µESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 µm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]+ f [M + H - 116]+ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) and six were considered nonsmokers (three female and three male). Of the samples analyzed, six showed dG-C8-ABP levels above the limit of quantification for the method, five were considered to have levels that were undetectable, and one was discarded due to inconsistent internal standard signal. The age of the human subjects ranged from 17 to 63, and, in samples where adduct was present, levels ranged anywhere from 1 to 60/108 nucleotides. Although no correlation between smoking preference, age, or gender was proven with this particular sample pool, this report demonstrates that capillary LC-µESI-MS can provide a sensitive and definitive method for DNA adduct analysis in human tissue.
Introduction Cigarette smoke is known to be a contributing cause of lung, larynx, pharynx, esophagus, and bladder cancers (1). Further, it has been implicated in the development of a variety of other cancers, such as uterine, kidney, cervical, and pancreatic cancers (2). The analysis of cigarette smoke constituents has determined the presence of over 122 carcinogens, including polycyclic aromatic hydrocarbons and aromatic amines (3). About 30 aromatic amines have been detected in cigarette smoke, including the human carcinogen, 4-aminobiphenyl,1 which is present in nanogram quantities in mainstream smoke and even higher in sidestream smoke (4-6). * To whom correspondence should be addressed. Tel.: (617) 3732840. Fax: (617) 373-2693. E-mail:
[email protected]. † Northeastern University. ‡ National Center for Toxicological Research. § Thermo Electron Corp. 1 Abbreviations: 4-ABP, 4-aminobiphenyl; µESI-MS, micro-electrospray mass spectrometry; TRIZMA, tris(hydroxymethyl)-aminomethane; DMF, N,N-dimethylformamide; dG-C8-ABP, N-(deoxyguanosin-8-yl)4-aminobiphenyl; dG-C8-AsBP-D9, N-(deoxyguanosin-8-yl)-4-aminobiphenyl-D9; dG-N2-ABP, N-(deoxyguanosin-N2-yl)-4-aminobiphenyl; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.
Cancer of the pancreas is the fourth leading cause of cancer mortality in the United States and, as stated earlier, has been linked to smoking (7). Studies indicate that there is a 2-fold increase in risk of developing pancreatic cancer in heavy smokers (8, 9). The increased risk could indicate that the aforementioned carcinogens found within tobacco smoke physically affect pancreatic tissue and its related processes. As such, the capacity of the pancreas to metabolically activate aromatic amines has been previously investigated (10). Metabolism of 4-aminobiphenyl involves various cytochrome P450 enzymes. As with many aromatic amines, the initial step in metabolic activation involves hepatic Phase I pathway where the N-oxidation occurs to form an N-hydroxylamine. In the case of 4-aminobiphenyl, cytochrome P4501A2 is the predominant isoform catalyzing the oxidation to N-hydroxy-aminobiphenyl (1113). The N-hydroxy intermediates can then undergo Phase II conjugation in which enzymes such as Oacetyltransferases or O-sulfotransferases catalyze the formation of the highly reactive electrophiles such as N-acetoxy or N-sulfonyloxy-arylamine, which can then react with proteins or DNA (14-17).
10.1021/tx049692l CCC: $30.25 © 2005 American Chemical Society Published on Web 03/05/2005
Analysis of dG-C8-ABP Adducts in Human Pancreas
Figure 1. Structures of N-deoxyguanosin-C8-yl-ABP (MW 434) and the internal standard, N-deoxyguanosin-C8-yl-ABP-D9 (MW 443).
Covalent binding of carcinogens to DNA has long been considered a potential precursor to mutagenesis and cancer, and the measurement of these adducts is of critical importance as biomarkers. However, the low level of adduction (often 1 in 108 nucleotides or lower) and the low amounts of human DNA typically available have limited the methods of analyses. Primarily, immunoassays, 32P-postlabeling, and gas chromatography/mass spectrometry have been utilized with 32P-postlabeling chosen most often because of its high sensitivity despite its inability for structural elucidation (18-20). In the past decade, advancements in the combined technique of liquid chromatography/electrospray mass spectrometry have improved sensitivity for DNA adduct detection and quantification (21). Specifically, the use of mass spectrometry coupled primarily to micro- or nano-liquid chromatography has been demonstrated not only in the detection, but also the quantification of various DNA adducts including and not limited to those associated with heterocyclic aromatic amines (22, 23), melphalan (24), and oxidative stress (25). A thorough investigation of the state of mass spectrometry in DNA adduct analysis can be found in a full review by Koc and Swenberg (26). The presence of 4-aminobiphenyl DNA adducts has been studied both in vitro and in vivo by the various analytical methodologies described previously. The predominant adduct formed by 4-aminobiphenyl in vivo has been identified as N-(deoxyguanosin-8-yl)-4-aminobiphenyl (Figure 1), while a minor guanosine adduct has been identified as 3-(2′-deoxyguanosin-N2-yl)-4-ABP (27, 28). Research has primarily been conducted using immunochemical or 32P-postlabeling methods and primarily targeting bladder samples (29-32). Doerge and coworkers have developed a method using HPLC-ESI-MS to quantify the dG-C8-ABP adducts and have utilized this method in investigating DNA isolated from mice dosed with 4-aminobiphenyl (33, 34). In this study, capillary LC/micro-electrospray mass spectrometry is utilized for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8ABP) in human pancreas tissue. This method provides an alternative approach for the detection of DNA adducts in human tissues and illustrates the viability of electrospray mass spectrometry as a highly sensitive analytical technique for screening human specimen.
Materials and Methods Chemicals. Caution: 4-Aminobiphenyl and its derivatives are carcinogenic to humans and should be handled carefully. The following chemicals were obtained from Sigma Chemical Co. (St. Louis, MO): calf thymus DNA (ct DNA), potassium phosphate, monobasic (anhydrous, min. 99%), deoxyribonuclease I (type 2 from bovine pancreas), alkaline phosphatase (type IIIs),
Chem. Res. Toxicol., Vol. 18, No. 4, 2005 693 tris (hydroxymethyl)-aminomethane (TRIZMA) hydrochloride, 4-nitrobiphenyl, and magnesium chloride. Acetic acid (glacial, 99.99+%) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Phosphodiesterase 1 (Crotalus adamanteous venom) was received from Amersham Pharmacia Biotech (Piscataway, NJ). 4-Nitrobiphenyl-D9 (98%+) was obtained from Cambridge Isotope Laboratories (Andover, MA). Solvents were obtained from Fisher Scientific (Pittsburgh, PA) and were HPLC grade, unless otherwise noted. All water was prefiltered through a Millipore Milli-Q plus system (Millipore Co., Bedford, MA) and had a minimum resistance of 18 MΩ. Adduct Synthesis and HPLC Purification. The dG-C8ABP standard was synthesized by converting 4-nitrobiphenyl to its N-acetoxy derivative as follows. 4-Nitrobiphenyl (1 mmol) was dissolved in THF (5 mL) containing Pd/C catalyst (50 mg) and reduced to N-hydroxy-4-aminobiphenyl by adding hydrazine (6.4 mmol) while maintaining the temperature of the reaction at 0 °C for 45 min with gentle stirring every minute (35). Triethylamine (1.1 molar equiv) was then added, and the reaction mixture was allowed to cool to -30 °C. The N-hydroxy intermediate was subsequently converted to its O-acetyl derivative by adding pyruvonitrile (1.5 mmol) to the reaction mixture and allowing the reaction to proceed 30 min while maintaining the temperature at -30 °C (36). The mixture was then poured into 100 mL of ice-cold water, and the white precipitate was collected and washed with water (37). After drying, the molecular weight of the product was confirmed by mass spectrometry. The product, N-acetoxy-4-aminobiphenyl, was reacted with 2′-deoxyguanosine (2′-dG) in a 10:1 molar ratio to produce dGC8-ABP. The N-acetoxy derivative (0.3 mmol) was dissolved in 2 mL of dry THF and maintained at 0 °C. The N-acetoxy solution was then added to a 30 mmol (5 mL total volume) solution of 2′-dG in N,N-dimethylformamide (DMF)/water (1/1) at 0 °C. After being stirred at room temperature for 24 h, the reaction mixture was extracted four times with 5 mL of water-saturated diethyl ether, and the aqueous phase was lyophilized to dryness under vacuum. The deuterated internal standard, N-(deoxyguanosin-8-yl)-4-aminobiphenyl-D9 (dG-C8-ABP-D9), was prepared using 4-nitrobiphenyl-D9 in a similar manner, except on a smaller scale due to the limited availability of 4-nitrobiphenylD9. After reconstitution with 0.5 mL of 1:1 methanol/water, purification of both standards was performed by reverse phase HPLC (column: Supelco, 15 cm × 4.6 mm, C18 column) using a 20 min linear gradient of 20-70% methanol (aqueous portion: 10 mM ammonium acetate). Standard and internal standard identification was performed by infusing effluent from the HPLC diode array detector to a TSQ 700 triple quadrupole mass spectrometer for analysis in the product ion scanning mode. Mass spectrometric analysis of the internal standard indicated ∼95% isotopic purity. The concentration of dG-C8-ABP and dGC8-ABP-D9 in each stock solution was quantified spectrophotometrically (Spectronic Genesis 5 model 336008) based on a molar extinction coefficient of 305 ) 31 000 (in CH3OH) (38). The final stock solution concentrations were 12 and 9.5 µg/mL for dGC8-ABP and dG-C8-ABP-D9, respectively. Preparation of dG-C8-ABP Standard Curves. 300 µg of calf thymus DNA was added to each of six vials containing 250 µL of 5 mM Tris-HCl/10 mM MgCl2 (pH 7.2). Next, 18-fmol of dG-C8-ABP-D9 was added to the solution (20 µL of working solution) along with 30 µL of a previously prepared serial dilution solution of dG-C8-ABP, such that the mass range spanned an order of magnitude. Specifically, the masses of dGC8-ABP added were 68.3, 54.7, 34.1, 20.1, 10.0, and 5.00 fmol. A solution of DNase I (dissolved at 10 mg/mL in 5 mM TrisHCl/10 mM MgCl2 (pH 7.4)) was added at 770 units/mL and incubated at 37 °C for 5 h. Alkaline phosphatase (straight, 4.0 units/mL) and 0.3 units/mL phosphodiesterase (1.0 mg/mL in 5 mM Tris-HCl/10 mM MgCl2, pH 7.40) were introduced to the reaction solution, and the digest was incubated for 18 h. The digestion was terminated with three volumes of ice-cold ethanol (-20 °C). The digestion reaction was then centrifuged for 15
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min at 5000g (Fisher Scientific, model Marathon 21000R) to pellet and discard any insoluble material, and the supernatant was evaporated to approximately 100 µL. The digestion mixtures were then diluted with 1.5 mL of 5 mM potassium phosphate, monobasic (pH 3.40), in preparation for further sample enrichment. Preparation of Human Pancreas Samples. Tissues were obtained from donated organs made available for research from the U.S. Cooperative Tissue Network or from the University of Minnesota (six current smokers and six current nonsmokers, i.e., lifetime nonsmokers and past smokers). There were six female samples (three smokers and three nonsmokers) and six male samples (three smokers and three nonsmokers). The age range was 17-63; however, complete clinical information on sample donors was unavailable. After procurement, organs were placed in University of Wisconsin cold storage preservation solution (39). They were subsequently trimmed of fat, rinsed in cold saline, snap-frozen in liquid nitrogen, and stored at -80 °C. For DNA isolation, all tissues were thawed at the same time, nuclear pellets were prepared as previously described (10), and DNA was isolated according to the method described by Gupta (40). Once DNA was isolated from tissue, 300 µg was added to a siliconized microcentrifuge tube. Depending on the DNA solution concentration, 5 mM TRIS-HCl/10 mM MgCl2 (pH 7.2) was added to produce a sample volume of 280 µL. Additionally, 20 µL of the internal standard working solution used to prepare the standard curve was spiked into the solution, totaling a 300 µL DNA solution. The enzymatic digestion continued as previously described with the addition of DNase I and incubation for 5 h at 37 °C. Adduct Enrichment and Sample Purification Using Solid-Phase Extraction. Solid-phase extraction was accomplished utilizing a vacuum manifold (Alltech Associates, Deerfield, IL). Disposable, Teflon manifold needles (Alltech Associates, Deerfield, IL) replaced standard valves to limit cross contamination. Isolute C18 (EC), 100 mg sorbent cartridges (Argonaut Technologies, Foster City, CA) were conditioned with 3 mL of methanol followed by 3 mL of 5 mM potassium phosphate, monobasic (pH 3.40, adjusted with glacial acetic acid). Digestion mixtures were then applied to the conditioned cartridges and permitted to flow through the cartridge at approximately 1 mL/min. Washes of 3 mL of 10:90 (v/v%) methanol:5 mM potassium phosphate, monobasic (pH 3.40), and 3 mL water were used to elute any extraneous material, including unadducted nucleosides and other polar materials. Adducted DNA nucleosides were then eluted from the sorbent with 1 mL of methanol and evaporated to dryness under vacuum. Prior to analysis, the sample was reconstituted in 90 µL of 10:90 (v/v%) methanol:water. Data from spiked calf thymus DNA indicated approximately 80% recovery. Capillary Liquid Chromatography and µESI Mass Spectrometry. Chromatographic separations were performed on a Beta Basic, C18 capillary column (0.32 mm ID, 50 mm length, 3 µm particle size) (Thermo Hypersil-Keystone, Bellefonte, PA). Solvent was delivered using a Finnigan Surveyor MS HPLC System (Thermo, San Jose, CA) at a flow rate of 200 µL/min that was split down to 10 µL/min using a simple PEEK tee (Upchurch Scientific, Oak Harbor, WA). Using a Finnigan Surveyor Autosampler (Thermo, San Jose, CA), 4.0 µL of sample was injected on-column. The desired analyte was separated from other components using a binary mobile phase composed of 0.05% acetic acid in water and methanol. The mobile phase was initially set at 10% methanol (0.05% acetic acid in methanol). To allow for sample concentration at the head of the column, the initial conditions were held for 6 min after injection. The % methanol was subsequently increased linearly to 90% over the next 10 min and then held isocratic at 90% for another 5 min. This was followed by a step gradient down to 10%, which was held for 10 min to allow for reequilibration of the column. All mass spectrometric analyses were performed on a Finnigan TSQ Quantum AM triple quadrupole mass spectrometer
Ricicki et al. equipped with the microspray interface (Thermo, San Jose, CA). The mass spectrometer was calibrated with a 1,3,6 poly-tyrosine solution. Prior to any sample analysis, a 1 pmol/µL solution of the purified dG-C8-ABP standard was used to auto-tune the instrument to determine optimal instrument parameters. Capillary temperature was set to 400 °C, while the capillary voltage was set at 4.5 kV and sheath gas pressure at 60 PSI. All sample analyses were performed in the positive mode utilizing selected reaction monitoring. In the methodology, the first quadrupole was set to transmit [M + H]+ ions, which corresponded to 435 and 444 for dG-C8-ABP and dG-C8-ABP-D9, respectively. The ions then entered the second quadrupole, where they were fragmented using a collision energy of -27 eV. The third quadrupole transmitted only the aglycone fragments [M + H 116]+, corresponding to m/z 319 and m/z 328 for dG-C8-ABP and dG-C8-ABP-D9, respectively. The response factors for the labeled and unlabeled analyte were essentially identical for this transition. Quantification of dG-C8-ABP Present in Human Tissue. The standard curve for dG-C8-ABP was prepared by spiking synthetic preparations of the adduct and deuterated internal standard into a solution of calf thymus DNA followed by the previously described enzymatic digestion and analyzed in triplicate over a 2-day period for full method validation. The curve was then constructed by plotting the analyte to internal standard peak area ratio at each mass level versus the mass of dG-C8-ABP in each 300 µg of DNA digested. Linear regression data derived from the plot (m ) 0.251, R2 ) 0.983) enabled the quantification of dG-C8-ABP in the human samples. The lowest standard of 5.00 fmol spiked into 300 µg of DNA was the limit of quantification (LOQ) resulting in a signal-to-noise ratio of 10. Standards spiked into the DNA at the LOQ were calculated to have 4.8 fmol of dG-C8-ABP, indicating a 4% error with an RSD of 6.6% (n ) 3).
Results Mass Spectrometric Analysis of ABP-DNA Adduct Standard and Internal Standard. The identities of the synthetic standard and its deuterium-labeled analogue were confirmed by direct infusion of their respective solutions (0.330 and 1.17 µg/mL) into a TSQ 700 mass spectrometer at a rate of 0.3 µL/min. The ESI/ MS/MS mass spectra of the unlabeled and labeled adduct are presented in Figure 2. Panels A and B show spectra collected utilizing a collision offset voltage of -20 eV. These spectra verify the respective protonated [M + H]+ mass-to-charge ratios of 435 and 444 for dG-C8-ABP and dG-C8-ABP-D9 as well as the facile formation of the aglycone fragment ion [M + H - 116]+ at m/z 319 and 328, respectively, for both standards. Subjecting the [M + H]+ ions to an increased collision energy (-70 eV) produced a wide variety of additional fragment ions, many of them exhibiting 9 Da shifts, further confirming the identity of the synthetic products (Figure 2C and D). Liquid Chromatographic Analysis of ABP-DNA Adduct Standard and Internal Standard. Although nucleosides commonly tend to elute from C18 reverse phase columns with a relatively low percentage of organic mobile phase, the biphenyl moiety present on the adduct adds additional hydrophobicity that promotes a greater interaction with the C18 chain in the stationary phase. After examination of various liquid chromatographic methods, the compound was determined to demonstrate the best chromatographic peak characteristics, including peak width, plate number, and Gaussian distribution using a chromatographic system consisting of 10% methanol held isocratic for 6 min, 10 min gradient from 10% methanol to 90% methanol, and another 5 min isocratic
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Figure 2. Infusion analysis of synthesized dG-C8-ABP (A) and dG-C8-ABP-D9 (C) internal standard. (A) and (C) represent product ion spectra of the [M + H]+ ion of the standard and internal standard, respectively, utilizing a collision offset voltage of -20 eV. (B) and (D) represent product ion spectra of the [M + H]+ ion of the standard and internal standard at a collision offset voltage increased to -70 eV.
Figure 3. (A) Total ion current (TIC) of 108 amol (47.0 fg) of dG-C8-ABP and 29.7 amol (12.9 fg) of dG-C8-ABP-D9 injection (on-column) of synthetic standard (prior to digestion). This analysis was utilized to evaluate LC/MS methodology. (B) Extracted ion chromatogram of m/z 435.0 f 319.0, illustrating the detection of 108 amol of dG-C8-ABP. (C) Extracted ion chromatogram of the 444.1 f 328.1 transition representing the detection of 29.7 amol of dG-C8-ABP-D9. It should be noted that the deuterium-labeled analogue consistently eluted slightly ahead of the unlabeled analyte.
at 90% methanol. Figure 3 shows three chromatograms from an LC/MS analysis of aliquots of 108 attomol (47.0 fg) and 29.7 attomol (13.2 fg) injected on-column of dGC8-ABP and dG-C8-ABP-D9, respectively, using the selected LC method. Trace A in Figure 3 represents a total ion current for the analysis and shows a single peak at retention time of 15.39 min. Traces B and C show the extracted ion chromatograms of 435.0 f 319.0 and 444.0
f 328.1, demonstrating that, as expected, both the standard and the internal standard have similar retention times, with the deuterated analyte eluting slightly ahead of the nondeuterated dG-C8-ABP. The molar ratio of dG-C8-ABP analyte to internal standard injected was 3.64. Automated integration by the Xcalibur software illustrates a peak area ratio of 3.59, thus discounting any mass spectrometric bias for either compound. Detection of Adduct in Human Pancreas Samples. A set of 12 samples was analyzed by mass spectrometry for the presence of 4-aminobiphenyl adducted to DNA. Specifically, the investigation centered on the major adduct of dG-C8-ABP. The samples were divided according to gender and smoking preference and were placed into four subcategories: female nonsmokers, male nonsmokers, female smokers, and male smokers. Each of the four subcategories consisted of three samples. Prior to analysis of the human DNA samples, system blanks and procedure blanks were processed and analyzed along with the standard curve used in the quantification of the adducts. Figure 4 shows representative chromatograms from these studies. In row A, the system blank shows no interferences above the noise level present in the TIC in transition 1, extracted ion chromatogram of the 435 f 319 in transition 2, or the extracted ion chromatogram of the 444 f 328 in transition 3. A procedure blank was developed in a manner similar to that of the standard curve with the substitution of the magnesium chloride/Tris-HCl buffer for the synthetic standard. Row B shows the results of the analysis of the latter sample. In the extracted ion chromatogram of 435 f 319 (trace 2 in row B), there is a small peak at the retention time of the dG-C8-ABP adduct. This is suspected to be a trace amount found in the calf thymus DNA possibly from environmental exposure. Consequently, the results of the lowest standard in the calibration curve (row C of Figure 4) were compared to the blank digest. In the aforementioned standard, where 96.7 fg of dG-C8-ABP was injected on-column, the measured peak
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Figure 4. Row A: (1) TIC of system blank; (2) extracted ion chromatogram (435 f 319) of system blank; (3) extracted ion chromatogram (444 f 328) of system blank. Row B: (1) TIC of procedure blank (300 µg of ct DNA followed by addition of internal standard, enzymatic digestion, and SPE workup); (2) extracted ion chromatogram (435 f 319) of procedure blank; (3) extracted ion chromatogram of internal standard transition (444 f 328) of procedure blank. Row C: (1) TIC of lowest mass standard on calibration curve, 5 fmol in 300 µg of DNA (96.7 fg of dG-C8-ABP and 355 fg of dG-C8-ABP-D9 injected on column); (2) extracted ion chromatogram of dG-C8-ABP (435 f 319) of the lowest mass standard; (3) extracted ion chromatogram of dG-C8-ABP-D9 (444 f 328).
Figure 5. Extracted ion chromatograms of 435 f 319 from two different male human sample analyses. Panel A is a male nonsmoker (identified as #41) with a calculated adduct presence of 540 fmol in 300 µg of DNA (10.4 pg injected) or 60 adducts/108 nucleosides. In panel A, chromatogram 1 is the TIC, chromatogram 2 is the 435 f 319 extracted ion chromatogram representing dG-C8-ABP, and chromatogram 3 is the extracted ion chromatogram of 444 f 328 representative of the internal standard (dG-C8-ABP-D9) transition. Panel B is a male smoker (identified as #159) containing 219 fmol of adduct in 300 µg of DNA (4.23 pg injected) or 24 adducts/108 nucleosides. Chromatograms 1, 2, and 3 mimic the traces in panel A for the male smoker.
area in the extracted ion chromatogram of the dG-C8ABP (chromatogram 2 in row C) was 4.4 times greater than the minimal amount found in the blank digest. Further, the signal-to-noise ratio of the analyte in the procedure blank was determined to be 3/1, while the lowest standard had a signal-to-noise ratio of 10/1. It was thus concluded that any sample signal resulting in an
analyte to internal standard ratio corresponding to less than that of the lowest point in the standard curve was considered to be nonquantifiable with the current methodology. The results from the human sample analyses demonstrated the presence of dG-C8-ABP in a limited number of subjects. Representative chromatograms from four
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Figure 6. Extracted ion chromatograms from 435 f 319 from two male human analyses. Panel A are results from a male nonsmoker (identified as #28), while panel B are results from a male smoker (identified as #152). Trace 1 is the TIC of the corresponding sample, while trace 2 shows a small dG-C8-ABP peak. In both samples, the presence of dG-C8-ABP was found to be less than the set minimum detection of 5 fmol in 300 µg of DNA. Trace 3 shows the corresponding extracted ion chromatogram of the internal standard (444 f 328). Table 1. Quantification of dG-C8-ABP in Human Pancreatic Tissue Samples sample ID
gender
smoker/ nonsmoker
age
IS area
analyte area
analyte/ IS ratio
fmol of dG-C8-ABP
78 160 3 8 28 41 26 59 62 152 159 163
F F F M M M F F F M M M
NS NS NS NS NS NS S S S S S S
17 20 42 22 59 37 41 63 51 23 24 23
63 972 41 342 49 010 55 601 50 629 55 747 50 777 51 422
18 670 88 712 0 46 704 11 414 770 654 0 86 159
0.292 2.15 0 0.840 0.225 13.8 0 1.68
54 460 49 054 20 932
16 532 270 382 12 366
0.304 5.51 0.591
