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Chem. Res. Toxicol. 1999, 12, 722-729
A Gas Chromatography/Electron Capture/Negative Chemical Ionization High-Resolution Mass Spectrometry Method for Analysis of Endogenous and Exogenous N7-(2-Hydroxyethyl)guanine in Rodents and Its Potential for Human Biological Monitoring Kuen-Yuh Wu,† Nova Scheller,† Asoka Ranasinghe,† Ten-Yang Yen,† Ramiah Sangaiah,† Roger Giese,‡ and James A. Swenberg*,† Laboratory of Molecular Carcinogenesis and Mutagenesis, Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7400, and Department of Pharmaceutical Sciences in the Bouve College of Pharmacy and Health Professions, Barnett Institute of Chemical Analysis and Chemistry Department, Northeastern University, Boston, Massachusetts 02115 Received April 1, 1999
A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-13C4]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C21H9N4O3F10, resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [13C4]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N ) 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [13C4]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 µg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7HEG varied from 0.2 to 0.3 pmol/µmol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 µg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.
Introduction Ethylene is ubiquitous in the environment and is the most used petrochemical, with an annual production of 47 billion pounds in the United States in 1995 (1, 2). People are exposed to ethylene indoors and outdoors. Ethylene oxide is also an important industrial chemical, a sterilant gas, and a metabolite of ethylene (1, 3, 4). Ethylene oxide is classified by the International Agency for Research on Cancer as a known human carcinogen, and occupational exposure is regulated at 1 ppm (time weight average) in many countries (1). N7-(2-Hydroxyethyl)guanine (N7-HEG)1 is the major DNA adduct produced from in vitro reactions between ethylene oxide and calf thymus DNA (5, 6). Repeated exposures to * Corresponding author. Telephone: (919) 966-6139. Fax: (919) 9666123. E-mail:
[email protected]. † University of North Carolina. ‡ Northeastern University.
ethylene oxide caused significant accumulation of N7HEG (7). N7-HEG was also detected in rodents exposed to ethylene (8-10).2 In addition to exogenous sources, ethylene oxide has been reported to be derived from ethylene produced in turn by lipid peroxidation and by oxidation of methionine, hemin, and by intestinal bacteria (6, 11). N7-HEG is not promutagenic itself, but could cause mutations through the formation of apurinic (AP) sites if these are not repaired prior to cell proliferation (12). 1 Abbreviations: N7-HEG, N7-(2-hydroxyethyl)guanine; GC/EC/ NCI-HRMS, gas chromatography coupled with electron capture negative chemical ionization high-resolution mass spectrometry; HPLCECD, HPLC coupled with electrochemical detection; HPLC/ESI-MS, HPLC coupled with electrospray ionization mass spectrometry; PFBBr, pentafluorobenzyl bromide; PFB, pentafluorobenzylated. 2 V. E. Walker, T. R. Craft, N. L. Clement, and T. R. Skopek, In vivo mutagenicity at the hprt locus in splenic lymphocytes of B6C3F1 mice following parenteral exposures to ethylene oxide. Submitted for publication in Carcinogenesis.
10.1021/tx990059n CCC: $18.00 © 1999 American Chemical Society Published on Web 07/29/1999
GC/MS Quantitation of N7-(2-Hydroxyethyl)guanine
Several methods have been used to analyze N7-HEG in tissues of rodents and in human lymphocytes (7, 11, 13-15). Due to the limitation of sensitivity and/or specificity of these assays, important issues relevant to risk assessment for chronic exposures of ethylene oxide or ethylene have been difficult to study. These issues include the dose responses for N7-HEG at repeated exposures to low doses of ethylene oxide or to ethylene and the amount of endogenous N7-HEG in tissues of animals and humans. To study these issues, a highly specific and sensitive assay is required for biological monitoring. A general method for analysis of N7-alkylated guanines (16) was modified for the quantitation of N7-HEG in animal tissues using a 13C isotopically labeled internal standard and gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS). The assay was used to measure the endogenous amount of N7-HEG in rodent tissues (0.2 pmol/µmol of guanine) and human liver and lymphocyte DNA (1-3 pmol/µmol of guanine), as well as higher numbers of adducts in animals exposed to ethylene and ethylene oxide. The quantitative accuracy of the assay was validated for DNA samples obtained from animals exposed to ethylene oxide using an independent method involving high-pressure liquid chromatography/ electrospray ionization mass spectrometry (HPLC/ESIMS).
Experimental Procedures Caution: Ethylene oxide is classified as a known human carcinogen and should be handled with extreme caution. Chemicals. Ethylene oxide (99% pure) was purchased from National Welders Supply Co. (Raleigh, NC). N7-HEG standard (>98% pure) was obtained from Chemsyn Science Laboratories (Lenexa, KS). Pentafluorobenzyl bromide (PFBBr), potassium hydroxide (KOH), tert-butylnitrite, and tetrabutylammonium sulfate (Bu4NHSO4) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Gas chromatography quality hexane, dichloromethane, and ethyl acetate were obtained from Baxter Diagnostic Inc. (McGaw, IL). The sources of DNA, purificationgrade sterilized Dulbecco’s phosphate-buffered saline (PBS) solution, lysis buffer [100 mM Tris (pH 8.0), 0.2 M NaCl, 0.5% N-lauroylsarcosine, 4 M urea, and 10 mM 1,2-diaminocyclohexane-N-tetraacetic acid], 70% phenol/water/chloroform reagent, RNase T1, RNase A, and other DNA isolation and HPLC reagents have been listed previously (7). Synthesis and Purification of the Internal Standard. [4,5,6,8-13C4]-N7-HEG was synthesized from the reaction of 5 mg of [4,5,6,8-13C4]guanine with 5 mL of a 0.2 M ethylene oxide solution in a capped brown vial overnight at 37 °C (17). Unreacted ethylene oxide was removed by evaporation of this product solution in a vacuum for 4 h. Chromatographic separation of [13C4]-N7-HEG was carried out using an SCX precolumn (15 mm × 3.2 mm; Brownlee Labs, Santa Clara, CA) and an ES Industries (Marlton, NJ) hybrid RP-SCX column (4.6 mm × 250 mm, 5 µm, lot no. 199055VW) eluted with 130 mM ammonium formate (pH 2.2, rate of 0.8 mL/min) and 80% acetonitrile/water solution (rate of 1.2 mL/min) isocratically delivered by two Waters 510 HPLC pumps. N7-HEG was monitored using a Perkin-Elmer LS40 fluorescence detector (Norwalk, CT) with excitation at 290 nm and emission at 370 nm (7). The fraction containing [13C4]-N7-HEG was collected and dried in a Speed Vac (Savant model SVC100, Farmingdale, NY). The residue was dissolved in 500 µL of HPLC grade water and desalted using reverse phase solid phase extraction. A disposable 13.5 cm borosilicate Pasteur pipet column was plugged with siliconized glass wool and filled with a 2 cm height of C18 gel (70-230 mesh ASTM, EM Science, Gibbstown, NJ). The column
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 723 was washed with 2 mL of methanol, 2 mL of HPLC grade water, and 2 mL of methanol. After being loaded into the column, the sample was washed with 10 mL of HPLC grade water. [13C4]N7-HEG was eluted using 4 mL of methanol. The sample was dried in a vacuum and redissolved in 500 µL of HPLC grade water. UV Spectrophotometry. Both N7-HEG and [13C4]-N7-HEG aqueous solutions were characterized at pH 1, 7, and 13 using a Shimadzu (Columbia, MD) 160U UV spectrophotometer. Characterization and Quantitation of [13C4]-N7-HEG Using Liquid Chromatography/Electrospray Ionization Mass Spectrometry (HPLC/ESI-MS). A similar method has been described in a previous study (18). Briefly, N7-HEG and [13C4]-N7-HEG solutions were delivered by a water and methanol (HPLC grade) gradient using a Beckman Gold liquid chromatographic system (Beckman Instruments, Arlington Heights, IL) coupled to a Finnigan 4000 quadrupole mass spectrometer (Finnigan Mat, San Jose, CA) retrofitted with an Analytica electrospray source (Analytica of Branford, Branford, CT). Samples were characterized using HPLC/ESI-MS by injecting 4 µL of a [13C4]-N7-HEG aqueous solution through a C18 capillary column (150 mm × 0.3 mm i.d., Hypersil, 3 µm particle size; LC Packings, San Francisco, CA). A 30 cm fused-silica capillary column (50 µm i.d. × 375 µm o.d.) directed the eluent from the capillary column to the electrospray needle. The sample injection volume was controlled by a Rheodyne model 7725I injector (20 µL external loop). The mobile phase changed from 100% water to 25% water/75% methanol over the course of 18 min, then to 5% water/95% methanol over the course of 4 min, and finally to 100% methanol over the course of 1 min. A voltage of 3.6 kV was applied to the electrospray needle, and 70 psi of nebulizer gas (nitrogen) was used to stabilize the spray. The voltage difference between the exit of the glass capillary and the first skimmer in the differential pump region was optimized at 130 V for the detection of the (M + H)+ ion of N7-HEG. The mass spectrometer was calibrated using a mixture of arginine, adenosine, gramicidin S, and sodium chloride (1:1:1:1) with total concentration 0.5 mM. Data were acquired and processed by a Technivent Vector data system (Teknivent, Maryland Heights, MO). Full scan spectra were achieved by scanning from m/z 20 to 350 in 1 s. For selected ion monitoring (SIM) analyses, two ions (m/z 196 and 200) were monitored with a dwell time 0.70 s. N7-HEG was weighed using a microbalance and dissolved in 20 mL of HPLC grade water in a poly bottle to prepare a standard solution with a concentration of 327 µM. The standard solution was diluted to a concentration of 32.7 µM. [13C4]-N7HEG (20 µL) was mixed with 20 µL of the diluted standard solution. The mixture (4 µL) was injected into the LC/ESI-MS system. The isotope-labeled standard was analyzed by monitoring the ions at m/z 196 (N7-HEG) and 200 ([13C4]-N7-HEG). The concentration of the [13C4]-N7-HEG solutions was determined by the ratio of peak areas between the N7-HEG standard and [13C4]-N7-HEG and comparison with a calibration curve, assuming the relative molar response factor was equal to 1.00. Derivatization of Synthesized N7-HEG. The method for analysis of N7-alkylguanine adducts by GC/MS was modified for derivatization and analysis of N7-HEG using GC/HRMS (16). The N7-HEG (0.1 µmol) solution was dried in vacuo before 50 µL of 6 N HCl (degassed with N2) and 20 µL of tert-butylnitrite were added to the residue and mixed at 4 °C (nitrosation) to convert N7-(2-hydroxyethyl)guanine to N7-(2-hydroxyethyl)xanthine. After 4 h, the sample was dried under vacuum. The residue was subjected to liquid-liquid extraction using 150 µL of ethyl acetate and 50 µL of water. After the aqueous phase was dried in a Speed-Vac, 5 mg of K2CO3 (dehydrated at 60 °C for at least 1 h) was added to the samples, followed by the addition of 150 µL of 10% PFBBr in acetonitrile and mixing at room temperature for 20 h (the first derivatization). The samples were evaporated under nitrogen at 85 °C before 150 µL of methylene chloride, 10 µL of PFBBr, and 50 µL of Bu4NHSO4 (50 mg in 50 mL of 1 N KOH) were added to its residue and
724 Chem. Res. Toxicol., Vol. 12, No. 8, 1999 mixed at room temperature for 20 h (the second derivatization). Samples were then dried under nitrogen at 45 °C and extracted three times with 200 µL of ethyl acetate. The combined supernatants for each sample were dried under nitrogen at 85 °C. The residues were dissolved in 100 µL of 50% ethyl acetate/ hexane solution, and cleaned up by solid phase extraction using Scientific silica gel (particles sizes of 0.063-0.2 mm). The columns were set up by using disposable 13.5 cm borosilicate Pasteur pipets plugged with siliconized glass wool and filled with silica gel (60-A, 40 fm of irregular particles in hexane solution, 200 mg/column). The columns were washed with 2.0 mL each of hexane, ethyl acetate, and hexane, and the sample solution was transferred to the silica solid phase extraction columns, washed with 4 mL of hexane and 8 mL of 10% ethyl acetate in hexane, and eluted with 2 mL of ethyl acetate. The eluent was evaporated under nitrogen at 85 °C and dissolved in 50 µL of toluene. Characterization of the Pentafluorobenzyl Derivative of N7-HEG Using GC/EC/NCI-HRMS. The derivatized sample (1 µL) was injected into a Hewlett-Packard 5890 gas chromatograph coupled with a VG 70 250SEQ mass spectrometer (Manchester, England) operated in the full-scanning mode and tuned to a resolving power of 1000 or 10 000. Perfluorotributylamine (CF43; Scientific Instrument Services, Ringoes, NJ) was used as the calibration and reference compound (lock mass). The gas chromatography was performed using a J & W Scientific (Folsom, CA) ∼15 m, 0.32 mm, and 0.1 µm film thickness DB-5 MS capillary column. Helium was used as a carrier gas, and the head pressure was set at 10 psi. The source temperature was set at 250 °C, and methane (5 × 10-5 mbar) was used as the reagent gas for electron capture negative chemical ionization. The GC temperature was increased from 70 to 300 °C over the course of 10 min. Preparation of Calibration Curves. Three calibration curves were prepared for quantitation of N7-HEG. The first one was prepared for HPLC/ESI/MS analysis. Six solutions with relative concentrations of 0.05, 0.2, 1, 5, 10, and 50 were prepared by spiking 50 µL of [13C4]-N7-HEG (640 pmol) into 100 µL of 32, 128, 640, 3200, 6400, and 32 000 pmol of N7-HEG. Each calibration standard solution (2 µL) was injected into the LC/ESI-MS system to build a calibration curve for the quantitation of [13C4]-N7-HEG. The second calibration curve for GC/ EC/NCI-HRMS was constructed by using 50 µL of each standard solution for derivatization following the procedure for derivatizing synthesized N7-HEG. To evaluate the reproducibility of this method, the third calibration curve was built with solutions having relative concentrations of 0.5, 2, 2.5, 8, and 10, each with four replicates. Determination of Endogenous and Ethylene-Induced N7-HEG. F-344 rats and B6C3F1 mice were exposed to 0 or 3000 ppm ethylene for 6 h/day for 5 days by inhalation. After the last exposure, animals were immediately sacrificed, and liver, spleen, lung, and brain were harvested and stored at -80 °C. DNA was extracted from the whole lung, spleen, and brain, or up to 2.0 g of liver from rats and mice using an automated phenolic extraction procedure (7). Briefly, DNA was isolated by two phenol/chloroform extractions, one chloroform extraction, and 2-propanol precipitation using a 340A Nucleic Acid Extractor (Applied Biosystems, Foster City, CA). DNA pellets were dissolved overnight in 3-4 mL of sterilized distilled deionized water at 4 °C. These DNA solutions were homogenized by shearing with 20 gauge needles and 5 mL syringes. DNA concentrations were examined using a UV 160U spectrophotometer and calculated by using calf thymus DNA as a standard with the equation [DNA] ) 50 µg/mL × the absorbance at 260 nm × the dilution factor. Derivatization of in Vivo Samples. DNA samples (about 100-300 µg of rodent DNA, dependent on ethylene exposures) with a known volume (from which 20 µL aliquots were taken for guanine analysis) were dried in vacuo and dissolved with 160 µL of HPLC grade water and 40 µL of [13C4]-N7-HEG (a total of 0.1 pmol) at 4 °C overnight. The DNA solution was boiled
Wu et al. Scheme 1. Experimental Scheme for the Analysis of N7-HEG
at 100 °C for 15 min (neutral thermal hydrolysis), then immediately placed in an ice bath, treated with 150 µL of cold 1 N HCl solution, and centrifuged at 1200g at 0 °C for 15 min (cold acid precipitation) using a Sorvall Instrument RC5C centrifuge (Dupont Co., Wilmington, DE). The DNA backbone was washed with 100 µL of a cold 1 N HCl solution with a second centrifugation. Supernatants were combined and dried in a Speed Vac. Then, the procedure for derivatization of synthesized N7-HEG was followed. These experimental procedures are summarized in Scheme 1. Derivatization of DNA from Lymphocytes of Unexposed Humans. Ten samples of human lymphocytes were kindly provided by V. Walker (New York State Department of Health, Albany, NY). DNA was extracted from these lymphocyte samples using the DNA isolation procedures outlined earlier. Thirty-two DNA samples (25-30 µg) from human lymphocytes were generously provided by D. Bell (National Institute of Environmental Health Sciences, Research Triangle Park, NC). [13C4]-N7-HEG (0.25 pmol) was spiked into each DNA sample. After being incubated at 4 °C overnight, these DNA samples were processed following the procedures for derivatization of in vivo samples. Determination of Guanine Content. HCl (0.1 N, 500 µL) was added into 20 µL of DNA solution from each sample and heated at 70 °C for 30 min. Guanine concentrations were quantitated with HPLC using an AllTech (Deerfield, IL) strong cation exchange column (250 × 4.6 mm, 10 µm) and 0.1 mM ammonia formate (pH 2.8) in 10% methanol with a flow rate of 2 mL/min. Absorbance was measured using an Applied Biosystems UV detector (model 757, Ramsey, NJ) with the wavelength set at 254 nm (7). A calibration curve was constructed by injecting 0.36, 1.8, 9.0, and 18 nmol of guanine into this HPLC system and plotting peak area versus the amount of guanine. Quantitation of N7-HEG with GC/EC/NCI-HRMS. The derivatized samples were quantitated using the Hewlett-Packard 5890 gas chromatograph coupled with a VG 70 250SEQ mass spectrometer (Manchester, England) operated in selectedion-monitoring (SIM) mode and tuned to achieve a resolving power of 10 000 using perfluorotributylamine (CF43; Scientific Instrument Services, Ringoes, NJ) as the calibration and reference compound (lock mass). The GC temperature was increased from 70 to 300 °C over the course of 10 min and held for 3 min at 300 °C. Other settings were described above in Characterization of the Pentafluorobenzyl Derivative of N7-HEG Using GC/EC/NCI-HRMS. The derivatized sample (1 µL) was directly injected through a Restek Uniliner instrument (#20355, Belfonte, PA). The isotope dilution method was used to quantitate N7-HEG by monitoring m/z 555.0515 (analyte) and 559.0649 (internal standard). Each day, 100 fmol of a wellcharacterized PFBBr-derivatized N7-HEG standard was in-
GC/MS Quantitation of N7-(2-Hydroxyethyl)guanine
Chem. Res. Toxicol., Vol. 12, No. 8, 1999 725
jected before the samples were analyzed to evaluate the performance of the HRMS instrument. A solvent blank was frequently injected between samples to check for carryover from the previous sample, and a standard was injected after every five samples to evaluate the continued performance of the instrument. Quantitation of N7-HEG in each sample was based on the ratio of the peak area of the analyte to that of the internal standard (relative response ratio) and normalized to the amount of guanine in each sample. Comparative Quantitation of N7-HEG by HPLC-ESIMS and GC/EC/NCI-HRMS. [13C4]-N7-HEG (10 pmol) was spiked into a sample of liver DNA (1 mg, approximately 1 mL) obtained from a rat exposed to 33 ppm ethylene oxide for 4 weeks (6 h/day, 5 days/week) (7). After neutral thermal hydrolysis for 30 min and cold acid precipitation, 50 µL of this sample solution was used for derivatization with PFBBr and quantitation of N7-HEG using GC/EC/NCI-HRMS. The remaining solution was subjected to the same HPLC conditons that were used for the purification of [13C4]-N7-HEG. Note that the guanine concentration in the original sample was estimated according to the method described in Experimental Procedures. Therefore, the volume taken for subsequent GC or LC/MS analysis (after spiking the labeled internal standard) does not affect the quantitative accuracy. The fraction containing N7HEG was collected, dried in a Speed Vac, desalted using C18 disposable columns, and rehydrated in 50 µL of HPLC grade water. Four microliters of this sample solution was injected into the HPLC/ESI-MS instrument for quantitation of N7-HEG. The HPLC/ESI/MS assay used in this study does not have the required sensitivity for monitoring endogenous N7-HEG in animal and human tissues.
Results and Discussion DNA adducts are considered risk-associated biomarkers for monitoring the biologically effective dose of genotoxicants in exposed or unexposed humans and animals (19). Determination of a particular DNA adduct in human lymphocytes requires methods capable of detecting one adduct in 107-109 unmodified bases with a high degree of specificity (20). 32P-Postlabeling has been employed in biological monitoring with superior sensitivity, but chemical identification and specificity have been of concern for this assay. Alternatively, derivatization of modified DNA bases with PFBBr and quantitation using gas chromatography coupled with negative ion chemical ionization mass spectrometry have demonstrated comparable sensitivities and excellent specificity, but also provide information about chemical structure (21, 22). In this study, HRMS was used to quantitate N7-HEG to further improve specificity. Accurately determining the number of DNA adducts using MS techniques primarily relies on the use of an internal standard and the isotope dilution method for quantitation. A previous study has suggested that internal standards require stable isotope analogues with a difference of at least 3 atomic mass units (amu) from the native analyte (23, 24). Characterization and quantitation of the internal standard are very critical for method validation. Ethylene Oxide Reaction with 13C4-Labeled Guanine. HPLC analysis of the reaction product yielded two major peaks. The retention time of [13C4]-N7-HEG was 5.38 min, immediately after the unreacted guanine peak at 4.75 min. The [13C4]-N7-HEG peak had a UV spectrum that was identical to that of N7-HEG at a λmax of 249.9 nm at pH 1. These data are consistent with a previous study on the characterization of various N7-alkyl-
Figure 1. (A) HPLC/ESI-MS CID spectra of N7-HEG. The molecular ion (M + H)+ (m/z 196) corresponds to the protonated N7-HEG. m/z 152 is the protonated guanine due to the loss of the hydroxyethyl group. (B) HPLC/ESI-MS CID spectra of [13C4]N7-HEG. The molecular ion (M + H)+ (m/z 200) corresponds to the protonated [13C4]-N7-HEG. m/z 156 is protonated [13C4]guanine due to the loss of the hydroxyethyl group.
guanines whose spectra demonstrated a λmax at 250 nm at pH 1 (25). Characterization and Quantitation of the Synthesized Standard Using HPLC/ESI-MS. The ESI mass spectrum at a low voltage for N7-HEG yielded one major peak at m/z 196, corresponding to the molecular ion of protonated N7-HEG (M + H)+. The loss of the hydroxyethyl group gave the protonated guanine fragment (guanine + H)+ at m/z 152 (not shown). Under the ESI conditions that cause collision-induced dissociation (CID) fragmentation, the relative intensity of the ion at m/z 152 increased significantly compared to that of the molecular ion at m/z 196 (Figure 1A). The isotope-labeled standard had the same fragmentation pattern, except for a difference in 4 mass units. The mass spectrum of the synthesized standard exhibited a major peak at m/z 200, representing the protonated [13C4]-N7-HEG (M + H)+, and a minor peak at m/z 156, representing the protonated [13C4]guanine (not shown). Under the CID conditions, the peak at m/z 156 significantly increased (Figure 1B). These results combined with UV absorption data demonstrate that the synthesized standard is [13C4]-N7-HEG. By monitoring m/z 196 and 200, we determined concentrations of [13C4]-N7-HEG on the basis of the ratio between the N7-HEG peak area and the [13C4]-N7-HEG peak area and comparison with a linear calibration curve with an R2 of 0.99 (Figure 2). Characterization of the Pentafluorobenzyl Derivative of N7-HEG by GC/EC/NCI-HRMS. The chemical structure of the derivatized N7-HEG was characterized using NMR (27). N7-(Hydroxyethyl)xanthine was benzylated at the N-1 and N-3 positions of xanthine during the first derivatization (26). The third PFBBr attacked the hydroxyl group of N7-HEG during the second derivatization (26). When the HRMS system was tuned to a resolving power of 1000, the EC/NCI-MS spectra exhibited no molecular ions, with the most abundant fragment ion being (M - 181)- at m/z 555, due to the cleavage of one pentafluorobenzyl (PFB) group from the N-1 or N-3 position of xanthine (16). The further loss of one PFBOH formed the (M - 379)- ion at m/z 357 (Figure 3). The full-scan spectrum analyzed using the GC/ HRMS tuned to a resolving power of 10 000 showed that
726 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Figure 2. Calibration curves for quantitation of N7-HEG using HPLC/ESMS and GC/EC-HRMS.
Figure 3. Full-scan spectrum of the derivatized N7-HEG. There is no molecular ion, but the most abundant fragment is m/z 555 for derivatized N7-HEG. (M - 181)- ) 555.
the elemental composition of the prominent fragment ion was C21H9N4O3F10 with the loss of one PFB group. Therefore, the most abundant fragment of derivatized N7-HEG was monitored at m/z 555.0515 for the analyte and m/z 559.0649 for the internal standard, to achieve maximum sensitivity and specificity. The detection limit was less than 0.1 fmol of derivatized N7-HEG, assuming the yield of derivatization is 10%, which is equivalent to less than 1 fmol of N7-HEG (16). The full-scan spectrum of the derivatized N7-HEG shows that the common loss of one PFB generates the most abundant fragment at m/z 555 (Figure 3). The absence of the molecular ion, due to ionization via a dissociative electron capture process, is a common feature of EC-MS of the PFB derivatives (21). Characterization using EC/NCI-HRMS further confirmed the structural information by resolving the elemental composition of this most abundant fragment. Quantitation of N7-HEG using GC/EC/NCI-HRMS to monitor the exact mass of the most abundant fragments from derivatized N7-HEG and [13C4]-N7-HEG can achieve the maximum sensitivity with excellent specificity. Although the structural information is lost when using selective ion monitoring (SIM), this method still provides superior specificity since the spectra of the derivatized analyte and isotope-labeled standard have been well characterized. Electrophore labeling of N7-HEG with three PFBBr enhances the volatility of this modified base and significantly improves the sensitivity of this method. Derivatization of this analyte molecule can also be used to raise m/z values of molecular or fragment ions as high as
Wu et al.
possible to elevate sensitivity, without adversely affecting the GC properties of the analyte because interferences are more abundant than the ions with high masses (27). Compared with PFBBr, N-methyl-N-(trimethylsilyl)trifluoroacetamide is one of the most commonly used derivatization reagents in GC/MS. The trimethylsilyl derivatives of N7-HEG are easy to form, and have excellent GC properties and useful spectrometric fragmentation, but are not very stable (28). Calibration Curves. The calibration curves generated from GC/EC/NCI-HRMS fit a linear curve very well with an R2 of 1.00 over a range of 1-1000 fmol of N7HEG versus 20 fmol of [13C4]-N7-HEG. The same calibration standards assayed using HPLC/ESI-MS also fit a linear response with an R2 of 0.99 (Figure 2). Both calibration curves revealed a relative response factor approximately equal to 1.0. The assumption of the same molar response for N7-HEG and [13C4]-N7-HEG was appropriate for HPLC/ESI-MS and GC/EC/NCI-HRMS. A third calibration curve was used to evaluate the reproducibility of this GC/EC/NCI-HRMS method. A linear curve was also fit for the third calibration curve with an R2 of 0.99 (data not shown). The standard deviation at the low end of the calibration curve was less than 15%, showing the excellent reproducibility of the GC/EC/NCI-HRMS assay (29). Comparative Quantitation of N7-HEG Using HPLC/ESI-MS and GC/EC/NCI-HRMS. After analysis of N7-HEG in liver DNA from a rat exposed to 33 ppm ethylene oxide for 4 weeks (6 h/day, 5 days/week) using both methods, the N7-HEG content was 9.1 pmol/fmol of guanine from GC/EC/NCI-HRMS and 8.7 pmol/fmol of guanine from HPLC/ESI-MS. The difference between these two methods was about 4%. These results indicate that no chemical interferences were generated or converted from the unknown compounds in biological samples during sample derivatization (30). Quantitation of Endogenous and Chemically Induced N7-HEG in Tissues of Rodents. This GC/EC/ NCI-HRMS assay was able to analyze endogenous and ethylene-induced N7-HEG with excellent sensitivity and specificity. Figure 4 displays two chromatograms: one from control rat spleen DNA and the other from brain DNA of an ethylene-treated rat. Quantitation of endogenous N7-HEG in liver, spleen, lung, and brain of rats and mice showed that the amounts ranged from 0.2 to 0.3 pmol/fmol of guanine (Table 1). Repeated exposures to 3000 ppm ethylene caused accumulation of N7-HEG in all four tissues of rats and mice (Table 1). The data in Table 1 show much lower amounts of endogenous N7-HEG in tissues of rats and mice than have been previously reported. Quantitation using HPLC coupled with fluorescence detection (HPLC-FD) and gas chromatography connected with an electron impact mass spectrometer (GC/EI-MS) (7, 31) reported up to 6 pmol/ mg (8.7 pmol/µmol of guanine) of DNA. HPLC-FD exhibited a detection limit of about 20 pmol and could not quantitate the numbers of endogenous N7-HEG in rodents without using 3 mg of DNA (7). N7-HEG was also derivatized with N-methyl(trimethylsilyl)trifluoroacetamide and analyzed using GC/EI-MS. The detection limit of this GC/EI-MS assay was reported to be about 35 fmol on column (30). HPLC combined with electrochemical detection (HPLC-ECD) and immunoassays have also been used to analyze N7-HEG (14, 15). Immunoassays have a detection limit close to that of the
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Chem. Res. Toxicol., Vol. 12, No. 8, 1999 727
Table 1. Amounts of N7-HEGa in Tissues of Control and Ethylene-Exposed Rats and Miceb
a
rodent (exposure)
liver
spleen
lung
brain
rat (control) rat (3000 ppm) mouse (control) mouse (3000 ppm)
0.30 ( 0.20 (n ) 12) 3.92 ( 0.46 (n ) 9) 0.30 ( 0.20 (n ) 9) 1.98 ( 0.67 (n ) 4)
0.22 ( 0.13 (n ) 16) 3.6 ( 1.06 (n ) 14) 0.20 ( 0.10 (n ) 8) 1.88 ( 0.96 (n ) 4)
0.20 ( 0.10 (n ) 8) 3.29 ( 0.42 (n ) 4) 0.30 ( 0.20 (n ) 9) 1.84 ( 0.32 (n ) 4)
0.20 ( 0.10 (n ) 9) 2.41 ( 0.33 (n ) 5) 0.30 ( 0.10 (n ) 8) 1.61 ( 0.22 (n ) 4)
Picomoles per micromole of guanine. b At 3000 ppm for 6 h/day for 5 days.
Figure 4. Representative chromatograms of (A) spleen DNA of a control rat and (B) brain DNA of a rat exposed to 3000 ppm ethylene for 6 h/day for 5 days.
HPLC-ECD varying from 1 pmol to several hundred femtomoles. However, the antibody that was used was made to detect the imidazole ring-opened adduct and cross reacted with N7-methyl- and N7-ethylguanine (14). Both methods were not sensitive and/or specific enough to quantitate endogenous N7-HEG in rodents. Endogenous N7-HEG analyzed using 32P-postlabeling was reported to be at a level of 2.94 ( 0.4 pmol/µmol of guanine in liver and 2.32 ( 0.88 pmol/µmol of guanine in lymphocytes of male Sprague-Dawley rats (9). Although the 32P-postlabeling assay is extremely sensitive, N7-HEG could not be separated from N7-methylguanine using thin-layer chromatography (13). Recently, N7-HEG has been isolated using a combination of TLC plates and HPLC (13), and the amounts of endogenous N7-HEG in rodents were very close to those shown in Table 1. Therefore, the previously reported concentrations of endogenous N7-HEG in rodents that were much higher than those shown in Table 1 were likely to have been overestimated due to the limited sensitivity and/or specificity of these methods. Sensitivity and specificity are very important when analyzing trace amounts of DNA adducts arising endogenously or from very low levels of exposure. The specificity and sensitivity of this GC/EC/NCI-HRMS assay were affected by the yield of derivatization. Estimated by the comparison of the peak area of spiked 0.1 pmol of [13C4]N7-HEG in each sample with that of 500 µg of the derivatized standard, the yield of derivatization for real DNA samples varied from about 2.5 to 15%. Conversion
of N7-HEG to N7-(2-hydroxyethyl)xanthine by nitrosation was employed in this study because it previously had been shown to increase the derivatization efficiency about 2.5-fold (26). For real DNA samples, the derivatization efficiency decreased with the presence of salt (such as ammonium formate and ammonium phosphate). The derivatization efficiency went up with the increase of derivatization time or the amount of PFBBr that was used. Meanwhile, the extent of reaction between undesirable compounds and PFBBr was also increased so that noise levels also went up. These chemical interferences might overlap with the analyte or internal standard peak so that sensitivity and specificity could drop dramatically. PFBBr (10% in acetonitrile, 150 µL) and 10 µL of PFBBr in the first and second derivatization for 20 h were appropriate for the analysis of endogenous N7-HEG. Other factors also affecting the sensitivity of this assay include sample loss and purity of the analyte. To increase the recovery and purity of released N7-HEG from the DNA backbone, cold acid precipitation and filtration with a Centricon-30 concentrator (Amicon, Danvers, MA) were used to recover 50 pmol of N7-HEG spiked into 100 µL of calf thymus DNA solution (300 µg) after neutral thermal hydrolysis. Nuclear base solutions were quantitated using HPLC (7). Samples filtered through Centricon-30 recovered 90-95% of N7-HEG. Ninety-eight percent of N7-HEG was recovered from cold acid precipitation. The recovered N7-HEG was derivatized and analyzed with GC/EC/NCI-HRMS. The chromatograms from samples filtered through Centricon-30 concentrators exhibited more interference and higher background signals than those of samples processed by cold acid precipitation. This might be due to the presence of unknown chemicals released from the Centricon units while the N7-HEG solution was being filtered. Therefore, cold acid precipitation was considered superior and used in this assay. The use of neutral thermal hydrolysis permits the analysis of small to large amounts of DNA. Increased chemical noise in the selected ion chromatograms can originate from large amounts of DNA (milligram amounts) being used. However, the increased specificity of GC/HRMS eliminates nominal mass interferences, allowing the use of large amount DNA samples without sacrificing the S/N of the analyte signal. As far as injection was concerned, direct injection, guard column, and a split splitless injection were evaluated. The intensity of the background signal increased with the number of samples analyzed when samples were injected directly on column or through a guard column. After replacement with a Restek direct injection liner (31), the level of accumulation of background signals was reduced when rat and mouse samples were analyzed. However, background signals still accumulated when N7HEG was quantitated in human DNA even though a solvent blank was injected immediately after analysis of each human sample. In addition, chromatography characteristics and sensitivity were restored by cutting the
728 Chem. Res. Toxicol., Vol. 12, No. 8, 1999
Figure 5. Representative chromatogram from 25 µg of DNA from lymphocytes of an unexposed human.
GC column (6 cm from the injector side) after about 50 injections. Although this can change the GC retention time of the analyte signal, coelution of labeled internal standards and the use of calibration standards maintain the GC/MS quantitative accuracy. Repeated injections of the derivatized standard with a known relative response ratio were used to evaluate not only instrument performance but also carryover. Relative response ratios from the standard chromatograms were compared with the originally prepared relative response ratio. If the acquired relative response ratio deviated 15% from the prepared relative response ratio or three standard deviations from the mean (after a series of injections of the same standard on the same and different days), the HRMS instrument was tuned again until the acquired relative response ratio fell in the acceptable range to ensure minimal instrument variation. Although analysis of small amounts of N7-HEG in tissues of rodents did not carry over between runs, the instrument was checked carefully by the injection of solvent blanks before and after the standard was injected. Quantitation of N7-HEG in Lymphocytes from Unexposed Humans. N7-HEG was quantitated in 2530 µg of lymphocyte DNA from unexposed humans. Figure 5 displays a representative chromatogram generated from 25 µg of lymphocyte DNA. N7-HEG in lymphocytes of 23 unexposed humans varied from 0.9 to 7.4 pmol/µmol of guanine with a mean of 2.5 pmol/µmol of guanine. The method LOD was about 600 amol (S/N ) 5) injected on column. Therefore, 25-30 µg of DNA, an amount contained in approximately 5 mL of blood, was sufficient, making this assay useful for biological monitoring of N7-HEG in human blood. Note that the method LOD of the GC/EC/NCI-HRMS is estimated as an amount injected on column (600 amol) rather than as a N7-HEG amount per guanine. The latter unit changes with the starting amount of DNA. Although the method LOD remains the same, the assay has successfully been implemented in quantitating lower concentrations of N7HEG in rodents (0.2 pmol/µmol of guanine) because of higher amounts of DNA (300 µg) being processed.
Wu et al.
The derivatized human DNA samples often had high background signals that reduced the sensitivity of this method. Background signals can be reduced using HPLC cleanup of derivatized samples with 80% methanol in water delivered isocratically through an AllTech C18 column. The disadvantages of using HPLC for sample cleanup include the likely loss of sample and potential contamination or carryover, especially in the injector (32). A satellite HPLC method has been presented in an attempt to overcome this problem (33). Molecular dosimeters of DNA adducts are considered better parameters for risk extrapolation from high to low doses than external exposures since they are capable of integrating the effect of such factors as absorption, distribution, biotransformation, and DNA repair (34). Ehrenberg indicated that two problems constituted the main components of research in ethylene oxide-induced adducts: the sensitivity of the analytical methods and the lack of knowledge of the shape of dose-response curves at the very low doses (35). Hemminki also emphasized that specificity is a problem in quantitation of DNA adducts (36). To directly address these issues, this GC/EC/NCI-HRMS assay has been used to quantitate N7-HEG in tissues of rodents exposed to ethylene or ethylene oxide with excellent sensitivity and specificity. This method has also been successfully adopted to quantitate the N7-alkylguanine adducts induced by propylene oxide (37), butadiene monoxide (38), and styrene oxide (39) in tissues of rodents with minimal modification.
Acknowledgment. Samples of human lymphocytes were kindly provided by Drs. Doug Bell (NIEHS, Research Triangle Park, NC) and Vernon Walker (Wadsworth Laboratory, Albany, NY). This research was supported in part by grants from the Chemical Manufacturers Association and the EPA/NIEHS Superfund Basic Research Program (P42-ES05948).
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