Analysis of DNA Adducts in DNA Hydrolysates by Capillary Zone

Once the stack limit current was reached, the normal electrophoresis was started (applying an electrophoresis voltage of ...... Cornelis E. C. A. Hop ...
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Anal. Chem. 1996, 68, 3575-3584

Analysis of DNA Adducts in DNA Hydrolysates by Capillary Zone Electrophoresis and Capillary Zone Electrophoresis-Electrospray Mass Spectrometry Dieter L. D. Deforce, Filip P. K. Ryniers, and Elfriede G. Van den Eeckhout*

Laboratory for Pharmaceutical Biotechnology, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium Filip Lemie`re and Eddy L. Esmans

Department of Chemistry, Nucleoside Research & Mass Spectrometry Unit, University of Antwerp (RUCA), Groenenborgerlaan 171, B-2020 Antwerp, Belgium

The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2′-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified and modified 2′-deoxynucleotide adducts were analyzed by capillary zone electrophoresis (CZE), CZE-electrospray mass spectrometry (CZE/ES-MS) and CZE-electrospray tandem mass spectrometry (CZE/ES-MS/MS) using sample stacking. For the CZE analyses, a homemade system was developed in order to enhance the reproducibility of the retention times. This modification enabled the total comparison of the electropherograms obtained for the analysis of 2′deoxynucleotides mixtures with the electropherograms obtained for the DNA hydrolysates both treated with PGEs. The assignment of adducted and nonadducted 2′deoxynucleotide peaks was unambiguous. Analysis of the CZE/ES-MS data gave the necessary structural information and revealed the presence of mono- and dialkylated 2′-deoxynucleotides. Interpretation of the CZE/ES-MS/ MS data of the monoalkylated products allowed differentiation between purine or pyrimidine alkylation and alkylation of the 5′-phosphate moiety. Recording of fullscan mass spectra during CZE/ES-MS/MS analysis of 2′deoxynucleotide reaction mixtures and DNA hydrolysates was possible, using the described CZE sample stacking technique. DNA is the carrier of genetic information and acts as the mold for protein synthesis. Therefore, it is of major importance to study its modifications and the consequences on DNA replication. A major factor responsible for these changes is chemicals that covalently bind to DNA, resulting in the formation of so-called DNA adducts, which may be responsible for mutations and carcinogenesis. In this context, aromatic polycyclic hydrocarbons have been studied extensively. This is illustrated by a literature search covering this topic over the period from 1980 to 1995, for which 2391 references were found.1-5 This is in contrast to the results obtained for simple unsaturated epoxides, with only 30 references.6-10 * To whom correspondence should be addressed. Tel.: +32-9-221-99-43. Fax: +32-9-220-66-88. E-mail: [email protected]. (1) Peltonen, K.; Dipple, A. J. Occup. Environ. Med. 1995, 37, 52-58. (2) Strickland, P. T.; Groopman, J. D. Am. J. Clin. Nutr. 1995, 61, 710S-420S. S0003-2700(96)00001-7 CCC: $12.00

© 1996 American Chemical Society

One of the classes of important industrial epoxides belonging to these unsaturated epoxides are the phenyl glycidyl ethers (PGEs), which have only been studied to a limited extent.11-14 They are used mainly in the resin and paint industries and were selected as examples to develop an analytical methodology applicable in a study investigating their effect on DNA. Until now, most of the studies on the interaction of DNA with xenobiotics have been accomplished by using the 32P postlabeling technique.15-18 Although this technique is very sensitive, it lacks structural information, especially in the absence of suitable reference compounds. Because the concentration of 2′-deoxynucleotide adducts present in modified DNA material isolated from biological material is estimated to be a few picomoles per milligram of DNA, there is no doubt that, if an alternative methodology is developed, not only its sensitivity but also its ability to give structural information should be addressed. In view of these requirements, it is of no surprise that mass spectrometry has been investigated for the analysis and structure (3) Wellemans, J.; Cerny, R. L.; Gross, M. L. Analyst 1994, 119, 497-503. (4) Ross, J. A.; Nelson, G. B.; Wilson, K. H.; Rabinowitz, J. R.; Galati, A.; Stoner, G. D.; Nesnow, S.; Mass, M. J. Cancer Res. 1995, 55, 1039-1044. (5) Wise, S. A.; Sander, L. C.; May, W. E. J. Chromatogr. 1993, 642, 329-349. (6) Van den Eeckhout, E.; De Moerloose, P.; Sinsheimer, J. E. J. Chromatogr. 1985, 318, 343-349. (7) Hooberman, B. H.; Chakraborty, P. K.; Sinsheimer, J. E. Mutat. Res. 1993, 299, 85-93. (8) Sinsheimer, J. E.; Hooberman, B. H.; Das, S. K.; Brezzell, M. D.; You, Z. Mutat. Res. 1992, 268, 255-264. (9) Kumar, R.; Staffas, J.; Forsti, A.; Hemminki, K. Carcinogenesis 1995, 16, 483-489. (10) Das, L.; Das, S. K.; Chu, E. H. Y.; Sinsheimer, J. E. Mutat. Res. 1993, 299, 19-24. (11) Greene, E. J.; Friedman, M. A.; Sherrod, J. A.; Salerno, A. J. Mutat. Res. 1979, 67, 9-19. (12) Van den Eeckhout, E.; De Bruyn, A.; Pepermans, H.; Esmans, E. L.; Vryens, I.; Claereboudt, J.; Claeys, M.; Sinsheimer, J. E. J. Chromatogr. 1990, 504, 113-128. (13) Van den Eeckhout, E.; Coene, J.; Claereboudt, J.; Borremans, F.; Claeys, M.; Esmans, E.; Sinsheimer, J. E. J. Chromatogr. 1991, 541, 317-331. (14) Lemie`re, F.; Esmans, E. L.; Van Dongen, W.; Van den Eeckhout, E.; Van Onckelen, H. J. Chromatogr. 1993, 647, 211-218. (15) Szyfter, K.; Hemminki, K. Scand. J. Work, Environ. Health 1992, 18, 2226. (16) Szyfter, K.; Krueger, J.; Ericson, P.; Vaca, C.; Forsti, A.; Hemminki, K. Mutat. Res. 1994, 313, 269-276. (17) Amin, S.; Misra, B.; Desai, D.; Huie, K.; Hecht, S. S. Carcinogenesis 1989, 10, 1971-1974. (18) Reddy, M. V.; Hemminki, K.; Randerath, K. J. Toxicol. Environ. Health 1991, 34, 177-186.

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identification of many DNA adducts. In this context, it is worth mentioning that an excellent review article covering different mass spectral approaches has been published by Chiarelli and Lay.19 Since the development of electrospray ionization by Fenn et al.,20 a new dimension has been added to the mass spectral analysis of both low and high molecular weight polar compounds. Recently, we have shown that direct coupling of electrospray (ES) to capillary HPLC greatly improves the detection limit: bisphenol A diglycidyl ether (BPADGE) adducts of 2′-deoxynucleosides were detected in the low picogram region under full-scan conditions.21 Within this framework, experiments were initiated with the aim to investigate the utility of CZE, CZE/ES-MS, and CZE/ESMS/MS for PGE-DNA adducts. To detect a few DNA adduct molecules in a huge pool of nonadducted nucleotides after DNA hydrolysis, a preconcentration technique was needed. Using sample stacking22,23 as a concentration technique, concentrations of 5 × 10-7 M could still be analyzed using UV detection. This sample stacking technique, which implies applying a high voltage with reversed polarity immediately after loading the sample, was for the first time used in a combination with CZE/ES-MS and CZE/ES-MS/MS. Using this technique, a signal enhancement of a factor of several hundred was obtained as compared to the classical use of CZE/ES-MS. Sample stacking was recently described by Wolf and Vouros,24 using continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS). This technique offers the same preconcentration potential as transient capillary isotachophoresis (CITP) CZEMS,25,26 but in contrast to transient CITP, only one buffer system is used in CZE/ES-MS. This buffer system is the same as that used in normal CZE, without stacking. Furthermore, the sample stacking technique is very flexible and allows comparison of electropherograms obtained with or without stacking. Another preconcentration technique, combining liquid-liquid electroextraction-isotachophoresis (EE-ITP), with CZE/ES-MS, was described by Van der Vlis et al.27 The sample stacking technique described here makes CZE a suitable technique to get mass spectrometric (MS) results of dilute samples and even tandem mass spectrometric (MS/MS) results of real-world DNA adduct samples. EXPERIMENTAL SECTION Instrumentation. CZE separations were done on a Lauerlab Prince system equipped for on-column detection with a Kontron UV detector (type HPLC 332) at a wavelength of 270 nm. Data collection was performed with the PC Integration Pack version 3.90 (Kontron Instruments). CZE/ES-MS and CZE/ES-MS/MS experiments were done on a VG Quattro II triple-quadrupole system (Fisons Instruments), equipped with a Mass Lynx data system. (19) Chiarelli, M. P.; Lay, J. O., Jr. Mass Spectrom. Rev. 1992, 11, 457-493. (20) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-50. (21) Vanhoutte, K.; Esmans, E. L. J. Mass Spectrom. Rapid Commun. Mass Spectrom. 1995, S143-S152. (22) Chien, R.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (23) Beckers, J. L.; Ackermans, M. T. J. Chromatogr. 1993, 629, 371-378. (24) Wolf, S. M.; Vouros, P. Anal. Chem. 1995, 67, 891-900. (25) Thompson, T. J.; Foret, R.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65, 900-906. (26) Zhao, Z.; Wahl, J. H.; Udseth, H. R.; Hofstadler, S. A.; Fuciarelli, A.; Smith, R. D. Electrophoresis 1995, 16, 389-395. (27) Van der Vlis, E.; Mazereeuw, M.; Tjaden, U. R.; Irth, H.; Van der Greef, J. J. Chromatogr. A. 1995, 712, 227-234.

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Chemicals. 2′-Deoxycytidine-5′-monophosphate (dCMP) and 2′-deoxyadenosine-5′-monophosphate (dAMP) were obtained from Janssen Chimica (Beerse, Belgium). Thymidine-5′-monophosphate (TMP), 2′-deoxyguanosine-5′-monophosphate (dGMP), highly polymerized DNA Type I from calf thymus, deoxyribonuclease I (DNA-ase I), and nuclease P1 were obtained from Sigma (St. Louis, MO). (()-2,3-Epoxypropyl phenyl ether (PGE), N-(2,3epoxypropyl)phthalimide and (()-(2,3-epoxypropyl)benzene were obtained from Aldrich (Steinheim, Germany). HPLC-grade water was obtained from an Elga Maxima Ultrapure Water treatment device. Other products and solvents were of analytical grade and were used without further purification. Procedure. All preparations and manipulations were carried out in a laminar air flow (LAF) system. HPLC-grade water, all glassware, and other recipients were sterilized in an autoclave for 20 min. All buffer solutions were filter-sterilized using 0.2 µm syringe filters from Nalgene. These sterile conditions were necessary to prevent contamination by and growth of bacteria and/ or molds, since the enzymatic hydrolysis reactions were carried out in a buffer solution at 37 °C for 12 h. Nucleotide Adduct Formation. Aqueous solutions (2 mg/ ml) of each 2′-deoxynucleotide were prepared. To 1 mL of this solution were added 1 mL of methanol and 1 mL of a 1 M methanolic solution of the above-mentioned epoxides in separate test tubes. The mixtures were reacted for 24 h at 37 °C. Reactions were terminated by evaporating the methanol using a stream of nitrogen gas. To the remaining solution was added 1 mL of HPLCgrade water. This mixture was extracted three times with 3 mL of chloroform (to remove unreacted epoxide), and the aqueous phase was lyophilized. The residue was redissolved in 300 µL of HPLC-grade water. DNA Adduct Formation and Enzymatic Hydrolysis to 2′Deoxynucleotides. Calf thymus DNA was dissolved in HPLCgrade water at a concentration of 10 mg/mL. To 1 mL of this solution were added 1 mL of methanol and 1 mL of 1 M of the above mentioned epoxides in methanol in separate test tubes. The mixtures were reacted for 24 h at 37 °C. Reactions were terminated by evaporating the methanol using a stream of nitrogen gas. To the remaining solution was added 1 mL of HPLC-grade water. This mixture was extracted three times with 3 mL of chloroform (to remove unreacted epoxide). The aqueous phase (400 µL) was used to isolate DNA by ethanol precipitation. The DNA pellet was then dissolved in 1 mL of 20 mM Tris/HCl (pH 7.5), 50 mM CaCl2, and 2.25 mM MgCl2. Then, 500 units of DNAase I was added and the mixture was incubated at 37 °C. After 12 h, 20 mL of a 1 M sodium acetate solution (pH 5.2) and 5 units of nuclease P1 were added, and the mixture was incubated another 6 h at 37 °C. Capillary Zone Electrophoresis. The CZE capillary was a 75 µm i.d. fused silica capillary. The distance of the inlet of the CZE capillary to the UV detector was 61.5 cm. The total length was 91.5 cm. At both inlet and outlet, the polyimide coating was removed. This was necessary since the ammonium-containing buffer system detached the polyimide coating from the fused silica. This led to current fluctuations, producing spikes in the electropherogram. The inlet tray containing the samples and the inlet buffers was kept at 5 °C to prevent evaporation of the volatile buffer components (this can greatly affect reproducibility) and to preserve the samples. The CZE capillary itself was kept at a

constant temperature of 30 °C. Before the start of a CZE run, the capillary was rinsed with 0.1 M sodium hydroxide (NaOH), water, and running buffer (using a pressure of 1000 mbar for 2 min). Electrophoresis was performed using a constant voltage of 17 kV. Coupling of the CZE system to ES-MS implies the use of volatile buffers. Optimal separation conditions, both for nucleotide adducts mixtures and mixtures obtained from hydrolyzed DNA, were realized by using a 100 mM ammonium carbonate buffer system (pH 9.68). Sample Stacking. This technique was used to preconcentrate samples on the CZE capillary. This method can be used when the conductivity of the sample solution is lower than that of the running buffer system.22,23 The technique is based on the principle that the local electrophoretic velocity of the ions inside the sample buffer is much faster than the bulk electroosmotic flow (EOF) of the solution. By applying a high voltage with reversed polarity immediately after loading the sample, the sample buffer was removed prior to separation of analytes. Since all molecules of interest were negatively charged (2′-deoxynucleotides and their adducts), this was a very straightforward approach. At first, the capillary was filled with 100 mM ammonium carbonate, pH 9.68, and the conductivity was measured by applying a voltage of -7 kV. A current in the range -45 to -52 µA was noted as the stack limit current. A sample plug of desired length (as described for each application in Results and Discussion) was then introduced using pressure. This plug was then concentrated using a reversed electrical field of -7 kV. This field causes the EOF to blow the sample plug out of the column toward the negative electrode, while the negatively charged molecules stack at the interface between buffer and remaining sample plug. When the conductivity of the solution in the column came to the stack limit current +0.5 µAsmeaning that the sample plug was nearly completely removedsa reversed voltage of -5 kV was applied for 30 s, in order to remove any of the remaining sample plug buffer. Normal electrophoresis was then started. Provided a sample plug was injected at 100 mbar for 7 min (approximately equivalent to a complete filling of the CE capillary with sample), the procedure described above gave an increase in sensitivity by a factor of 200 as compared to conventional CZE. When stacking the DNA hydrolysates a better stacking efficiency was obtained when the hydrolysates were diluted 1:1 in HPLC-grade water. This led to a broader difference in conductivity between the sample plug and the overall conductivity of the capillary filled with running buffer. Coupling CZE to ES-MS.28-31 To couple the CZE system to ES-MS, a fused silica capillary of 1 m × 75 µm i.d. (365 µm o.d.) was used, which was inserted inside the triaxial ES probe. The CZE capillary was positioned such that, at the tip of the ES probe, the CZE capillary protruded 200 µm with reference to the liquid sheath tube. The liquid sheath tube itself protruded 500 µm with reference to the capillary needed to introduce the nebulizing gas. The anode reservoir and the tip of the electrospray probe were (28) Fre´geau, C. J.; Fourney R. M. Biotechniques 1993, 15 (1), 100-119. (29) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A-584A. (30) Wahl, J. H.; Goodlett, D. R.; Udseth, H. R.; Smith, R. D. Electrophoresis 1993, 14, 448-457. (31) Perkins, J. R.; Parker, C. E.; Tomer, K. B. Electrophoresis 1993, 14, 458468.

positioned at the same height in order to prevent additional hydrodynamic effects during the sample stacking procedure. Using the Lauerlab Prince, it was possible to monitor the current during electrophoresis, even when coupled to MS, by using the current control at the inlet electrode. This is a prerequisite for sample stacking. A makeup flow of 80% 2-propanol, 15% water, and 5% 0.01 M ammonium carbonate buffer (pH 9.68) was introduced using the sheath capillary at a flow rate of 10 µL/min, using an auxiliary HPLC pump (herein referred to as HPLC pump 1). This makeup flow is necessary to obtain stable ES conditions and, at the same time, ensures electrical contact at the CZE capillary. The flow rate of the N2 bath gas was adjusted between 50 and 100 L/h. The flow rate of the N2 nebulizing gas was set at 30 L/h. During injection of the samples and during sample stacking, the voltages in the ionization probe were turned off to prevent the negative potential at the probe tip to cause sample discrimination during injection (in our case, the analytes of interest were negatively charged and were discriminated during injection). During the sample stacking procedure, the EOF is reversed (from the probe tip to the inlet); this causes makeup solvent to be drawn into the capillary. Due to the low conductivity of the makeup solvent (80% 2-propanol, 15% water, 5% 0.01 M ammonium carbonate), this would render the sample stacking procedure unusable when connected to ES-MS. To overcome this problem, a second makeup flow system was set up, using a second auxiliary HPLC pump (HPLC pump 2), which delivered 100 mM ammonium carbonate buffer (pH 9.68) at a flow rate of 10 µL/min. Using this flow rate, the amount of buffer presented at the outlet of the capillary was sufficient to allow the EOF to draw buffer into the capillary without introducing air bubbles in the capillary. Both makeup flow systems were connected to the line leading to the sheat tube using a Valco six-way switching valve. Before the sample stacking procedure was started, the valve was switched to HPLC pump 2. The electrospray efficiency was the determining factor to ascertain the moment when the 100 mM ammonium carbonate buffer reached the probe tip. The electrospray efficiency dropped tremendously when this buffer system reached the probe tip. The sample stacking procedure, as described above, was then carried out using -9 kV as stacking voltage. During the sample stacking procedure, the voltages at the probe tip were switched off. Once the stack limit current was reached, the normal electrophoresis was started (applying an electrophoresis voltage of 13 kV), the valve was switched to HPLC pump 1, and the voltages at the probe tip were switched on again. Negative ES ionization was performed using an ionization voltage of -4.5 kV at the probe tip. The cone voltage was set at 30 V, and the source temperature was kept constant at 75 °C. All CZE/ES-MS results were obtained using ammonium carbonate as the buffer system and an electrophoresis voltage of 13 kV. A scan range of 160-800 Da was covered at a scan speed of 320 Da/s for the detection of the 2′-deoxynucleotide adducts. To detect the products present in the DNA hydrolysate, a scan range of 300-900 Da was covered at a scan speed of 300 Da/s. Lowenergy collision-activated dissociation (CAD) product ion spectra were obtained of the [M - H]- ions. The collision gas (Ar) pressure was 3 × 10-3 mbar, and the collision energy was optimized for each component by introducing a sample plug through the capillary using pressure. The optimal collision energy varied between 20 and 35 eV, depending on the component. Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

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Table 1. Detection Limits Obtained with and without the Use of the Described Sample Stacking Technique, Using Different Detection Systemsa

without stacking with stacking without stacking with stacking without stacking with stacking a

detection system

injection pressure (mbar), time applied (s)

length of the plug injected (mm)

sample concentration (M)

amount injected (fmol)

signal to noise

UV detector UV detector ES-MS full scan ES-MS full scan ES-MS SIM ES-MS SIM

22, 12 220, 120 22, 12 440, 120 22, 12 440, 120

5 500 4.6 928 4.6 928

2.9 × 10-5 2.9 × 10-7 2.9 × 10-5 1.45 × 10-7 7.25 × 10-7 3.63 × 10-9

540 540 520 520 12 12

5/1 5/1 5/1 5/1 5/1 5/1

See text for conditions.

Figure 1. Leveling system, keeping the outlet buffer level at an identical level during consecutive runs and consequently improving the reproducibility of the migration times.

RESULTS AND DISCUSSION Reproducibility of CZE-UV. One of the problems encountered in CZE is the reproducibility of retention times,32,33 which is related to hydrodynamic leveling between the inlet and outlet vials. The reproducibility in retention times was investigated by analyzing the reaction mixture of dAMP with PGE (see Experimental Section, using normal pressure injection of 10 mbar for 6 s) using 100 mM ammonium carbonate buffer (pH 9.68) in 20 consecutive runs. For this purpose, seven inlet buffer vials were used, and each of these vials was used for three electrophoresis runs. The reproducibility was tested either at constant current (195 mA) or at constant voltage (17 kV). It was observed that the best data were obtained at constant current. However, one should realize that, during the time elapse of the 20 runs, the level of the buffer in the outlet vial gradually increased, due to the rinsing of the capillary between each run and the EOF. This increase in level at the outlet vial results in hydrodynamic backsiphoning effects. The back-siphoning causes a flow through the capillary opposite to the EOF, resulting in longer retention times and hence decreased reproducibility. This problem was overcome by building a system that kept the outlet buffer always at the same level throughout consecutive runs. As can be seen from Figure 1, this can be realized by adding an easy-to-build and budget-friendly system constructed out of plastic tubing and a glass capillary and applying compressed air. The glass capillary was bent at an angle of 90° about 0.5 cm from the end. The bent tip was then pushed through a piece of plastic tubing with a small diameter and connected at both ends (32) Strege, M.; Lagu, A. L. J. Liq. Chromatogr. 1993, 16, 51-68. (33) Smith, S. C.; Strasters, J. K.; Khaledi, M. G. J. Chromatogr. 1991, 559, 5768.

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with other wide-bore plastic tubing. At one end, a gentle flow of pressurized air is applied, and the inlet of the glass capillary is positioned just above the outlet buffer level. With this system, a reproducibility in retention times with a coefficient of variation (CV) of 0.0092 was obtained. Without the introduction of the former device, a CV of only 0.0248 was obtained. These CVs were calculated using the retention time of the nucleotide peak having the largest k′ value. Detection Limits of CZE-UV and CZE/ES-MS. The detection limits were determined using a stock solution of dAMP and TMP (10 mg of each dissolved in 10 mL of HPLC-grade water; further dilutions were also prepared in HPLC-grade water). The results of these tests are given in Table 1. Using CZE/ES-MS under full-scan conditions (scanning from 100 to 700 Da at a scan speed of 300 Da/s), detection limits similar to those for CZE using UV detection were obtained. Sensitivity could easily be enhanced by a factor 40 under selected ion monitoring (SIM) conditions (scanning the masses 321.14 and 330.01 Da, each with a dwell time of 0.3 s, span 0.00 Da). Using the sample stacking technique, a 200-fold increase in sensitivity could be obtained as compared to the normal injection, leading to a detection limit of 1.45 × 10-7 M using full scan. This detection limit is comparable to the detection limits obtained by Thompson et al.25 using transient CITP/CZE-MS. They report a detection limit using full scan (600-1850 Da) of 5 × 10-7 M. The detection limit obtained during our experiments was 1 order of magnitude better than the detection limit reported by Wolf and Vouros24 using CZE/CF-FABMS. They report a detection limit using full scan of 5 × 10-6 M. Similar detection limits were obtained with the monoalkylated and dialkylated adducts of PGE with 2′-deoxynucleotides. Optimization of CZE for MS Detection. CZE was optimized using UV detection, in view of coupling CZE to ES-MS. To determine the optimal separation conditions, the mixture obtained by reaction of PGE with the four main 2′-deoxynucleotides (dAMP, dCMP, TMP, and dGMP, see Experimental Section) was analyzed using pressure injection (10 mbar, 6 s). The ammonium carbonate buffer system (pH 9.68) was used as running buffer at different molarities (12.5, 25, 50, 100, and 200 mM). The effect of buffer concentrations on the separation of dansyl amino acids has been described by Issaq et al.34 Consistent with their results, a linear relationship was observed if retention times were plotted versus the square root of the buffer concentration. Separation efficiency and resolution increased with buffer concentration (Figure 2). A 100 mM ammonium carbonate buffer concentration was optimal; (34) Issaq, H. J.; Atamna, I. Z.; Muschik, G. M.; Janini G. M. Chromatographia 1991, 32, 155-161.

Figure 2. Effect of buffer concentration on the separation efficiency. (A) Effect of buffer concentration on the retention times of the analytes: [, (bottom to top) EOF (the EOF could be determined thanks to the presence of PGE-diol, which migrates with the EOF, in the samples); +, dAMP with PGE adduct on the heterocyclic moiety; f, dCMP with PGE adduct on the heterocyclic moiety; b, dGMP with PGE adduct on the heterocyclic moiety; 9, dAMP; 2, dCMP. (see text for conditions; electrophoresis was performed at 17 kV). (B) Number of theoretical plates obtained for the peak of PGE-dGMP using the different buffer concentrations (see text for conditions).

Figure 3. Structures of the epoxides used in these studies.

above this concentration, the current generated was too high which led to complications related to Joule heating. A pH range was tested from 9 to 10.5. The pH influenced the sequence of elution and the resolution of the nucleotides and their PGE adducts. An optimal electrophoretic resolution was obtained using the ammonium carbonate buffer system at a pH of 9.68 (the number of theoretical plates, N, for this buffer system was 200 000). The 2′-deoxynucleotide reaction mixtures of PGE, (epoxypropyl)phthalimide, and (epoxypropyl)benzene (Figure 3) were analyzed by CZE (conditions and preparation see Experimental Section). Direct pressure injection (10 mbar, 6 s) of these samples

overloaded the column; therefore, samples were diluted by a factor of 20 in HPLC-grade water. The electropherogram for the four 2′-deoxynucleotides reacted with PGE is shown in Figure 4. Analysis of these mixtures was accomplished within 22 min. The DNA hydrolysates obtained after the treatment of calf thymus DNA with PGE, (epoxypropyl)phthalimide, or (epoxypropyl)benzene were analyzed in an analogous way. However, the sample stacking technique had to be used to load enough sample on the CZE column (pressure injection of 44 mbar, 30 s) after dilution 1:1 with HPLC-grade water. As such, a better stacking efficiency was realized because of the decreased ion concentration, i.e., conductivity in the sample plug which was too high in the original hydrolysate (due to the hydrolysis buffer, see Experimental Section). The electropherogram of the DNA hydrolysate reacted with PGE is shown in Figure 4. Because of the good reproducibility in retention times, the adducts in the DNA hydrolysates could be located by comparison with the corresponding electropherograms obtained for the 2′deoxynucleotide-epoxide mixtures. It was observed that PGE and (epoxypropyl)phthalimide reacted intensively with both 2′-deoxynucleotides and calf thymus DNA. In the case of (epoxypropyl)benzene, adduct formation was observed with the 2′-deoxynucleotides but not with DNA. It is furthermore noteworthy that the DNA hydrolysates of the reactions with PGE and (epoxypropyl)phthalimide show the presence of adducts that were not formed in the reaction with the 2′-deoxynucleotides (Figure 4). These differences in reaction between nucleotides and DNA were expected in view of the molecular structure of DNA and the greater hindrance due to the presence of a double strand in the DNA molecule. CZE-Electrospray Mass Spectrometry. To obtain additional structural information on the adducts formed by treatment of the different 2′-deoxynucleotides and calf thymus DNA, CZE was coupled to ES-MS. In the scope of this paper, the results obtained with one of the epoxides, i.e., PGE, are given as an example. Other data resulting from the treatment of calf thymus DNA with other epoxides will be outlined in another paper. Because of the low electroosmotic flow rates in CZE (e1 µL/ min), stable electrospray conditions were created by the coaxial introduction of the makeup flow. Four makeup flow mixtures were tested for their performance during ES-MS: (A) 50:50 methanol/0.01 M ammonium carbonate, (B) 50:50 2-propanol/0.01 M ammonium carbonate, (C) 80:20 2-propanol/0.01 M ammonium carbonate, and (D) 80:15:5 2-propanol/HPLC-grade water/0.01 M ammonium carbonate. Ammonium carbonate was necessary to ensure electrical contact at the probe tip. To investigate the influence of these makeup mixtures on the ES-MS data, the PGE-dAMP reaction mixture was analyzed (conditions in Experimental Section) under full-scan conditions (160-800 Da, 320 Da/s). Each time a plug was injected by applying a pressure of 40 mbar for 30 s. When the composition of the coaxial flow was altered from A to B, the signal intensity was enhanced by a factor of 4 (Table 2). This emperical observation was in agreement with the general experience that 2-propanol works better when using electrospray in the negative mode. The introduction of system C enhanced the signal intensity by a factor of 2 compared to system B. The use of more 2-propanol improves the evaporation efficiency and the droplet formation in the spray (due to 2-propanol’s lower boiling point Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

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Table 2: Influence of Makeup Flow System on the Peak Areas Obtained for the Products of the PGE-dAMP Reaction Mixturea makeup flow system m/z

A

B

C

D

630 480 480 330

256 1652 1729 3367

814 8392 9557 15604

1354 15709 17871 32120

1406 15108 17985 32325

a See text for conditions. Dialkylated product of dAMP at m/z 630; monoalkylated product of dAMP at m/z 480 and dAMP at m/z 330. The numbers in columns 2-5 represent ion counts.

Figure 4. (A) In all electropherograms depicted, the EOF is visible because of the elution with the EOF of PGE-diol. The identity of all numbered peaks was determined by CZE/ES-MS and CZE/ES-MS/ MS. (1) Electropherogram of the dCMP-PGE reaction mixture (a, dCMP alkylated on the 5′-phosphate moiety; b, dCMP alkylated on the heterocyclic moiety; c, unmodified dCMP). (2) Electropherogram of the dGMP-PGE reaction mixture (a, dialkylated dGMP; b, dGMP alkylated on the 5′-phosphate moiety; c, dGMP alkylated on the heterocyclic moiety; d, unmodified dGMP). (3) Electropherogram of the TMP-PGE reaction mixture (a, TMP alkylated on the 5′phosphate moiety; b, unmodified TMP). (4) Electropherogram of the dAMP-PGE reaction mixture (a, dialkylated dAMP; b, dAMP alkylated on the 5′-phosphate moiety; c, dAMP alkylated on the heterocyclic moiety; d, unmodified dAMP). (B) Electropherogram of the hydrolysate of DNA reacted with PGE: a, dAMP; b, dCMP; c, dGMP; and d, TMP.

and lower surface tension). However, prolonged use of system C resulted in the formation of small crystals at the ES probe tip. 3580 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

This problem could be overcome by using system D as makeup flow for (-)-ES. Due to the lower concentration of buffer, no more crystals were formed at the ES probe tip. To obtain a stable electrospray, it was necessary to remove the polyimide coating over the complete length of the sheath capillary. If the coating was not removed, it came loose during operation. Before starting a CZE/ES-MS separation, a sample plug (40 mbar, 30 s) was introduced into the ES source by applying a pressure of 150 mbar on the CZE capillary (full scan 100-1500 Da, 280 Da/s). From these (-)-ES data, it was possible to get an idea of the ions to be expected in the mixture. Once this was known, a complete CZE (-)-ES-MS separation was started, and the analysis was followed in real time by looking at the extracted ion electropherograms of the compounds of interest. This procedure was followed for the analysis of both the 2′-deoxynucleotide-PGE mixtures and the hydrolysate obtained after treatment of calf thymus DNA with PGE. The 2′-deoxynucleotide-PGE mixtures were analyzed after a pressure injection of 40 mbar for 30 s. This was the maximal plug length that could be injected without the use of sample stacking, and that resulted in an acceptable number of theoretical plates (N ) 35 156). To be able to introduce such a long sample plug, ammonium carbonate buffer (at a pH of 9.68) was added to the sample solution to a final concentration of 10 mM. Because these samples were dissolved in a buffer where the electrolyte concentration was only one-tenth of the running buffer concentration, general stacking effects occurred. As an example of the CZE-MS analysis of a 2′-deoxynucleotide-PGE mixture, the electropherogram of the PGE-dAMP analysis is shown in Figure 5. In this dAMP-PGE mixture, the following adducts were detected and assigned: an adduct of adenine base with PGE [(M - H)- at m/z 284], a dialkylated adduct of dAMP with PGE [(M - H)- at m/z 630], and two monoalkylated adducts of dAMP with PGE [(M - H)- at m/z 480], the first eluting with the PGE group on the phosphate moiety and the last eluting with the PGE group on the base moiety. This order of elution can be easily explained on the basis of the structure of the adducts. When alkylation occurs on the heterocyclic moiety, the charged phosphate group is still free (leading to later elution). When alkylation occurs on the phosphate group, a less polar compound is created (leading to faster elution). Two other compounds, characterized by the presence of a deprotonated molecule, [M - H]-, at m/z 377 and 227, were also found. These ions correspond with the MW of alkylated and nonalkylated 1-methoxy-D-deoxyribofuranose-5′-monophosphate (Figure 6).

Figure 5. Mass electropherogram of the reaction mixture of dAMP with PGE using full-scan conditions (160-800 Da, at a scan speed of 320 Da/s). (see text for conditions). (M - H)- in order of increasing retention time: 167, PGE-diol; 284, adduct of adenine with PGE; 630, dialkylated dAMP; 480, dAMP alkylated on the 5′-phosphate moiety; 377, deoxyribose methoxylated on C-2′ and phosphorylated on C-5′ with PGE adduct on the phosphate moiety; 480, dAMP alkylated on the heterocyclic moiety; 330, unmodified dAMP; and 227, deoxyribose methoxylated on C-2′ and phosphorylated on C-5′.

The structures of the above compounds were assigned by CZE/ES-MS/MS. To obtain more structural information, lowenergy CAD spectra were obtained using Ar as the collision gas at a pressure of 3 × 10-3 mbar. Product ion scan spectra of the respective precursor (M - H)- ions of monoalkylated and dialkylated 2′-deoxynucleotide-PGE adducts gave additional structural information. For the monoalkylated adducts, a normal injection was used (40 mbar, 12 s). In the case of the 2′-deoxynucleotide-PGE mixtures, monoalkylation was observed at the 5′-phosphate or the heterocyclic moiety.

As an example, the product ion spectra of both monoalkylated adducts of dAMP and PGE are given (Figure 7). Alkylation of the 5′-phosphate group could easily be recognized by the presence of a product ion at m/z 247; this ion represents the phosphate group with PGE bound to it. This phenomenon was already observed in earlier experiments during a study of the interaction between 2′-deoxynucleotides and bisphenol A diglycidyl ether.21 Alkylation of the heterocyclic moiety could be recognized by the presence of a product ion at m/z 195; this ion represents the phosphorylated sugar. In this context, it is worth mentioning that, in the TMP-PGE reaction mixture, only one monoalkylated product was found, i.e., the TMP adduct, where alkylation occurred at the 5′-phosphate group, in contrast to the other 2′deoxynucleotides where monoalkylation occurred both on the phosphate moiety and on the heterocyclic moiety. To obtain product ion spectra of the dialkylated adducts (which were present in lower amounts than the monoalkylated products), the sample stacking procedure had to be used (100 mbar, 30 s). In general, acceptable MS/MS data were obtained from an injection of about 2.5 ng of product (S/N 5:1 for the major significant peaks in the spectra). Analysis of the product ion spectra of the dialkylated dAMP, dCMP, and dGMP adducts in the respective 2′-deoxynucleotidePGE mixtures showed that dialkylation was the result of alkylation at both the phosphate and base moiety. In Figure 8, the MS/MS spectra of the dialkylated product of dAMP with PGE are shown. The product ion at m/z 247 indicates alkylation of the 5′-phosphate group, and the product ion at m/z 284 corresponds with alkylation of the heterocyclic moiety. In the case of TMP, however,

Figure 6. CZE/ES-MS/MS elucidation of the structures of the compounds detected in the mass electropherogram of Figure 5 (dAMP-PGE mixture).

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Figure 7. CZE/ES-MS/MS spectra of the daughters of monoalkylated dAMP with PGE (mother ion mass (M - H)- ) 480). (A) Mass spectrum of dAMP with alkylation on the 5′-phosphate moiety. (B) Mass spectrum of dAMP with alkylation on the heterocyclic moiety. Both spectra were obtained after a normal pressure injection (40 mbar, 30 s) and an electrophoresis voltage of 13 kV. The collision energy in the gas cell was set at 25 eV.

Figure 8. CZE/ES-MS/MS spectrum of the daughters of dialkylated dAMP with PGE (mother ion mass (M - H)- ) 630).

dialkylation was only the result of reaction at the 5′-phosphate group. For the analysis of DNA hydrolysates, sample stacking was a prerequisite to detect the adducts. However, these DNA hydrolysates were characterized by the presence of a high concentration of electrolytes (see Experimental Section). General stacking effects did not occur as efficiently as for the nucleotide adduct 3582 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

samples. Therefore, it was necessary to use the sample stacking technique described above, allowing for a much larger sample plug to be injected and removal of the sample buffer prior to normal electrophoresis. The DNA hydrolysates were analyzed after sample stacking of a pressure-injected plug using 100 mbar for 30 s (the DNA hydrolysates were first diluted 1:1 with HPLCgrade water, leading to a better stacking efficiency). In the PGE-

Figure 9. Mass electropherogram of the hydrolysate of the reaction of DNA with PGE using full-scan conditions (300-900 Da, at a scan speed of 300 Da/s). (M - H)- (see text for conditions): (A) m/z 480, monoalkylated dAMP; m/z 471, monoalkylated TMP; m/z 496, monoalkylated dGMP. (B) m/z 745, 760, 769, 775, 784, 785, 793, 800, and 809, monoalkylated dinucleotides (see text). (C) m/z 330, dAMP; m/z 306, dCMP; m/z 346, dGMP; m/z 321, TMP.

Figure 10. CZE/ES-MS/MS spectrum of the daughters of monoalkylated dAMP (mother ion mass (M - H)- ) 480), present in the hydrolysate of the reaction of DNA with PGE. Here the sample stacking technique was used following an injection of 100 mbar for 30 s. The collision energy was set at 25 eV.

DNA-treated hydrolysate, the following mononucleotide adducts were detected: the monoalkylated adduct of TMP with PGE [(M

- H)- at m/z 471], the monoalkylated adduct of dAMP with PGE [(M - H)- at m/z 480], and the monoalkylated adduct of dGMP Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

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with PGE [(M - H)- at m/z 496] (Figure 9). These compounds were unambiguously identified by using CZE/ES-MS/MS. No adduct of dCMP with PGE was detected. The following monoalkylated dinucleotide adducts were found: PGE-dCMP-dCMP adduct [(M - H)- at m/z 745], PGE-dCMP-TMP adduct [(M - H)- at m/z 760], PGE-dCMP-dAMP adduct [(M - H)at m/z 769], PGE-TMP-TMP adduct [(M - H)- at m/z 775], PGE-TMP-dAMP adduct [(M - H)- at m/z 784], PGE-dCMPdGMP adduct [(M - H)- at m/z 785], PGE-dAMP-dAMP adduct [(M - H)- at m/z 793], PGE-TMP-dGMP adduct [(M - H)- at m/z 800], and PGE-dAMP-dGMP adduct [(M H)- at m/z 809] (Figure 9). The structures of these compounds were also unambiguously assigned by CZE/ES-MS/MS. Product ion scan spectra were obtained using low-energy CAD (using argon gas as the collision gas at a pressure of 3 × 10-3 mbar). The mononucleotide adducts present in the DNA hydrolysate were analyzed by CZE/ES-MS/MS using the sample stacking technique (injection of 100 mbar, 30 s). For all the mononucleotide adducts present in the DNA hydrolysate, only reaction on the base moiety was observed. As an example, the MS/MS spectrum of the monoalkylated adduct of dAMP with PGE is shown in Figure 10. Note the presence of the ion with m/z 195, which is typical for alkylation on the heterocyclic moiety. The PGE-DNA mixtures can be perfectly structurally analyzed by the powerful combinations of CZE/ES-MS and CZE/ES-MS/

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MS. More detailed structural assignment for the other epoxides will be given elsewhere. CONCLUSIONS Coupling of CZE to MS offers unique possibilities for the analysis of 2′-deoxynucleotide adducts in both 2′-deoxynucleotide mixtures and in the corresponding DNA-hydrolysates. Full-scan data and MS/MS data can be obtained, provided that sample stacking techniques are used. The advantage of this sample stacking technique compared to CITP/CZE is that this methodology does not require the use of different buffer systems. When CZE-MS is performed under selected ion monitoring using the sample stacking technique, it is estimated that adducts can be detected in a concentration down to 10-8-10-9 M. In view of the results described in this paper, it is worthwhile to pursue other experiments using CZE-MS for the detection of DNA adducts in vivo. ACKNOWLEDGMENT This work was supported by NFWO Grant No. 32013394.

Received for review January 3, 1996. Accepted July 22, 1996.X AC9600013 X

Abstract published in Advance ACS Abstracts, September 1, 1996.