Anal. Chem. 1996,67,891-900
Incorporation of Sample Stacking Techniques into the Capillary Electrophoresis CF-FA9 Mass Spectrometric Analysis of DNA Adducts Susan M. WOW and Paul Vouros* Department of Chemistry and Bamett Institute of Chemical Analysis, Northeastem Univetsity, Boston, Massachusetts 02 1 15
Sample stacking is used to improve the detection limits of capillary mne electrophoresis coupled to continuous flow fast atom bombardment mass spectrometry for the analysis of DNA adducts. It was found that, with stacking, the concentration detection limit of deoxynucleotide adducts could be improved by as much as 3 orders of M range. In magnitude, thereby bringing it into the addition, the mass spectrometric mass detection limits of a model acetylaminofluorenedeoxyguanosine 5'-monophosphate adduct were found to be in the low picomole range for full scanning and the low femtomole range for multiple reaction monitoring of a selected fragmentation. The techniques have been applied to the analysis of adducts formed in the in vitro reaction of N-acetoxy-Nacetyl-2-aminofluorenewith DNA. Capillary zone electrophoresis (CZE) enjoys a combination of features such as fast separation times, superior efficiencies, ease of use, and low cost which make it unique among analytical separation techniques. Interestingly, its development as an indispensable technique for the analysis of labile polar biomolecules has paralleled a similar advancement in mass spectrometry with the development of ionization methods such as continuous flow fast atom bombardment (CF-FAB) and electrospray ionization @SI)which operate in a flow regime compatible with that of CZE. In attempts to capitalize on the obvious advantages associated with the coupling of the two techniques, several groups have investigated interfacing strategies and the application of the CZE-MS technique to the analysis of biomolecules such as peptides,lv2 drugs? proteins,Iq4 etc. Both CF-FAB and ESI have been successfully employed for this purpose. Monodeoxynucleosides and monodeoxynucleotides contain both acidic and basic functionalities which permit the CZE separation of these components under both acidic and basic pH conditions. In addition to nucleobase ionization sites, monodeoxynucleotides also contain a phosphate group which bears a negative charge over most of the pH range. Several reports have appeared in the literature detailing the use of CZE for a variety of separations of monomeric nucleic acid constituents, given their t Current address: Department of Chemistry, MIT, Room 56.028, Cambridge, MA 02139. (1) Smith, R D.; Loo, J. A; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R]. Chromatogr. 1989,480, 211-232. (2) Moseley, M. A; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1991,63, 109-114. (3) Perkins, J. R; Parker, C. E.; Tomer, K B.]. Am. SOC.Mass Spectrom. 1992,
3, 139-149. (4) Tsuji, IC; Baczynskyj, L.; Bronson, G. E. Anal. Chem. 1992,64, 18641870.
0003-2700/95/0367-0891$9.00/0 0 1995 American Chemical Society
favorable structural features. These include studies concerned with fundamental separations mechanisms5and applications such as the quantitation of mononucleotides generated from the hydrolysis of RNA6 free nucleotide profiling of human blood lymphocyte^,^ and quantitation of nucleotides in fish tissue as a measure of fish freshness.8 Micellar electrokinetic chromatographic techniques, in which analytes are resolved, at least in part, on the basis of their ability to partition into micelles, have been employed to enhance the separability of these types of comp o u n d ~ . ~In- ~addition, ~ reports have appeared which detail the application of CZE techniques to the separation of modified nucleobases/nucleosides/nucleotides from one another and from unmodified s p e ~ i e s . ~ J ~ - ~ ~ In recent years, we have been interested in the analysis of adducts formed by the covalent bonding of polycyclic aromatic hydrocarbon @'AH) metabolites with DNA. The use of capillary LC in combination with CF-FAB-MS has figured prominently in the development of our analytical methodology, and concurrently the utility of CZE-CF-FAB-MS for this application has been evaluated.I5 Since PAH-deoxynucleoside adducts exhibit dissociation behavior similar to that of the parent base, CZE should be able to provide analytically useful separationsfor a wide variety of adduct applications. In addition, it is likely that CZE could be used in a diagnostic fashion to provide information as to the site of adduct formation by allowing for the pKa determination of ionizable sites.16 Adduct pK, data are currently most frequently determined from pH-dependent changes in CD or W spectra or analyte partitioning into organic phases.17J8 While our initial results concerning the analysis of PAH-deoxynucleoside adducts (5) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988,60, 2642-2646. (6) Huang, X.; Shear, J. B.;Zare, R N. Anal. Chem. 1990,62, 2049-2051. (7) Ng, M.; Blaschke, T. F.; Arias, A A; Zare, R N.Anal. Chem. 1992,64, 1682-1684. (8) Nguyen, A-L.;Luong, J. H. T.; Mason, C. Anal. Chem. 1990,62, 24902493. (9) Lecoq, A F.; Leuratti, C.; Marafante, E.; DiBiase, S. J. High Resoluf. Chromafogr. 1991,14, 667-671. (10) Cohen, A S.; Terabe, S.; Smith, J. A; Karger, B. L. Anal. Chem. 1987,59, 1021-1027. (11) Row, K H.; Griest, W. H.; Maskarinec, M. P. J. Chromafogr. 1987,409, 193-203. (12) Lee, T.; Yeung, E. S.; Sharma, M.]. Chromatogr. 1991,565, 197-206. (13) Jackim, E.; Norwood, C. ]. High Resoluf. Chromatogr. 1990,13, 195-196. (14) Li,W.; Moussa, A; Giese, R W. J. Chromatogr. 1993,633, 315-319. (15) Wolf, S. M.; Vouros, P.; Norwood, C.; Jackim, E.]. Am. SOC.Mass Spectrom. 1992,3, 757-761. (16) Smith, S. C.; Khdedi, M. G. Anal. Chem. 1993,65, 193-198. (17) Babson, J. R; Russo-Rodriguez, S. E.; Rastetter, W. H.; Wogan, G. N. Carcinogenesis 1986,7, 859-865. (18) Agarwal, S. IC; Sayer, J. M.; Yeh, H. J. C.; Pannell, L. K.; Hilton, B. D.; Pigott, M. A; Dipple, A; Yagi, H.; Jerina, D. M. ]. Am. Chem. SOC.1987, 109, 2497-2504.
Analytical Chemistty, Vol. 67, No. 5, March 1, 1995 891
by CZE-CF-FAB-MS15 indicated absolute mass detection limits in the range of 50-100 fmol, the practical applicability of the technique was limited because of the low sampling volume limitation of CZE. In an attempt to circumvent this sampling efficiency problem, several research groups have developed strategies which allow for the increased loading of analytes from dilute solutions onto the CZE column. These include on-linelg and, more recently, transient oncolumn ITPpreconcentration,20-22 the inclusion of stationary phase materials at the head of the CZE c o l ~ m n , and ~ ~ -sample ~ ~ stacking method~.~~*27 In its simplest incarnation, sample stacking merely requires that the analyte be introduced onto the column in a solution of lower conductivity than the CZE buffer.27 When high voltage is applied to the column, a higher field is experienced across the higher resistivity analyte solvent than across the rest of the CZE buffer. Under this enhanced field, negative ions rapidly stack at the anode boundary between the analyte solvent plug and the CZE buffer, while cations stack at the cathode boundary. A normal CZE separation then follows. While in many cases this technique can provide a decrease in concentration detection limits by up to an order of magnitude, stacking of larger volumes is typically limited by (a) the laminar flow broadening which can result from the electroosmotic velocity difference between the analyte solvent and CZE buffer and (b) the need for an adequate field strength across the CZE buffer to effect a good separation of the stacked ions. The stacking of larger sample plugs has recently been accomplished by Chien and Burgi2'jqZ7and involves the removal of the analyte solvent prior to analysis. The method, which takes advantage of the electroosmotic flow generated across uncoated capillaries under most conditions, involves the following steps: (a) the introduction of the analyte in a low-conductivity buffer, (b) the application of a high voltage which is reversed in polarity to that which will be used for the separation and causes the stacking of ions on either side of the sample solvent plug while simultaneouslyelectroosmoticallypumping the sample solvent out of the column, (c) the reversal of the polarity of the high voltage back to the conditions used for analysis, and (d) separation and detection of the stacked ions. In some cases, the entire column can be filled with the analyte solution prior to stacking, allowing for the sampling of microliter volumes. Hence, sampling efficiencies of > 100 times that normally possible can be accommodated for analysis of trace constituents. It is apparent from the above discussion that when large volume stacking is performed in uncoated capillaries under most pH conditions (electroosmotic flow toward the cathode), this method is suited to negative ion analysis as the positive ions are pumped out of the column at the head of the sample solvent. In addition, the larger the negative electrophoretic mobility of an analyte ion, the larger the volume of analyte solution which can be effectively stacked. It seemed likely, therefore, that DNA (19) Stegehuis, D. S.;Irth, H.; Tjaden, U. R; van der Greef, J. J. Chromatop. 1991,538, 393-402. (20) Schwer, C.; Lottspeich, F. J. Chromatogr. 1992,623, 345-355. (21) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65,900-906. (22) Foret, F.; Szoko, E.; Karger, B. L. J. Chromafogr. 1992,608, 3-12. (23) Debets, A J. J.; Mazereeuw, M.; Voogt, W. H.; van Iperen, D. J.; Lingeman, H.; Hupe, K-P.; Brinkman, U. A Th.J. Chromatop. 1992,608,151-158. (24) Swartz,M.E.;Merion, M. J. Chromatogr. 1993,632, 209-213. (25) Cai, J.; El Rassi, 2.J.Liq. Chromatogr. 1992,15, 1179-1192. (26) Chien, R L.; Burgi, D. S. Anal. Chem. 1992,64, 1046-1050. (27) Chien, R L.; Burgi, D. S. Anal. Chem. 1992,64,489A-496A 892 Analytical Chemistry, Vol. 67, No. 5,March 1, 1995
adducts in deoxynucleotide form would be ideal candidates for this type of analysis, since all such adducts would contain the phosphate group and therefore possess a negative charge in water. It should be mentioned that, depending upon the type of enzymatic digestion employed, adducts can be isolated from DNA in several forms, including 3' or 5' monodeoxynucleotides, monodeoxynucleosides, or bases. With the above considerations in mind, we proceeded to evaluate the utility of ion stacking techniques for the analysis of PAH-DNA adducts by CZE-CF-FAB-MS. Toward this end, we redesigned our earlier CZE-MS interface to facilitate the execution of the ion stacking experiments. Following optimization of the system for performance and sensitivity, we evaluated the practical applicability of the technique using as a model system the in vitro reaction of N-acetoxy-N-acetyl-2-aminofluorene (AAAF, 1) with DNA EXPERIMENTAL SECTION
Chemicals and Reactions. DeozynucleosideAdduct Standard. N-Acetyl-N-(deoxyguanosin-8yl)-2-aminofluorene (CSAAFdG, 2 ) was prepared according to the method of Heflich et alez8by Mohamed Itani Pepartment of Medicinal Chemistry, Northeastem University, Boston, MA). The structures of all adducts are given in Figure 1. N-Acetyl-2aminofEuoreneAdduct of .?-Deoxyguanosine T-Monophosphate (CSAAF-dGMP, 3). In 1 mL of 10 mM pH 6 citrate buffer plus 250 p L of ethanol was dissolved 960 pg of 2'deoxyguanosine 5'-monophosphate (dGMP, Sigma, St. Louis, MO). To this solution was added a total of 3.5 mg of AAAF (-1 mg/100 pL in ethanol) in three aliquots. The progress of the reaction and the isolation were monitored using CZE (10 mM ammonium acetate, pH 9.4, UV detection at 260 nm, 75 cm x 75 pm column). Before each new addition of AAAF, an ether extraction was performed to remove any AAAF byproducts. When the productreactant peak area ratio had reached 20:l as monitored by CZE-UV, the reaction mixture was loaded onto a polystyrene divinylbenzene column (Bio-Beads SM-2 100-200 mesh packing, Bio-Rad, Richmond, CA) and washed with 1mL of water to remove salts and dGMP, and the product was eluted in 1 mL of 20% methanol plus 3 mL of 40%methanol. After drying and redissolution of the product (AAF-dGMP) in 10%methanol, the product concentration was determined by UV using CSAAF-dG standard solutions. The molar absorptivity of CSAAF-dGMP at 274 nm (A,& was taken to be the same as that of the deoxynucleoside analogue of the adduct. In Vitro DNAReaction.29 In 1.2 mL of 83% pH 6 10 mM citrate/ 17%EtOH was reacted 1.0 mg of calf thymus DNA (type I) with 1.0 mg (in 200 pL of EtOH) of N-acetoxy-N-acetyl-2-aminofluorene at 37 "C for 4 h, after which unreacted materials were removed by ether extraction. After precipitation with 20 p L of 5 M NaCl and 2 mL of ice-cold ethanol, the DNA was pelleted by centrifugation at l2000g and -10 "C for 20 min. Upon removal of the supernatant, the DNA was washed with ethanol and redissolved in 1 mL of pH 7.1, 5 mM Bis-Tris. Enzymatic cleavage to deoxynucleotide 5'-monophosphates was accomplished using 100 pg of DNase I (type II) and 0.1 unit of snake venom phosphodiesterase (type VII). CZE-W was employed to monitor (28) Heflich, R H.; Djuric, 2.; Zhuo, Z.; Fullerton, N. F.; Casciano, D. A; Beland, F. A Environ. Mol. Mutagen. 1988,11, 167-181. (29) Fu, P.P.;Miller, D. W.; Von Tungeln, L. S.; Bryant, M. S.; Lay, J. O., Jr.; Huang, K; Jones, L.; Evans, F. E. Carcinogenesis 1991,12, 609-616.
$=O I
-3
I I
1
L
................................
.....
..................'
'..':
U Figure 2. CZE-UV-MS setup employing the following components: 1, CF-FAB solution reservoir; 2, 30x magnifier; 3, CF-FAB capillary; 4, CF-FAB probe; 5, CZE cathode; 6, liquid junction interface; 7, UV detector; 8, CZE capillary; 9,anode buffer; 10, highvoltage power supply, and 11, switch box.
2 0
3
Q '0
for distilled deionized water, which was obtained from a Sybron/ Barnstead NanopureII/Organicpure system (Boston, MA) or a Milli-Q Plus apparatus (Millipore,Bedford, MA). Glycerol (99.9%) was purchased from J. T.Baker. HPLC grade ammonium acetate, ammonium hydroxide, and acetic acid were obtained from Fisher. Instrumentation. Capillary Electrophoresis. The capillary electrophoresis system used in the following studies consisted of a high-voltage power supply, an ultraviolet detector, and a switch box. A Series IvlJ high-voltage power supply was obtained from Glassman High Voltage (Whitehouse Station, NJ) and was tunable from 0 to 30 kV. The ultraviolet detector was an Isco V4 model (Lincoln, NE) outfitted for capillary on-line detection. Separations were carried out at 15-20 kV (10-15 pA) employing a 75 cm x 75 pm i.d. (375 pm 0.d.) polyimidecoated fused silica capillary obtained from Polymicro Technologies Phoenix, AZ). On-line detection was achieved by burning a -0.5 cm length of the polyimide coating, thereby creating a UV window, and inserting the capillary in line with the UV lamp. Unless otherwise noted, the capillary electrophoresis buffer consisted of a 10 mM solution of deliquescentammonium acetate in water adjusted with NHdOH to pH 9.4. Mass Spectrometry. Continuous flow fast atom bombardment mass spectrometrywas conducted on a VG Fisons Quattro triple quadrupole mass spectrometer (Altrincham, England), outfitted with a cesium ion gun. Full scan spectra were acquired over an appropriate mass range at a rate of 1s/scan, while constant neutral loss (CNL) spectra were acquired at scan rates of approximately 1 s/Am/z 150/scan. For the CNL and MRM analyses, a collision energy of 50-70 eV was employed, and argon was used as the collision gas. The collision gas pressure was adjusted to a level sufficient to attenuate the m/z 373 (aglycon) ion of an acetylaminofluorene-deoxyguanosine adduct standard to 30%. The dynamic FAB matrix consisted of 1%glycerol and 20-40% methanol in water. For deoxynucleotide analysis, the water was replaced by a 50-100 mM ammonium acetate solution. Stainless steel twilled Dutch weave wire cloth (mesh size 325 x 2300) was employed as the CF-FAB probe screen material (Small Parts, Miami, FL). CZE-MS System. A schematic of the CZE-CF-FAB-MS setup is shown in Figure 2. The design of the CZE-MS interface
3%py$ 4
Figure I.Structures of reactive metabolite and DNA adducts AAAF (l),C8-AAF-dG (2), C8-AAF-dGMP (3), and N2-AAF-dGMP(4).
the progress of the reaction. When the digestion was complete, an Amicon (Beverly, MA) MW 3000 cutoff centrifuge filter was employed to remove enzymes and undigested oligonucleotides. A 100 mg polystyrene divinylbenzene solid phase extraction cartridge was employed for adduct isolation from the DNA digest. After the DNA digest was loaded onto the column, fractions were eluted in MeOH/HzO solvents, as will be discussed later in the text. Each of the fractions obtained was dried under vacuum, and the contents were redissolved in 100 pL of HzO and subjected to CZE-W-MS analysis to determine the elution prolile of the AAFdGMP adducts. Calf thymus DNA, digestion enzymes, and buffer salts were obtained from S i a . Other. All solvents were HPLC grade and were purchased from J. T. Baker (Medford, MA) or Fisher (Medford, MA) except
Analytical Chemistiy, Vol. 67, No. 5, March I, I995
893
r CFFAB Solution Inlet- Teflon Tubing Plexigiasa Block
I CZE Capillary
CFFAB Capillary
i '
/
+ Prearure Releaas Channel Flgure 3. Liquid junction CZE-MS interface constructed out of Plexiglas.
employed was of the liquid junction type,30s31modfied for facile operation with ion stacking. The 17 x 25 mm (8 mm thick) Plexiglas interface is shown in Figure 3. The channels were l / in. ~ in diameter and allowed for a tight fit of the Teflon sleeves which held the capillaries and the cathode in place and permitted the introduction of the CF-FAB solution. One channel was left open to prevent pressure buildup within the interface. Approximately three-fourths the length of each of the l/16 in. 0.d. x 0.3 mm i.d. Teflon capillary sleeves (Rainin, Woburn, MA) was drilled out using a pin vise fitted with a 0.0145 in. (-368 pm) drill bit to allow introduction of the 375 pm capillaries. Once a capillary had been pushed through the undrilled portion of the Teflon sleeve, it was held within the sleeve in a hfitting which was tight enough to prevent any leakage but loose enough to allow for the movement of the capillary back and forth withii the sleeve. This allowed for the capillary positions to be changed quickly and easily and, as discussed below, was especially convenient for stacking work. These sleeves also provided for the facile alignment of the two capillaries, whose ends were squarecut using a diamond point cutting tool. The cathode was located downstream of the liquid junction such that bubbles formed from gaseous electrolysis products would not interfere with the analysis. The CF-FAB and CZE buffer solutions were degassed by sonication under vacuum and were filtered daily using a Luerlok 10 mL R D syringe fitted with a 0.2 pm Gelman Acrodisc CR disposable filter cartridge from Fisher. An additional filter cut from the twilled Dutch weave stainless steel wire cloth was included in the bottom of the CF-FAB syringe as an added precaution against the introduction of particulates into the interface. The interface was incorporated into the system shown in Figure 2. The interface block and the anode buffer were clamped in place such that their liquid levels were at the same height. The CFFAB reservoir was mounted at 1-2 cm above the height of the interface to ensure that the CF-FAl3 solution was continually provided to the interface. In most studies, on-line ultraviolet detection was conducted at a distance of 15-20 cm from the cathode end of the capillary (total capillary length was typically 75 cm). Injections were made by hydrodynamically siphoning (30) Caprioli, R M.; Moore, W. T.; Martin, M.; DaGue, B. B.; Wilson, IC;Moring, S. J. Chromatogr. 1989,480,247-257. (31)Reinhoud, N.J.; Niessen, W. M. A; 'Tjaden, U. R; Gramberg, L. G.;Verheij, E. R; van der Greef, J. Rapid Commun. Mass Spectrom. 1989,3,348-351. 894
Analytical Chemistry, Vol. 67,No. 5,March 1, 1995
sample solutions into the capillary. Typical conditions involved raising the sample reservoir to 3 cm above the height of the anode buffer for 10 s. For a 75 cm x 75 pm capillary, this resulted in the introduction of 3.5 nL of sample solution. The amount injected was determined by elevating a solution of deoxyguanosinein water to a height of 3 cm and monitoring the W response to determine the amount of time required for this solution to travel a known length from the anode buffer to the detector cell. The volumes of solution then introduced under various other conditions were determined using Poiseuille's Law (V/t = d ( P 1 - Pz)/8q(YzYi)) and P = Qgh. A Micronta 30x lighted magnifer (Radio Shack) clamped over the interface and a small piece of white paper taped to the underside of the interface assisted in the viewing of capillary positions. Typically, a -20 pm distance was employed between the outlet of the CZE capillary and the inlet of the CF-FAB capillary. A 40 cm x 50 pm i.d. capillary was used to transfer materials eluting from the CZE capillary along with a makeup flow of CF-FAB matrix solution into the CF-FAB probe. Vacuum induced flow through this capillary provided a -2-2.5 pL/min flow of solution from the interface into the mass spectrometer. A source temperature of 30 "C was employed to maintain a steady volatilization of matrix components in the ion source. It was observed that special care was required to prevent excessive peak tailing at the continuous flow FAB probe tip. For instance, the meshes used in the tip (-1 mm diameter) had to be hand-cut from a piece of wire mesh to provide the snug fit required at the tip for low dead volume applications. Also, special care had to be taken to ensure that wicking materials at the tip were in close contact with the probe tip screen and that the capillary was firmly pressed against the screen. RESULTS AND DISCUSSION Performance of the CZE-MS System. Transfer efficiency
through the interface was evaluated using a solution of methylene blue pumped through the CZE capillary into the interface at 500 nWmin by a syringe pump. This flow rate was roughly that generated by the electroosmotic flow induced across the CZE capillary under the 10 mM ammonium acetate, pH 9.4, conditions typically employed. It was visually observed that, under these conditions, the vacuum-induced flow rate of -2-2.5 pL/min generated through the CF-FAB column permitted the complete transfer of the dye solution from the interface even at capillary separations well in excess of 100 pm. As expected, the peaks detected in the mass spectrometer were significantly broader than those observed by on-line ultraviolet detection before the interface. The contributionsto the observed peak variance can be summarized in the equation uZpe*= d P r e - ~ uZWst.w, where u z p r eincludes -~ contributions to peak variance between the injection point and the W detector and u~,,~.w includes all contributions to peak variance arising between the W detection point and the tip of the CF-FAB probe. The magnitude of U~W , is readily observed in the CZE-W profile. Since it was impractical to incorporate a detector between the CZE-W detector and the CF-FAB probe tip, contributions to broadening arising after the W detector could not be differentiated. It is clear, however, that given the observed loss in efficiency (N) of the CZE peaks, most of the broadening could be attributed to post-CZE column factors involving the interface, CF-FAB transfer line, and CF-FAB probe tip. Under optimized interface and probe tip conditions, values for aWst.wwere determined to
+
110 100
j "1
373
El
60
1'
Figure 5. Positive ion FAB spectrum from the flow injection of 60 ng of C8-AAF-dGMP. CF-FAB solution: 1% glycerol, 24% methanol, and 75% 50 mM ammonium acetate.
FI
115 110 105 100 1s
M 85 M 7s
Figure 4. CZE traces obtained for a 3.5 nL, 1.O ng injection from a standard solution of C8-AAF-dG. CZE conditions: 10 mM ammonium acetate adjusted to pH 9.4 with ammonium hydroxide, 15 kV, 10 PA, UV detection at 274 nm. (a) CZE-UV; (b) CZE-MS.
be on the order of -2.2-2.7 s. Figure 4 demonstrates the typical broadening associated with use of the interface for an average injection of 3.5 nL (1ng) of a standard C8AAFdG solution. Under these particular conditions, there is a %foldloss in efficiency (NMS has been calculated for the estimated time of elution of the peak from the CZE column so as not to give an inflated estimation of efficiency). It should be noted that while excessive broadening of the CZE peaks was considered unacceptable, a small amount of broadening was actually necessary so that a reasonable number of mass spectral scans could be collected for each eluting peak, especially in full scanning normal and MS/MS modes. Deoxynucleotide Adduct Detection. As stated in the introduction, ion stacking techniques are expected to work only with negatively charged species under typical CZE conditions. For DNA adducts, this means that use of the nucleotide forms was warranted. However, given that almost all of our prior work using CF-FAB-MS had been conducted with deoxynucleoside add u c t ~ ,the ~ ~mass , ~ ~spectral behavior of the nucleotide analogues had to be examined first. A reference sample of the standard C8AAF-dGMP (3) was used to assess the detectability of nucleotide adducts. It was observed that substitution of 250 mM ammonium acetate for water in typical CF-FAB solutions (1% glycerol and 20-40% methanol) was required to effect a detedability similar to that of the nucleoside analogue in positive ion CF-FAB. Figure 5 shows the positive ion FAB spectrum of C g AAFdGMP generated by flow injection of a 60 ng sample. Other than the shift of the protonated molecule peak from m/z 489 to 569 (m/z 591 is derived from [M Na]+), the spectrum of the nucleotide resembles substantially that of the nucleoside both quantitatively and qualitatively. In particular, the spectrum is
+
(32) Wolf, S. M.; Vouros, P.Chem. Res. Toxicol. 1994, 7, 82-88
dominated by the ion at m/z 373, formed by cleavage of the glycosidic bond, a process typical of deoxynucleotides and their adducts. This fragmentation provides a useful diagnostic process for constant neutral loss (CNL) and multiple reaction monitoring (MRM) for selective detection of DNA a d d ~ c t s . ~ ~ - ~ ~ Mechanics of Stacking with Interface. As indicated in the introduction, the stacking process requires a step in which the sample solvent is pumped out of the CZE capillary under the influence of a reversed high voltage. During this time, buffer solution flows from what would ordinarily be the cathode reservoir into the CZE column. In the typical CZE-CF-FAB-MS setup, the cathode solution has been replaced by the CF-FAB solution in the interface. Adaptation of the stacking technique to CZEMS analysis requires the inclusion of a true cathode buffer reservoir in the instrumental setup and the employment of offline stacking prior to on-line CB-MS analysis, as shown in Figure 6. In step a, the sample solution in water was hydrodynamically introduced into the anode end of the CZE capillary. Subsequently, in step b, a high voltage opposite in polarity to that normally employed was applied to cause stacking of sample in the CZE capillary until the current was 295%that of the normal operating value. Finally, the cathode end of the CZE capillary was carefully transferred to the liquid junction interface and the electrodes reversed back to their normal configuration as shown in step c. Upon activation of the high voltage, CZE-MS was conducted as usual. No deleteriouseffects were noted as a result of the manual manipulation of CZE capillary or time delay (typically less than -30 s) required for step c. In addition, the ability to perform this type of analysis demonstrates the convenience of using the Teflon sleeve interface design. Since alignment of the capillaries is automatic, only their proximity needed adjustment, and, with practice, this could be accomplished in a few seconds. Stacking-CZE-CF-FAB-MS with AAF-dGMP Standard. Efficient stacking required that the anode end of the CZE capillary be rinsed carefully with water prior to each sample injection to prevent CZE buffer ions from contaminating sample solutions and thus degrading the stacking process. Typically, this was accomplished by dipping the CZE capillary into a vial filled with distilled deionized water, swirling the solution, and then wiping the capillary briefly with a Kimwipe before injection. It was determined that a 3.5 nL injection through the CZE-CF-FABMS system of a 5 x M C8AAF-dGMP solution (or 1.0 ng) provided a good quality normal full scan signal under the (33) Bryant, M. S.; Lay, J. O., Jr.; Chiarelli, M. P. J. Am. SOC.Muss.Spectrom. 1992, 3, 360-371. (34) Nelson, C. C.; McCloskey, J. A Proceedings of the 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA June 1988; pp 145-146.
Analytical Chemistry, Vol. 67, No. 5,March 1, 1995
895
10
a) Injection Sample VIal
.......................................... CZE CapIHw : *
Anod. Buffer
50
30
40
20
10
30 Tj
0
9:OO
Power
j supply 10
70
::j
U
F
e
40
Cathode Buffer
n-
313 I
50 40
30
Interlace
20
CFFAB Solutlon
30 20 10 0
CFFAB PROBE 373
b) Stacking ......................................... 50 40
30
i-
20
30 20
LO 0
Figure 7. Sampling of AAF-dGMP at three different solution concentrations. The analyte profiles are shown as ion electropherograms of m/z373 on the left, while full scan spectra obtained in each case are shown on the right. CZE conditions: 10 mM ammonium acetate adjusted to pH 9.4 with NHIOH, 20 kV, 14 PA. CF-FAB solution: 1% glycerol, 24% MeOH, 75% 100 mM ammonium acetate.
U
Cahode Buffer
Table 1. Stacking Efficiency for C8.AAF-dQMP Solutlon
CFFAB Capillary
{
1
amount injected (nW
I
CFFAB PROBE
3.5 (5 10-4 M) 35 (5 10-5 M) 350 (5 x M)
c) Analysis
65.0 (3.0) 65.4 (2.9) 68.5 (1.8)
recovery (%) 101 105
Each volume injected at the concentration given in parentheses number given in corres onds to 1.0 ng of C8-AAF-dGMP. ”e paren&eses is the percent relative standard deviation.
CZE Caplllay
Anod. Buffer
+
AAF-dGMP, resulted in spectra of equivalent quality. Hence, as shown in Figure 7, the stacking method lowered the concentration detection limit by 2 orders of magnitude for this analyte. CZEW and MS peak areas obtained from the stacked samples compared well with those obtained from the “standard” 5 x 10-4 M solution, as shown in Table 1. Figure 8 shows the MRM analysis conducted to determine the concentration sensitivity obtainable for known or suspected adducts. In this case, the loss of deoxyribose 5’-monophosphate (m/z 569 373) was monitored. Again, stacking volumes of up to 350 nL were accommodated. Given the mass detection limit of -25 pg, solutions as dilute as 1.25 x M could be sampled. The MS peak area for these data was found to vary linearly with the absolute amount of adduct sampled (r > 0.99). Additionally, it was found that even full-column stacking conditions provided a high recovery of this analyte. For example, when stacking was performed on a CZE column filled with 3.3 pL of a 6.3 x M solution of CSAAF-dGMP, recoveries in excess of 50%of the injected amount were realized. However, in practice, stacking
M;............................................................................................
IntWiK.
Salutlon CFFAB
CZE-UV peak ar;a (arb units, n = 3)
CFFAB Capillmy
CFFAB PROBE
Figure 6. Utilizationof the large volume sample stacking technique involving (a) hydrodynamic sample injection of the desired sample volume (HV off), (b) application of a high voltage opposite in polarity to that normally employedfor analysis in order to effect off-line sample stacking, and (c) transfer of the CZE capillary from the cathode buffer to the liquid junction interface for CZE-UV-MS analysis.
conditions employed. Using the stacking technique, it was found that a 35 nL injection of a 5 x M solution and a 350 nL injection of a 5 x M solution, both providing 1.0 ng of C S 896 Analytical Chemistfy, Vol. 67, No. 5,March 7, 1995
7.n
7.
a
1.25 X 1 0 7 M 350 nL
6.Ap A
25 PS
Figure 8. Stacking of picogram amounts of C8-AAF-dGMP from dilute solutions with MRM detection.
under these conditions was found to give irreproducible recoveries and frequently resulted in peak broadening. It was observed that worse results were obtained for solutions that had been sampled multiple times and that good performance was restored for freshly diluted sample solutions. Therefore, poor results were likely due to an increase in sample solution conductivity through contamination by the CZE buffer during injection. It has been proposed that, assuming that yx >> 1 and that the electroosmotic flow during stacking is equivalent to that generated
over the background electrolyte alone, the maximum length of CZE column, x, which can be filled with analyte solution and stacked without analyte loss can be estimated as -pj/peo, where y is the ratio of the background electrolyte to analyte solution conductivities and l i and peoare the analyte and electroosmotic mobilities, respectively.26 In more general terms, the limitations of stacking will be d e h e d by the ratio of analyte mobility to bulk electroosmotic flow during the stacking step, which in turn depends upon a variety of factors, including analyte and background electrolyte solution pH and conductivity. It should be possible to optimize the stacking efficiency for a given analyte by either increasingthat analyte's negative mobility (e.g., if possible, by increasing the pH of the analyte solution) or decreasing the electroosmotic flow during the stacking step (e.g., through the use of coated capillaries). The extent to which these principles can be applied to a given system will determine the usefulness of the stacking technique for analytes other than those considered here. Analysis of Deoxynucleotide Adducts Generated from an in Vitro DNA Reaction. Having established the utility of the stacking-CZE-CF-FAB-MS technique for analysis of a representative standard, the application of this methodology to the determination of adducts generated from an in vitro DNA reaction mixture was considered next. Specifically, the reaction of AAAF with calf thymus DNA was examined. This is a well-documented reaction which is known to yield two adducts, C8AAF-dGMP (3) and, to a lesser extent the isomeric adduct, N2-AAF-dGMP(4).35 It was evident that two closely related criteria had to be met in order for stacking methods to be used for this application: (a) adduct isolates had to be relatively free of interferences (especially
CE-CFFABMS MRM 564 --> 373
CE.UV (274 nm)
L Fraclon #7
FracIbn LB
Fnclm L5
/ Figure 9. MRM monitoring for the establishment of the SPE elution profile for AAF-dGMP adducts from the DNA digestion mixture. Elution solvents (all 1.OO mL): fraction 1 (unretained materials), fractions 2 and 3 (100% HzO), fractions 4 and 5 (loo/, methanol), fractions 6 and 7 (50% methanol), fractions 8 and 9 (100% methanol). CZE: 10 mM ammonium acetate, pH 9.4, 20 kV, and 14 PA; UV at 274 nm. CF-FAB: 1% glycerol, 39% methanol, 60% 100 m M ammonium acetate. Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
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2:OO
I ::! I
6:00
4:OO
8:OO
1O:OO
12:OO T I K C
569
E 0
2.00
4.00
6.00
8.00
10.00
12.00
TIME (minuter)
Figure 10. Comparison of CZE-UV (274 nm) profiles for the injection of (a) 7 nL and (b) 175 nL of fraction 6 from Figure 9.
isobaric adducts) with similar CZE migration times and (b) adducts had to be isolated in a lowconductivity matrix. The first requirement would apply even if stacking methods were not to be employed, as CZE suffers from a limited mass loadability, which results in broadening for overloaded zones. To meet the second criterion, the adducts had to be separated from such ionic species as buffer salts, normal deoxynucleotides, and enzymes present in a typical DNA digest. As these are essentially sample preparation considerations, the application of solid phase extraction techniques to accomplish the desired isolation was attempted. It was found that C8-AAF-dGMPwas retained on polystyrene divinylbenzene supports in water and 10% methanol, solvents which provided for the facile elimination of unadducted monodeoxynucleotides and buffer salts from the SPE column. Neverthe less, concentrated adduct fractions eluted in higher percent methanol solvents were invariably contaminated with residual undigested deoxyoligonucleotides and apparently nuclease-related breakdown products. It should be noted that the enzymatic degradation procedure employed here is typical of those used for amino-PAH adducts, in that the buffer pH of 7.1 was lower than that which would provide maximum enzyme activity in order to prevent the adduct degradation observed to occur for these analytes in alkaline media.36 Therefore, these conditions, including the long digestion times (> 20 h) , may have been especially likely to result in the presence of the aforementioned contaminants. It was found that filtration of the DNA digest solution using a MW 3000 cutoff centrifuge filter prior to solid phase extraction (35) Kriek, E. Cancer Lett. 1979,7, 141-146. (36) Shibutani, S.; Gentles, R G.; Iden, C. R; Johnson, F.].Am. Chem. SOC.1990, 112, 5667-5668.
898 Analflical Chemistry, Vol. 67, No. 5, March 1, 7995
sbo
ibo
do
960
M/IZ
Figure 11. (a) Mass electropherogram of the m/z373 ion from the CZE-CF-FAB-MS analysis of a 175 nL stacking injection of fraction 6 in Figure 9. (b) Full scan FAB mass spectrum of the C8-AAF-dGMP adduct. (c) Full scan FAB spectrum of unknown interference.
provided a substantial cleanup of adduct fractions and made stacking analysis possible. Given the low mass spectral detection limits achievable for the AAF-dGMP adducts, CZE-CF-FAB-MS of the individual SPE fractions provided a very useful method for establishing the elution profile of these compounds. The utility of this approach is demonstrated in Figure 9 for a typical adduct reaction mixture, along with the SPE conditions employed to elute the adducts. While UV detection gave an indication of the progress of the isolation, it was the selectivity of the mass spectral technique which allowed the adduct-containing fractions to be definitively targeted. Since the adduct identities were known in this case, normal hydrodynamic injection of small solution volumes with MRM detection (MH+ AH2+) was employed. If the adducts were of an unknown structure, stacking injections with constant neutral loss scanning (loss of dFUvlP) could be employed to identify adduct-containing fractions. As is evident in Figure 9, AAF-dGMP adducts were determined to be present in SPE fractions 6-8. In order to acquire more detailed mass spectral data, 175 nL stacking injections of fraction 6 were made. Figure 10 shows the profile of the UV electropherogram for fraction 6 under normal and stacking injection conditions. It was estimated that for the 175 nL stacking injections, approximately 7.5 ng of the C8-AAF-dGMP adduct was sampled. The peak broadening observed with stacking was predominantly due to overloading of the CZE column and was not an artifact of the stacking process. The mass electropherogramof the characteristic m/z 373 ion (aglycon ion) from the CZE-MS of the 175 nL stacking injection
-
interference whose molecular weight could not be ascertained from its full scan mass spectrum (Figure llc). Collision-induced dissociation (CID) of the m/z 373 ion was carried out to resolve any uncertainty regarding the identities of the species shown in the electropherogram of Figure lla. As shown in F i r e 12a and b, CID data collected for the m/z 373 ion were identical for C8-AAF-dGMP and the interference, indicating the presence of the C8AAF-guanine moiety in the interference and suggesting its origin as an undigested oligonucleotide. Under these conditions,with careful peak subtraction, a weak spectrum matching that reported previously for the N2AAF-dGMP adduct could be obtained (Figure 12c). The features of these spectra were discussed r e ~ e n t l y . ~Confirmation *,~~ of the identity of the interference was obtained by conducting a precursor ion scan for m/z 373, the results of which are shown in Figure 13. The first peak in the precursor ion electropherogram,Figure 13a, corresponds to that of C8AAF-dGMP and shows prominent [M HI+ and [M NHJ+ signals at m/z 569 and 586, respectively (Figure 13b). The signals at m/z 873 and 882 (Figure 13c) arising from the summation of scans over the interference peak (second peak in the electropherogram of Figure 13a) were almost certainly due to the presence of AAF adducts of the dideoxynucleotides GT (or T-G) and GA (or A-G), respectively. The peak at m/z 569 corresponds to the [M HI+ ion of the N2-AAFadduct; although based upon the observed fragmentation of the dinucleoside monophosphates?* it is possible that a fragmentation of the putative dinucleotides could also potentially give rise to an ion of the same mass. At this time, the origin of the signal at m/z 936 is not known. The relative areas and widths of the C8AAF-dGMP and dinucleotide W peaks indicate that similar quantities of the compounds were injected with each run, implying that low nanogram detectability of the putative dinucleotide adducts has been accomplished. While the above account discusses the application of the stacking-CZE-CF-FAl3-MS method to a particular DNAmutagen reaction mixture, the capabilities demonstrated and the challenges faced are expected to be general features of this sort
+
2p6
It
+
+
Figure 12. Collision-induceddissociation spectra of m/z 373 from (a) C8-AAF-dGMP, (b) the unknown interference, and (c) Nz-AAFdGMP.
is shown in Figure Ha. Constant neutral loss scanning (data not shown) of 196 Da (loss of deoxyribose monophosphate) resulted in the confirmation of the presence of the two expected deoxyguanosine monophosphate adducts, C8AAF-dGMP and N2-AAFdGMP, eluting at the positions indicated in Figure lla. Whereas a good quality full scan spectrum could be obtained for C8AAFdGMP (Figure llb), the signals arising from the expected NzAAF-dGMP adduct were obscured by a coeluting adduct-related
Figure 13. (a) Profile of the total ion electropherogram of the precursor ions of m/z 373 obtained over the course of the CZE-CF-FAB-MS analysis of fraction 6. (b) The precursor spectrum of mfz 373 obtained for the C8-AAF-dGMP peak (m/z 569 corresponds to [M H]+ and m/z 596 corresponds to [M NH4]+). (c) The precursor spectrum of m/z 373 obtained for the “interference”(m/z 569 corresponds to the N2-AAFdGMP adduct, while the proposed origins of the m/z 873 and 882 are AAF-dGMP adducts of the dinucleotides G-T (or T-G) and G-A (or A-G), respectively. The ion of m/z 936 is unidentified.
+
+
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of application. For instance, the presence of undigested dinucleotide adducts in adduct isolates is neither atypical nor unexpected and has frequently complicated the use of other less selective detection systems for DNA a d d u ~ t s . 3As ~ ~demonstrated ~ above, the stacking-CZE-MS technique could provide for a very rapid assessment of the content and quality of enzyme digests of DNA adducts. Such an approach should substantially decrease the amount of time required for the development of adduct isolation procedures.
CF-FAB has been demonstrated. It is anticipated that the approach described above will be applicable to a wide variety of PAH-DNA adducts and should be amenable to the characterization of in vitro samples if the compounds of interest can be isolated in a lowconductivity matrix. Moreover, the same methodology can be equally as readily incorporated into CZE-MS applications utilizing electrospray ionization, as discussed in a recent presentati~n.~~ ACKNOWLEDGMENT
CONCLUSIONS
In summary, the above studies illustrate the feasibility of interfacing large volume sample stacking techniques to CZECF-FAB-MS. In favorable cases, analyte concentration detection limits can be lowered by up to 3 orders of magnitude. Such a technique makes CZE-MS more competitive with LC-MS for trace detection and permits the analyst to take advantage of the fast analysis time of CZE. In addition, the mass spectrometric detection/characterization of low picomole to low femtomole amounts of PAH-DNA adducts in the deoxynucleotide form by (37)Chiarelli, M.P.;Lay, J. O., Jr.1. Am. SOC.Mass.Spectrom. 1994,5,58-63. (38) Phillips, D.R; McCloskey, J. A Int.1. Mass. Spectrom. Ion Processes 1993, 128,61-82. (39)Gupta, R C.Proc. Natl. Acad. Sci. USA. 1984.81,6943-6947. (40)Cheh, A M.; Yogi,H.; Jerina, D. M. Chem. Res. Toxicol. 1990,3,545-550. (41)Bany, J.; Norwood, C.; Vouros, P. F’roceedmgs of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago,IL, June 1994 p 117.
900 Analytical Chemistry, Vol. 67, No. 5, March 1, 7995
This work was supported by a grant from the US. Environmental Protection Agency (Grant No. 820113-01-0). We are indebted to Dr. Frederick Beland of the National Center for ToxicologicalResearch Uefferson, AEQ for his generous donation of the N-acetoxy-N-acetyl-2-aminofluorene and for helpful advice and to Curt Norwood and Eugene Jackim of the USEPA (Narragansett, RI) for their loan of CZE system components. We also gratefully acknowledge support from the NIH through an instrumentation grant (ISIORRO5602-01). This is contribution no. 626 from the Barnett Institute. Received for review July 20, 1994. Accepted December 19, 1994.8 AC9407330 Abstract published in Advance ACS Abstracts, January 15, 1995.
