Detection of DNA Adducts by Electron Capture Mass Spectrometry

MARCH 1997 VOLUME 10, NUMBER 3 © Copyright 1997 by the American Chemical Society

Invited Review Detection of DNA Adducts by Electron Capture Mass Spectrometry Roger W. Giese Department of Pharmaceutical Sciences in the Bouve´ College of Pharmacy and Health Professions, Barnett Institute, and Chemistry Department, Northeastern University, Boston, Massachusetts 02115 Received July 29, 1996

Introduction Electron capture mass spectrometry (EC-MS)1 for the detection of DNA adducts (covalent damage to DNA) has reached its “teenage years” and is growing in interest, so it is a good time to review it. The extremely high sensitivity of EC-MS explains why this form of MS often is favored over others for measuring the very tiny amounts of DNA adducts found in typical biological samples. Sample preparation for the detection of DNA adducts by EC-MS is the focus of this review. The main intent is to glean both guidelines and details from the literature to help the interested reader either set up or improve an EC-MS method for a DNA adduct. Also included is some background for those less familiar with the technique. After we discuss some general concepts, we will consider the more interesting details of the individual methods. At the end of the review are some thoughts about the future.

Related Reviews Other reviews have appeared which are related, more or less, to this one. Fedtke and Swenberg contributed a 1Abbreviations: BTFMBz, 3,5-bis(trifluoromethyl)benzyl; EC-MS, electron capture mass spectrometry; ECD, electron capture detection; HFBA, heptafluorobutyric anhydride; NNK, 4-(methylnitrosoamino)1-(3-pyridyl)-1-butanone; NNN, N1-nitrosonornicotine; PFB, pentafluorobenzoyl; PFBz, pentafluorobenzyl; PFBzal, pentafluorobenzaldehyde; PFP, pentafluoropropionyl; PFPA, pentafluoropropionic anhydride; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SPE, solid phase extraction.

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short review of MS techniques for DNA adducts, with an emphasis on EC-MS detection, especially in regard to their method for N2,3-ethenoguanine (1). Included in this work was a summary of earlier, more general reviews on the detection, by all types of MS, of DNA adducts as well as the normal constituents of DNA. Similarly, Giese et al. (2) emphasized their own work in a short review on the same general subject but with more attention to method development and instrumental aspects of ECMS. Detection of DNA adducts by all types of mass spectrometry has been reviewed recently (3-5). Similarly, another recent review has covered the detection of both DNA and protein adducts by general mass spectrometry (6).

Other Ways To Measure DNA Adducts This subject has been reviewed elsewhere (7-9). The other major techniques involve 32P-postlabeling, fluorescence, immunoassays, electrochemistry, and other forms of mass spectrometry. We can expect more blending of these methods, including EC-MS, in the future. Each technique has certain strengths as well as weaknesses, and the field of DNA adducts will progress most rapidly if we continue to take advantage of all of them.

The Meaning of DNA Adducts While the focus of this review is the measurement rather than the meaning (interpretation, significance) of © 1997 American Chemical Society

256 Chem. Res. Toxicol., Vol. 10, No. 3, 1997

DNA adducts, a brief comment on this “other half” of the story should be made. The general idea of DNA adducts is that they indicate a risk to health due to their relationship to mutations and cancer (10, 11). Many variables are involved related to differences in individual exposure, lifestyle, and metabolism, along with adduct details concerning dose, structure, and repair. Both natural and unnatural chemicals become attached to DNA, or unnatural DNA bonds form. Whatever detailed meaning the adducts have, it will be obscured unless we measure them accurately and precisely. Further, the full meaning of DNA adducts may become clear only after we are also able to measure them more comprehensively. Currently, just one type or class is measured in nearly all samples, and never has a complete picture been provided of all the DNA adducts in a given sample. Both a detailed and comprehensive analysis of DNA adducts may be necessary to fully learn their meaning. This is important not just for reducing the current burden of human cancer and other genetic diseases, but helps to determine the DNA legacy that we leave for future generations.

Slow Growth of EC-MS for Detecting DNA Adducts The growth of EC-MS for the detection of DNA adducts has been painfully slow for two general reasons, which vary in their importance in different laboratories. The first one is the equipment, which is expensive, requires a skilled operator, and provides a low sample throughput. Further, a typical mass spectrometer is used for different types of projects, some of which may involve samples that contaminate the instrument and thereby reduce its sensitivity for trace analysis. The ion source of such an instrument then needs to be cleaned more frequently. Thus, it can be important to set up a dedicated instrument, and ideally a spare ion source as well. Second, trace sample preparation in this area for ECMS has largely been a new frontier, and continues to be so, as we struggle especially with trace derivatization and purification of tiny amounts of analyte and encounter analyte losses, background interferences, and contamination. The latter is the presence of aberrant analyte in a blank sample and is a frequent problem in trace analysis, more than gets reported in the literature. This difficulty arises, depending on the analyte, either from environmental origins (including handling in the same laboratory of a larger quantity of the analyte) or from carryover of analyte in re-used components of the method from one sample to another. Extreme trace analysis, as required for the detection of DNA adducts, is difficult no matter what the analyte or technique, and this will always be true. Both the sample handling and equipment need to be pushed to their limit and maintained there.

Electrophores Electrophores in practice are compounds that are detected readily by EC-MS or electron capture detection (ECD), two gas-phase detection techniques. (Another common name for EC-MS is electron capture negative ion chemical ionization mass spectrometry.) Usually the compound is delivered to one of these detectors as a peak eluting from a gas chromatography column, but a direct insertion probe (12) and a liquid chromatography belt interface (13) to EC-MS also have been employed. In any

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case, it has been necessary for the electrophore to be volatile and thermally stable. With an ECD a gas phase current of electrons is monitored, and the electrophore reduces the current by capturing one of the electrons. Similar electron capture takes place in the EC-MS, but under partial vacuum conditions, and the ion(s) formed from the analyte are measured instead. For standards, the two detectors are similarly sensitive, but the greater specificity of the ECMS makes it considerably more powerful for “real” (e.g., biological) samples. Aside from special analytes that are unusually volatile (14) or nonvolatile (15), it is difficult to reach the low picogram level by GC-ECD when real samples requiring electrophore derivatization are tested. Largely this is because electrophoric contaminants are present or formed in the reagents and solvents during sample preparation (16). While some standards of derivatized DNA adducts have been detected by GC-ECD (see Table 1, which will be discussed later), only GC-EC-MS has been useful for measuring DNA adducts derived from biological samples. The measurement of 5-methylcytosine in DNA by GCECD (17) is not an exception, since this naturallyoccurring, minor nucleobase is not a DNA adduct and is present in DNA at a high concentration (about 1 in every 100 cytosines in human DNA) relative to the dose of DNA adducts generally encountered in “natural” biological samples (1 adduct in g106 normal nucleobases).

Conversion of DNA Adducts into Electrophores DNA adducts inherently are not electrophores for detection by GC-EC-MS, lacking in both volatility and thermal stability, so that one or more chemical reaction steps are necessary as part of sample preparation to make this conversion. Two kinds of electrophoric products have been formed: intact and released. The intact product is preferred since, by definition, it retains a component (e.g., the nucleobase) of the DNA where the damage took place, and thereby provides a more informative measurement. However, some adducts are too large or complex to be converted intact into an electrophore for detection by GC-EC-MS, and it is necessary or easier to release the extrinsic chemical from the DNA for this purpose. There may be an advantage as well to such released-adduct detection: an entire class of adducts might be detected in a single procedure, since the chemical properties of the released-chemicals may be more uniform than those of the parent adducts.

Electrophore Subtypes There are subtypes of electrophores in two respects: strength and fragmentation. While the basic idea is that an electrophore gives a strong response by EC-MS, sometimes it is useful to distinguish strong, medium, and weak electrophores, where the relative responses are in the range of 1:0.1:0.01, respectively. Some of the extraneous (background) peaks that are commonly encountered in EC-MS (and ECD) are probably due to the presence of weak- or medium-strength electrophores like plasticizers, nitroaromatics, and chlorinated compounds which are ubiquitous. It is important to convert a DNA adduct into a strong electrophore to maximize the sensitivity. In practical terms this means obtaining an electrophoric product

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Chem. Res. Toxicol., Vol. 10, No. 3, 1997 257

Figure 1. Structures of electrophoric derivatizing agents.

which gives a response similar to that of a known, strong electrophore. It must be kept in mind that the overall electrophore response in the GC-EC-MS reflects the recovery of the compound from the GC part into the EC ion source, the inherent EC properties of the compound, other events (e.g., wall effects; 18) in the ion source and the transmission efficiency (including the lifetime) of the electrophore anion to the detector. A strong electrophore can be detected as a pure standard at the low attomole level on a modern instrument (19). Throughout this review, reflecting common practice, the term “electrophore” will imply a strong electrophore, unless indicated otherwise. Once an electrophore captures an electron and becomes an anion radical, it may persist in this initial form long enough to reach the detector, or it may fragment first. Typically, a DNA adduct is converted into an electrophore that is detected either as a parent anion radical (nondissociative electron capture), or as an exclusive anion fragment which forms along with a corresponding neutral, thereby nondetected radical species (dissociative electron capture). Either way, the electrophore typically selected forms a single ion (aside from the associated isotope peak), which maximizes the sensitivity. Ideally, however, the electrophore-labeled DNA adduct would give two or three anion fragments in similar amounts, and their ratio would be checked for each sample as an extra guard against interferences. This would be worth the slightly higher detection limit.

(PFBzBr), introduced for nucleobase detection by Kok et al. (22), has been used the most, especially for the labeling of intact, small nucleobase adducts like O6methylguanine (e.g.: 23). 4-(Trifluoromethyl)tetrafluorobenzyl bromide (TFMTFBzBr) was introduced as an analogue of PFBzBr for two reasons (24). The first was to reduce derivatization side products, since such products might arise with PFBzBr due to the lability of the p-fluorine atom on PFBz (25). The second motivation for introducing this reagent was to form an electrophoric product of a DNA adduct with a different retention time by GC than that of a PFBz product. A change in the retention time of an electrophore is one way to overcome an interfering peak. 3,5-Bis(trifluoromethyl)benzyl bromide (BTFMBzBr) is another substitute for PFBzBr (26). Limited data is available, but a BTFMBz derivative may be somewhat more sensitive by GC-EC-MS than a corresponding PFBz derivative in some cases. A higher retention time can be expected on reversed-phase HPLC for a BTFMBz as opposed to a PFBz derivative, since the former group is more hydrophobic. One might select BTFMBzBr first when it is applicable, but not hesitate to switch to PFBzBr or TFMTFBzBr in order to overcome an interfering peak in the GC-EC-MS. If the interfering peak shifts as well to the new retention time, contamination by analyte in the method should be suspected. Pentafluoropropionic anhydride (PFPA) has been used to acylate 4-aminobiphenyl as a released chemical from DNA (21), and both heptafluorobutyric anhydride (HFBA) and pentafluorobenzoyl chloride (PFBCl) have been used, although not applied to a DNA sample, for the similarlymotivated detection of 1-aminopyrene and 2-aminofluorene (20). While the latter workers reacted the HFBA derivatives further with PFBzBr (replacing the residual amide hydrogen with a PFBz group), the product formed for detection by the other group (21) contained a single PFP group and a residual active hydrogen. Replacement of residual active hydrogens is discussed in more detail below. Pentafluorobenzaldehyde (PFBzal) provides a second, potentially more selective option for electrophore-labeling an aminoaromatic compound as a released DNA adduct (20), forming a pentafluorobenzylidine derivative. The authors concluded that this was the most attractive derivative of those tested as standards, with the HFBPFBz derivative as a close second choice. However, based on the data available currently, the above method with PFPA (21) is a safer choice, since it was shown to be successful when applied to a trace analyte derived from DNA (the other strategies were just tested on standards) and has the advantage of comprising a single-step derivatization procedure with a relatively volatile electrophoric reagent to make sample preparation convenient.

Residual Active Hydrogens Electrophore Labels Typically, the intact DNA adduct or released chemical is rendered electrophoric by substituting one or more of its active hydrogens with a polyfluorobenzyl or polyfluoroacyl electrophore label. The resulting derivatives tend to undergo dissociative and nondissociative electron capture, respectively, although HF loss can take place in the latter case (20, 21). Figure 1 shows representative electrophoric reagents. Pentafluorobenzyl bromide

It is not always essential to remove all of the “active hydrogens” (an oversimplified concept, since the pKa’s of NH and OH groups cover a broad range) when an analyte is derivatized for detection by GC-EC-MS. For example, the PFP derivative of 4-aminobiphenyl cited above contained a residual, active hydrogen as part of a primary amide (21). This and other examples of electrophorederivatized DNA components which are sensitive by GCEC-MS, and yet possess one or more active hydrogens,


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yl)-2′-deoxyuridine by GC-EC-MS after acetylation and pentafluorobenzylation (28, 32, 33). For the prior compound, the structure of the final derivative (3) is shown in Figure 2 as one of the products containing an active hydrogen. As pointed out by the authors (33), it is attractive to detect DNA adducts as derivatized deoxynucleosides since these compounds can be obtained from DNA by enzymatic digestion, and interference from an RNA form of the adduct is more directly avoided. Unfortunately, some of the larger DNA adducts as derivatized deoxynucleosides will fall outside the scope of GC-EC-MS due to their limited thermal stability and volatility, so one cannot always adopt this strategy. Other workers prepared and detected pentafluorobenzyl and cinnamoyl derivatives of O4-ethylthymidine and 3-methylthymidine by GC-ECD (34) and direct insertion probe-EC-MS (12), but this work did not fully define the detection limits for these products by GC-EC-MS.

Types of Derivatization Reactions

Figure 2. DNA adduct electrophores possessing one or more active hydrogens, along with a more typical compound (7) which gives no response by GC-EMS due to the presence of active hydrogens: 1 (17), 2 (27), 3 (28), 4 (21), 5 (29, 30), 6 (31), 7 (29).

are shown in Figure 2. Nevertheless, one must be cautious in committing to an electrophore containing an active hydrogen. While it may be sensitive when injected into a new GC column, the peak can degrade more rapidly in terms of tailing and area as the column ages relative to the behavior of a fully derivatized electrophore. For example, while 4 fmol of compound 2 was readily detected by GC-ECD, the peak was significantly tailed relative to those from related derivatives lacking an active hydrogen, which is a bad omen for the routine performance of the derivative. It is important not to just test a candidate electrophore at the picogram level on a new GC column, but also to inject a low femtogram amount, especially onto a somewhat aged column. If no response is seen at the femtogram level, one should repeat the injection after breaking off the first 15 cm of the column, since this can improve peak response significantly (e.g., by a factor of 100) even on a relatively new column for some femtogram-level electrophores. Also shown in Figure 2 is a representative compound (7) which contains active hydrogens and failed to give a response by GC-EC-MS, which is more the norm. Presumably an active hydrogen that is less acidic, more sequestered, or not part of a “polar footprint” (see below) is more likely to be tolerated as a component of a successful electrophore, saving an extra derivatization step.

Nucleoside Electrophores Mostly the intact, electrophoric products of DNA adducts have comprised derivatized nucleobases, but deoxynucleoside electrophores have been utilized as well. This is best demonstrated by the detection of 8-hydroxy-2′deoxyguanosine, thymidine glycol, and 5-(hydroxymeth-

The derivatization reactions used to convert DNA adducts into electrophores fall into two general categories: chemical transformation and electrophore labeling. Basically the purpose of a chemical transformation reaction is to convert the DNA adduct into a form, as necessary, which is ready for electrophore labeling. For example, the N2-G adduct of benzo[a]pyrene diol epoxide has been chemically transformed by reaction of DNA containing this adduct with hot acid to release benzo[a]pyrene 7,8,9,10-tetrahydrotetraol, which in turn was oxidized by potassium superoxide to yield 2,3-pyrenedicarboxylic acid (35). This latter product then was electrophore-labeled with pentafluorobenzyl bromide. More details are presented later on this method. The use of a chemical transformation reaction also can be motivated, as was partly the case here, by a goal of establishing a single method which measures an entire class of DNA adducts. In this example the class is diol epoxide polyaromatic hydrocarbon DNA adducts. Ideally the electrophoric product which is formed from the DNA adduct during sample preparation is stable both chemically and physically. Not only can this facilitate its purification and recovery at a trace level, but it makes it easier to maintain quality control, since a standard is always available for routine testing. Thus silyl derivatives, even the sterically hindered tert-butyldimethylsilyl ones, should be used with caution. They can break down not only during handling and storage, but also in the GCEC-MS. It was reported that the latter event can even deposit “de-derivatized” DNA adduct in the GC-EC-MS, so that subsequent injection of a reaction mixture containing residual silylating reagent gives rise to a ghost peak of the analyte (26). Nevertheless, certain silyl derivatives apparently can be fairly stable in a GC-ECMS; e.g., Hunt and Crow detected low femtograms of trimethylsilylphenol electrophores by GC-EC-MS (36).

Polar Footprint A hypothesis called “polar footprint” was introduced to explain the anomalously low response by GC-EC-MS of some electrophores (22). According to this concept, polar groups on an electrophore are more likely to cause loss of the compound on active sites in the GC-EC-MS (e.g., in the GC column) when these groups are adjacent and exposed on one side of the molecule, giving rise to a

Invited Review


Sample Cleanup

Figure 3. Related electrophores where the medium one gives a response by GC-EC-MS or EC-ECD which is in the range of 2- to 10-fold lower than the strong one. The postulated “polar footprint” is indicated by a wavy line (23). R ) -CH3 or -CH2CH2OR′, where R′ ) -CH2C6F5, -CH3, -C(CH3)3, -COC6H5, or -COC(CH3)3.

“polar footprint”. Examples of this concept are presented in Figure 3, where the difference between related strong and medium electrophores, in terms of response by GCEC-MS, is attributed to the latter possessing a polar footprint.

Derivatization Conditions It can be important to employ mild conditions (e.g., low temperature, mild catalyst, inert solvent) whenever possible for an electrophoric derivatization reaction to minimize the buildup of side products. Even the substitution of toluene for acetonitrile as a reaction solvent significantly reduced the noise for the detection of 2,3pyrenedicarboxylic acid, a chemical which can be released from the N2-G adduct of benzo[a]pyrene diol epoxide (37). This general strategy has been adopted by others (32). Severe reaction conditions may seem to be acceptable for some DNA adducts, since none of the side products may happen to interfere. However, at the trace level by GC-EC-MS there are always background peaks at various retention times in the different m/z ion channels, and the use of more severe reaction conditions increases the risk that a co-eluting peak will be encountered for the analyte of interest in one sample or another, or as new lots of reagents are employed. Once the analyte has been made electrophoric, it is better to derivatize any residual, active hydrogens on the analyte with a nonelectrophoric reagent. A single electrophoric group attached to an appropriate functional group on a DNA adduct usually is adequate to achieve efficient electron capture. Minimizing the quantities of the electrophoric and other reagents also is important, but optimizing this aspect should largely wait until real samples are analyzed. This is because background substances derived from real samples can consume some of the other derivatization reagents. Finally, reagents and solvents need to be selected which avoid the formation of a residue upon evaporation that is insoluble in the type of solvent needed for the next step in the procedure. Severe loss of an analyte can take place in this way. Analyte also can be lost upon evaporation by masking from insoluble, invisible (to casual inspection) residues derived from a solid phase extraction cartridge (38).

Solvent partitioning, solid phase extraction, HPLC, and antibody affinity columns have been used most frequently to purify a DNA adduct as part of sample preparation. The first three techniques have been applied to both the pre- and postderivatization stages of sample cleanup, whereas the latter has been used so far only in the first stage. This is an area of GC-EC-MS that is especially in need of further advances. Additional purification steps can reduce interferences and lengthen the lifetime of the capillary GC column (including a less frequent need to break off the top of the column where nonvolatiles accumulate and make the analyte peak smaller and broader). However, purification steps produce analyte losses. One should be suspicious of methods applied only to higher levels of DNA adducts and which employ little sample cleanup after derivatization: at lower adduct levels, interferences and column contamination may be encountered. Solvent partitioning is attractive when the analyte can be ionized by adjusting the pH (the analyte is acidic or basic), allowing some interferences to be extracted at one pH, and then the analyte at another pH. This technique was employed, for example, in an EC-MS method for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a basic chemical that can be released from adducted DNA by alkaline hydrolysis (39). This work is discussed in more detail later. Solid phase extraction (SPE) techniques typically are set up in one of two ways: simple and complex. In the simple approach, the analyst does not take much advantage of either the diversity of SPE packings available (beyond the usual C18-silica and silica choices), or the opportunity to optimize both the washing and elution steps. Most analysts practice SPE in this way for trace analytes like DNA adducts, largely because it is adequate (or may seem to be so), and optimization at the trace level is time consuming. In the complex approach, different packings, washing steps, and elution steps are studied and optimized, which can significantly enhance the degree of cleanup achieved. This was done, for example, in a method for detecting 5-methylcytosine by GC-ECD (17), where a cyano-silica packing was employed, a ternary solvent was used for washing, and an eluent was applied that eluted the analyte in the last two of four fractions. Switching from simple to complex SPE therefore is one way to enhance sample cleanup as needed. The increasing availability of robots may promote complex SPE. HPLC is attractive as a sample cleanup technique, since it provides high resolution with little effort needed to optimize the conditions. However, when higher amounts of analyte are injected first to establish retention times based on on-line detection such as absorbance, the HPLC system can become contaminated. Washing the outside of the injection needle may help to minimize this problem, as pointed out by one of the referees of this review. In one study it was shown that 99.9% of the sample carryover into subsequent blank samples was due to contamination of the injector (40). The authors pointed out that this problem can be overcome by setting up two injectors, one for high and one for low level analyte, and this technique has been adopted by others (28). As an extension of this approach, “satellite HPLC” (minimal HPLC comprising just a low-cost but high precision pump, injector, and temperature-controlled or -monitored


column) can be set up and dedicated to trace sample cleanup (41). Like HPLC, antibody affinity chromatography is attractive for sample cleanup since a high resolution separation is achieved with little or no effort needed to optimize the conditions. Increasingly, this powerful technique will be employed, at least for known DNA adducts whose measurement becomes common. Two of the entries in Table 1 employed an antibody: 4′-hydroxybutyl-O6-G and methyl-, ethyl-, propyl-, and butyl-O6-G.

Introduction to Applications Table 1 presents applications of electrophore labeling to DNA adducts and DNA monomers. In most cases, detection was by GC-EC-MS, but GC-ECD or GC-electron impact-MS was used when a GC-EC-MS was not available. In about half of the articles cited, only pure standards of electrophore products were detected, mostly reflecting the early years in this field. The DNA adducts listed arise from a chemical change to a nucleobase in each case except that of 2-phosphoglycolate, which is associated with oxidative damage to the sugar moiety on DNA. Since the use of stable isotope internal standards is routine in EC-MS, this aspect of the various methods is not discussed much here. One should avoid, whenever possible, an internal standard with a high deuterium content, since it can elute much earlier than the analyte (due, in part at least, to reduced van der Waals-driven adsorption). Such an internal standard thereby fails to monitor the instantaneous conditions in the ion source when the analyte is present. A heptadeuterio-substituted standard eluted 3 s earlier than the analyte in one case (42), and similarly, an octadeuterio internal standard eluted 3 s earlier (35). Nevertheless, sufficient isotope (e.g., 3-4 deuteriums) should be present to escape the natural isotopic peaks from the analyte. In the sample preparation column in Table 1, no listing of the type of sample indicates that only pure analyte was determined. When DNA is cited, it means that the analysis began with DNA containing adduct in one or more of the following ways: spiking of standard adduct into standard DNA, in vitro formation of DNA adduct in a chemical or metabolic reaction mixture, in vivo reaction of the DNA in a chemical-exposed animal, or “natural” occurrence of the adduct in a human or other species. Aside from the special challenge of a labile DNA adduct, or an adduct that can form artifactually during sample preparation, an electrophore-labeling method largely reaches maturity, in terms of the electrophore-preparation methodology, once the adduct is detected (reliably) in any of these types of DNA samples. This is because routine techniques are available to purify DNA from a biological sample. Unfortunately, a diversity of procedures is needed to measure a diversity of DNA adducts by electrophore labeling, as is made clear by the variety of procedures listed in Table 1. At least certain types of steps (e.g., pentafluorobenzylation) are used in many cases, and the increasing knowledge in this area is accelerating the ease with which new methods can be set up. Sometimes this diversity is also a strength, since unusual steps take advantage of the unique properties of a given adduct to provide a convenient or specific analysis. It is disappointing that the absolute yield column in Table 1 is basically empty for analyses that begin with a DNA sample. Analysts should strive to provide such

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data, since it helps to know whether the sensitivity of the overall method might be improved. Such information requires that an authentic sample of the final electrophore is prepared and employed as a calibrant. A repeated cycle of HPLC purification, careful drying, and weighing can provide such a calibrant for dilution purposes even when a small amount of an electrophore (e.g., 2 mg) is available. The molar absorptivity of the calibrant should be measured and reported in addition to its response by GC-EC-MS. Another disappointment for some of the articles is the absence of a chromatogram for the detection limit that is cited, and a chromatogram for the corresponding blank sample. In some cases only the peak of interest is displayed, rather than the entire chromatogram. A cleaner, overall chromatogram implies a more reliable and rugged method, as well as a broader potential of the method to detect other adducts. Key experimental details sometimes are omitted, including amounts of DNA analyzed. As authors and referees, we need to become more attentive to these shortcomings. Especially in trace analysis, details can be critical for success. Detection limit for an adduct in DNA is best established by measuring a known amount of adduct at this limit in the DNA. An independent technique (such as spiking in authentic adduct in a free or DNA-bound form, including the use of a radioactivity standard) is necessary to truly set up a known amount of the adduct. Some of the entrees for detection limit in Table 1 approximate this ideal by relying on an internal standard to estimate the smallest amount of inherent adduct that can be detected. It is clear from the variety of ways in which the detection limit is reported in Table 1 that no consensus has been reached on this aspect. There is little uniformity in reporting detection limits in other areas of analytical chemistry, so a diversity of units may continue here as well. The investigator is encouraged to report, along with whatever units are selected, adducts/total nucleotides a given amount of DNA, as exemplified by the detection limit data provided for PhIP in Table 1. This latter expression is similarly encouraged for the reporting of adduct levels found in real samples. A group of workers recommended that a standardized expression such as moles of adduct per mole DNA base should be employed for this latter purpose (43). We will next discuss several of these applications in more detail, focusing on interesting methodology aspects, and mainly giving attention to work where a DNA sample was tested.

Individual Applications (Refer to Table 1) 4-Aminobiphenyl (21, 44). Here, basic hydrolysis in hot sodium hydroxide (0.05 M, 130 °C, 18 h) was used instead of hydrazine (which also worked) to release 4-aminobiphenyl from its attachment to DNA. (Such alkaline hydrolysis has been extended as well to PhIP as cited below.) The DNA was obtained from human urinary bladder and lung. It is always helpful, when possible (the analyte is very nonpolar), and as practiced here, to employ a weak solvent like hexane for organic extraction of an analyte, since water, salts, and some side products will partition poorly into the hexane, which thereby enhances analyte purity. Acylation in hexane with pentafluoropropionic anhydride was done in the presence of triethylamine at room temperature, a good

C8-dG CH3 on T O6-G N7-G O6-G

N1,N2-G N1,N2-G N7-A, N1-A N3-Tdn O2-C O6-G

hydroxy hydroxy 4′-hydroxybutyl 2′-hydroxyethyl 2′-hydroxyethyl

(4′-hydroxypyridyl)butanone malondialdehyde malondialdehyde methyl methyl methyl methyl

C deamination C deamination

deoxyribose

BTFMBz/pivalyl acetyl/PFBz DNA/U DNAglycosylase/BTFMBz/H2O-isooctane PFB/Me

DNA/NaOH/hexane/PFP NH2NH2/PFBzal DNA/HCl/heat/KO2/PFBz/Si-SPE DNA/HCl/heat/CH3I/C6H6 PFB/Me/GC-ECD DNA/HCl/IEC/C18-Si-SPE/PFBz/Si-SPE PFBz/TLC/Si-FC PFBz/TLC PFBz/TLC/GC-ECD DNA/nucleases/TFA-acid/hydrazine/C18-SPE/acetyl/hydrolysis/ PFBz/Si-SPE urine/C18(OH)-Si-SPE/acetyl/PFBz/HPLC/EtOAc PFBz/TLC/Si-FC/PFBz/TLC DNA/HCl/Ab/PFBz/TMS DNA/heat/HCl/HNO2/PFBz/Si-SPE NaNO2-HBF4/Si-FC/PFBz/TLC PFBz/TLC DNA/HCl/CH2Cl2/PFB/HPLC/THF/ether DNA/nucleases/C18-Si SPE/NaBH4/HPLC/PFBz/HPLC/tBDMS/Si-SPE DNA/nucleases/C18-SPE/HPLC/HCO2H/PFBz/C18-Si-SPE urine/H2SO4/AgNO3/HCl/KOH/C18-Si-SPE/PFBz PFBz/TLC/GC-ECD PFBz/TLC NaNO2-HBF4/Si-FC/PFBz/TLC PFBz/TLC DNA/HCO2H/PFBz/CN-Si-SPE/pivalyl/Si-SPE/HPLC/GC-ECD DNA/HCl/Ab/PFBz/TMS HNO2/PFBz/Si-SPE DNA/NaOH/EtOAc/PFBz/EtOAc

procedure

30

0.25 43 72

9.7

56 57 55

35

22

yield (%)

1 pg in 100 µg of DNA

1 in 108 for 100 µg of DNA; 1 in 109 for 1 mg of DNA 10) 10 fg

1 in 2 × 107 (methyl)

25 fmol in 1 mg of DNA 1 pmol in 1 mg of DNA

0.12-3 µmol/mol of G 100 pg in 100 µg of DNA

1.8 pmol in 50 µL of urine

4 in 106 for 30 µg of DNA

60 fmol/µmol of G for 1 mg of DNA

5 in 107 (100 µg of DNA)

10 fmol (100 µg of DNA)

50 pg (S/N > 100)

DNA 0.32 in 108 (100 µg of DNA)

reported detection limit

150 pg (S/N ) 3; GC-EI-MS) 0.24 fmol

10 pg (30 fmol)

1.3 amol (S/N ) 10)

1.8 fmol 200 zmol (S/N ) 3)

0.44 fmol 1.8 fmol

25 amol (S/N ) 10) 14 amol (S/N ) 20) 1 fmol 7 fg 190 amol (S/N ) 10) 25 pg (S/N > 100)

standard

ref

71 33 26 57

28, 32 19, 27 61 19, 31, 62 23, 29 23 64 67 66 68, 69 34 29 23 29, 30, 56 13, 17, 29 42, 61, 70 62 39

21, 44 20, 45 19, 35 51 52, 53, 54 1, 55 29 29, 56 12, 34 28, 32

a Abbreviations: A, adenine; Ab, antibody; BTFMBz, 3,5-bis(trifluoromethyl)benzyl; dG, deoxyguanosine; ether, diethyl ether; EtOAc, ethyl acetate; G, guanine; IEC, ion exchange chromatography; Me, methyl; PFB, pentafluorobenzoyl; PFBz, pentafluorobenzyl; PFBzal, pentafluorobenzaldehyde; PFP, pentafluoropropionyl; Si-FC, silica flash chromatography; Si-SPE, silica solid phase extraction; T, thymine; tBDMS, tert-butyldimethylsilyl; Tdn, thymidine; TFA-acid, trifluoroacetic acid; TMS, trimethylsilyl.

2-phosphoglycolate thymidine uracil uracil

O6-G N7-G

N2,3-G O2-T O4-T O4-Tdn C8-dG

cytosine etheno ethyl ethyl ethyl hydroxy

5-methylcytosine methyl, ethyl, propyl, butyl methyl, phenyl, styrene oxide PhIP

C8-G (mostly) C8-G N2-G (mostly)

target on DNA

4-aminobiphenyl 2-aminofluorene benzo[a]pyrene

type of adduct

sample preparation

Table 1. DNA Adducts and Monomers Detected by Electrophore-Labeling GC-MS or GC-ECD (Detection Is by GC-EC-MS unless Indicated Otherwise)a

Invited Review Chem. Res. Toxicol., Vol. 10, No. 3, 1997 261


choice since mild conditions and volatile reagents were involved. Thus, high sensitivity was attained in this method without the use of any chromatographic steps for sample cleanup, which is impressive, although the favorable detection properties of the analyte helped to make this possible. 2-Aminofluorene (20, 45). The purpose of the hydrazine step (160 °C, 24 h in anhydrous hydrazine) used in the cited method was to liberate the 2-aminofluorene moiety from the N2 position of guanine. The technique was found to give an 86% yield of 2-aminofluorene starting from a deoxynucleoside form of the parent adduct. Independently, a pure standard of a pentafluorobenzylidine derivative of 2-aminofluorine was prepared, and as little as 10 fg (25 amol) was detected. This work encouraged the use of rigorous chemical conditions to transform a large DNA adduct into an electrophore (conventionally, the latter is volatile and thermally stable), and this general approach has been developed further as discussed below. Previously, it was known that hot hydrazine (60 °C, 16 h) can decompose pyrimidines (46). Benzo[a]pyrene (19, 35). The large size and structural complexity of benzo[a]pyrene diol epoxide DNA adducts makes it unlikely that an intact adduct (retaining the nucleobase or nucleoside moiety) can be converted into an electrophore for sensitive detection by GC-ECMS. In the cited method, (illustrated below) adducted DNA (from cultured human lymphocytes exposed to benzo[a]pyrene) was subjected to acid hydrolysis (0.12 M HCl, 90 °C, 3 h) in order to release the adducted chemical as benzo[a]pyrene 7,8,9,10-tetrahydrotetraol. Subsequent oxidation with potassium superoxide (KO2, 18-crown-6, dimethylformamide, 18 h, rt) gave 2,3pyrenedicarboxylic acid, which, in turn, was labeled with pentafluorobenzyl on the two carboxylate moieties. This latter reaction was conducted in toluene using triethylamine as a base with continuous vortexing for 5 h at 50 °C. Finally, the electrophore was trapped on a Si-SPE cartridge in hexane, and eluted with ethyl acetate prior to detection by GC-EC-MS. As will be seen, this latter step has been useful in general for purifying DNA adduct electrophores, since it takes advantage of their generally weak solubility in hexane relative to the polarity provided by a water-coated silica surface (the ordinary condition of silica). Chemical transformation with potassium superoxide provides a general way to convert a variety of polyaromatic compounds, when followed by pentafluorobenzylation, into electrophores. This is because KO2 converts such compounds, as long as they are partly oxidized in the first place, into aromatic or polyaromatic products containing carboxylic acid groups. A variety of such polyaromatic substrates have been oxidized in this way (47), including internal quinones (48) and nitropolyaromatics (49, 50). In a related approach, Melikian et al. (51) similarly subjected DNA to acid hydrolysis to release benzo[a]-

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pyrene tetrahydrotetraols as in step 1 above. After the tetraols were extracted into ethyl acetate and the solvent was removed by evaporation, the following sequence of steps was performed: methylation with methyl iodide/ methyl sulfinyl carbonion, water addition, extraction of the tetramethyl ether products into benzene, and injection of the resulting solution into a GC-EC-MS. Thus the authors took advantage of the inherent electrophoricity of PAHs. Four isomers of benzo[a]pyrene tetrahydrotetraols were studied, and they formed side products to different degrees in the methylation reaction. The tetramethyl ether product of each formed multiple fragment ions upon electron capture. For the trans-anti tetramethyl ether product, the detection limit was 1 fmol. The corresponding product of a chrysene tetrahydrotetraol gave a detection limit of 50 fmol. This method was motivated in part by an effort to maintain more of the structural details of the initial DNA adducts than in the prior approach using KO2. This was not fully successful, since the tetraols epimerized or decomposed to various degrees (depending on the isomer) during the acid hydrolysis step. Since no electrophoric reagents were employed in the derivatization reaction, the products could be detected simply by extracting them into benzene at the conclusion of this reaction. However, less purification after derivatization can lead to a faster buildup of nonvolatile chemicals at the front of the GC column, so that it needs to be cut more frequently. Cytosine (52-54). Cytosine has been converted into an electrophore by reaction with pentafluorobenzoyl chloride in acetonitrile using N-methylmorpholine as a base and heating at 35 °C for 4 h (65% yield of intermediate product, in which the PFB moiety is attached at N4) followed by methylation of the N1,N3 sites with dimethyl sulfate in acetonitrile with diisopropylethylamine (24 h at 35 °C gave an 87% yield of final product). Whether or not this product is a good electrophore (limited fragmentation upon electron capture) for GCEC-MS is unknown, since it was only tested by GC-ECD. Etheno (1, 55). Detection of the adduct N2,3-ethenoguanine in liver DNA from rats exposed to vinyl chloride has been accomplished by GC-EC-MS. Two aspects of the analyte made it favorable for such detection: (1) its stability to mild acid hydrolysis (30 min at 70 °C in 0.1 M HCl conditions) which releases it (and the other purines) from DNA; and (2) the fact that a single, mild derivatization reaction (pentafluorobenzylation in acetonitrile using solid potassium carbonate as a base for 1.5 h at 50 °C) converts it into an electrophore. Nevertheless, the background of purines that build up in the acid hydrolysis step make sample purification challenging. This was nicely accomplished, including removal of salt, by using a tandem combination of ion exchange and reversed-phase short chromatography columns (prepared in Pasteur pipets). On the strong cation exchange resin (Amberlite IR 120 AS 3545), the

Invited Review


Figure 4. Derivatization and detection by GC-EC-MS of N2,3-ethenoguanine. This DNA adduct was obtained from the liver (up to 1 g samples of liver were analyzed) from a vinyl chloride-exposed rat. The peak for the lower trace (m/z 354) is for II, and that above (m/z 358) is for [4,5,6,8-13C]guanine, the internal standard. Amount of the internal standard spiked into the DNA was 3.47 pmol, and 1 µL of the final 150 µL sample was injected into the GC-EC-MS. Reprinted with permission (55).

adduct eluted later than adenine and guanine in a mobile phase of 0.25 M NaCl. The eluted adduct was then directly trapped, followed by desalting (water wash) on C18-Si, followed by elution in methanol. It is not clear whether the pKa properties, hydrophobicity, or both of the analyte make it more retentive than adenine and guanine on the ion exchange packing. When commercially-available C18-Si-SPE columns (which come in plastic housings) were tested, interfering peaks were encountered, which is why this step was conducted as well in a Pasteur pipet plugged with silanized glass wool. As is typical with guanine adducts containing active hydrogens at both N7 and N9 positions, two isomeric pentafluorobenzyl derivatives were formed (N7-PFBz and N9-PFBz). 3,5-(PFBz)2-N2,3-ethenoguanine (isomer II in Figure 4; the etheno group changes the numbering of N7 and N9 to N3 and N1, respectively) apparently was selected for detection since its yield was 3 times higher than that of the other isomer. The detection of this adduct in liver DNA from a vinyl chloride-exposed rat is shown as well in this figure. Ethyl (12, 29, 34, 56). Two DNA adducts (O2-T and 4 O -T (with the latter also measured as the deoxynucleoside, O4-Tdn) were measured after electrophore derivatization. For the two nucleobases, the presence of the ethyl group facilitated such derivatization, since only a single, active NH hydrogen remained, which was derivatized by reaction with PFBzBr/K2CO3. Whereas O2ethylthymine was pentafluorobenzylated at N3 in CH3CN at room temperature, O4-ethylthymine was pentafluorobenzylated at 70 °C in a mixture of K2CO3 and PFBzBr. These reactions were done only at the milligram level. Thymine and cytosine can be released from DNA by strong acid hydrolysis, but such conditions hydrolyze O4ethylthymine to thymine. A reaction sequence is available which should overcome this problem. Minnetian et al. (30) showed that oxidation of O4-ethylthymidine with dimethylsulfoxide and acetic anhydride (40 °C, 3 h), followed by heating in 1 N ammonium hydroxide (80 °C, 1 h), gives a 96% yield of O4-ethylthymine. As pointed

out earlier, O4-ethylthymidine can be pentafluorobenzylated on its two active hydrogens (the two sugar OHs), and the product is sensitive by GC-ECD (34). By direct insertion probe-EC-MS, the base peak is m/z 178 (CH2C6F4O-), which only provides a nonspecific way to detect this analyte, but a specific peak at m/z 449, which has a relative intensity of 35%, is present as well. Hydroxy (C8-dG) (28, 32). This adduct has been detected in rat liver DNA as an deoxynucleoside electrophore. The structure of the electrophore (3) is shown in Figure 2, where it is seen to contain three acetyl groups (on the two sugar OHs and on the N2 amino group), and two PFBz moieties (one on the OH at C8, and one on N1). While this adduct apparently is present in a relatively high concentration in vivo in DNA, due to exposure of the DNA to endogeneous oxidizing agents, its accurate measurement is challenging for the same reason: it can easily form during sample preparation from exposure of the DNA to oxidizing contaminants in the reagents. The authors attempted to control this problem by using very clean reagents, and adding EDTA to complex metals that can catalyze oxidation reactions, with an awareness that the latter strategy can backfire. By using an internal standard of 15N-labeled DNA with a known content of hydroxy-C8-dG, the authors were able to monitor recovery of the analyte even starting with the enzymatic DNA hydrolysis step, which improved the reproducibility about 2-fold relative to the use of an isotopic [18O]hydroxy-C8-dG internal standard, which fails to monitor this step. Interestingly, removing RNA contamination from [15N]DNA by digestion with several ribonucleases was unsuccessful; finally, ultrafiltration solved the problem (59). Once the DNA was digested enzymatically to deoxynucleosides and ultrafiltered, the deoxynucleosides were dissolved in 90% trifluoroacetic acid and left for 5 min at room temperature to convert dG to G. After evaporation, pyrimidine deoxynucleosides were destroyed by heating the sample in hydrazine for 16 h at 60 °C. These conditions, which others had developed for purifying this


adduct as a nucleotide (46), left hydroxy-C8-dG intact, and were important to overcome interferences. The conversion of dG to G was motivated by the observation that the derivatization method (see below) converted dG to hydroxy-C8-dG. Destroying the pyrimidines by hydrazinolysis overcame an interference for the internal standard. After purification of the analyte by C18-SiSPE (see below, where a C18(OH)-Si-SPE column was tested on the urine samples and found to give a higher recovery), acetylation (acetonitrile/(dimethylamino)pyridine/60 min/80 °C), hydrolysis (to remove labile acetyls), and reaction with pentafluorobenzyl bromide (toluene/ triethyl amine/54 °C/3.5 h) was done prior to Si-SPE and GC-EC-MS. Since a 2-sector GC-EC-MS was available, detection was possible at high resolution (3300), enhancing the reliability of the method. Other interesting methodological details in this work are as follows: (1) plasticizers and other contaminants were removed from the plastic tubes by subjecting them to 80 °C acetonitrile for 3 h followed by rinsing with acetone; (2) dimethylformamide as a reaction solvent, while previously useful for higher analyte levels (33), was unacceptable for trace detection (32) (others have experienced problems with this solvent for derivatization purposes; 57), and so was replaced with acetonitrile; (3) only certain lots of methylene chloride, used for an extraction step after the acetylation reaction, were able to sustain a good yield of the final electrophore (not a surprising result since this solvent is prone to artifacts; 58); (4) use of slow flow rates in the C18-Si SPE step, set up by spinning the column at a low rpm, were important (59); (5) the use of a relatively high concentration of triethylamine (>0.5 M in the PFBzBr reaction was important); (6) triethylamine gave a higher yield of product (although with more background products) in the pentafluorobenzylation reaction than K2CO3 as a catalyst (tested on deoxythymidine; 33); (7) using dimethylpyridine alone, rather than combined with triethylamine, gave a much cleaner acetylation reaction (32); and (8) a side product, C6F5CH2N+ (CH2CH3)3, formed in the pentafluorobenzylation reaction, that previously had been identified by others (60), could be tolerated since it could be removed by Si-SPE. Hydroxy (CH3 on T; 19, 27). (Hydroxymethyl)uracil is the DNA adduct which arises when the 5-CH3 group on thymine is oxidized to 5-CH2OH. This adduct as a standard has been converted at the mg level to an electrophore by pentafluorbenzylation at N1 and N3 in the presence of K2CO3, and then the residual CH2OH was either left intact (compound 2 in Figure 2) or converted to a variety of final products (PFBz and several esters). These products were evaluated in terms of ease of formation, resistance to aqueous hydrolysis, and response by GC-EC-MS (27). The latter work was intended to learn the best derivative in general of an aliphatic hydroxyl group once an analyte already incorporated an electrophoric substituent. The pivalyl ester was recommended as the best choice, although the extra mass or steric bulk of this group could be a problem in some other cases. While as little as 150 attograms (200 zmol) of the O-PFBz derivative (with PFBz also at N1 and N3) has been detected as a standard by GC-EC-MS (19), there are two overlapping features associated with this derivative which will complicate its formation and detection at the trace level. The first is the need to employ strong conditions (a phase transfer reaction utilizing 1 M KOH and a tetrabutylammonium salt) to attach the third PFBz

Giese

group. This is because an aliphatic OH has a high pKa. As discussed before, strong conditions tend to produce interferences. Second, aside from the use of strong conditions, exposing the sample to an additional electrophoric derivatization reaction, after it already has become an electrophore, risks additional interferences. Thus, this derivative is not recommended. 4′-Hydroxybutyl (O6-G; 61). This adduct was released from rat DNA by acid hydrolysis (0.1 N HCl, 80 °C, 1 h), purified (after pH neutralization) on an antibody column (elution solvent: acetone/water, 95/5), pentafluorobenzylated (PFBzBr/KOH/anydrous ethanol/60 °C/1 h), reacted (directly after evaporation) with N,O-bis(trimethylsilyl)trifluoroaetamide (60 °C for 1 h), and injected into a GC-EC-MS. The final derivative which was detected apparently contained a single PFBz group at N7 and two TMS groups (N2G and 4-hydroxybutyl sites). Based in part on earlier work by this group in which O6-butyl G was similarly detected (ref 42, work which is discussed below), a corresponding minor product (with PFBz at N9) apparently forms as well in this procedure. Apparently the method was successful, in spite of the use of strong conditions for pentafluorobenzylation, and the formation of a trimethylsilyl derivative, since relatively high adduct levels were detected in relatively large (1-2 mg) amounts of DNA. The use of an antibody column, including its elution with a volatile solvent, is an attractive feature of this procedure. 2′-Hydroxyethyl (N7-G; 19, 31, 62). This adduct has been detected, after spiking 103 pg into calf thymus DNA, by the following sequence of steps: (1) heating an aqueous solution of the spiked DNA (100 °C, 15 min) to effect depurination of N7-substituted purines; (2) removal of the DNA by HCl precipitation (cold, to minimize additional depurination of the DNA) with centrifugation; (3) conversion of the analyte to a corresponding xanthine (NH2 f OH) with nitrous acid (6 M HCl, tert-butyl nitrite, 0 °C, 4 h); (4) double pentafluorobenzylation (first PFBzBr/ K2CO3/CH3CN for N7 and N3, then PFBzBr/phase transfer with 1 M KOH, Bu4NHSO4, CH2Cl2, 20 h, rt for hydroxyethyl); (5) silica SPE and then GC-EC-MS. It was important to use tert-butyl nitrite rather than sodium nitrite to generate nitrous acid from HCl when the method was conducted at the trace level, since the latter reagent left a residue of NaCl after evaporation. This interfered with the subsequent reaction in acetonitrile, since the NaCl residue was insoluble and trapped analyte, and it did not help either to proceed directly to the phase transfer reaction, involving 1 M KOH, where the NaCl dissolved but markedly inhibited the pentafluorobenzylation reaction. Efforts to remove the DNA by ultrafiltration rather than HCl precipitation produced interferences, but this was not studied in depth. The method has been adopted by others to measure endogenous levels of this adduct (approximately 1 adduct in 106 bases) in some biological samples (63), but this has required the use of a high resolution mass spectrometer in order to control interferences adequately on a routine basis. 2′-(Hydroxyethyl)-O6-G (23, 29). Several electrophoric standards of this adduct were prepared, each involving conversion of the 2-NH2 group to F with 48% tetrafluoroboric acid/sodium nitrite (0 °C, 6 h) and pentafluorobenzylation (K2CO3/acetone/acetonitrile) at N7 or N9 (competing sites). The related strategy cited above for chemically transforming N7-(2′-hydroxyethyl)G, in which nitrous is used to form a corresponding xan-

Invited Review

thine, could not be employed here since this would labilize the O6-hydroxyethyl group. The variation in derivatization focused on the residual hydroxyethyl group, where PFBz, methyl, tert-butyl, benzoyl, and pivalyl groups were introduced. The tert-butyl and pivalyl groups seemed to be the best choice. 4-(Hydroxypyridyl)butanone (64). The tobaccospecific nitrosamines 4-(methylnitrosoamino)-1-(3-pyridyl)1-butanone (NNK) and N1-nitrosonornicotine (NNN) undergo metabolic activation to form a common diazohydroxide intermediate that, in turn, reacts with DNA yielding adducts of unknown structure. At least some of these adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone upon acid hydrolysis (0.8 N HCl, 80 °C, 3 h). The protonated analyte stayed in the aqueous phase with CH2Cl2 extraction, and then partitioned into the CH2Cl2 after the pH was adjusted to 7.0. Evaporated, redissolved sample was derivatized with PFBCl/hexane/trimethylamine (2 h, rt), and the evaporated sample was purified by reversed-phase HPLC. Two UV markers (pentanophenone and hexanophenone) were included in the separation to sandwich the location of the UV-undetectable analyte. Evaporated, redissolved sample was retention gap-injected (5 µL of a 10 µL volume in ether) into a GCEC-MS. In the retention gap technique, which involves a nonretentive, initial precolumn connected to an ordinary column, a relatively large sample can be injected to increase peak height since on-column enrichment takes place. A danger is that the technique might reduce the recovery of a trace analyte, canceling out the enrichment factor. This analyte has features which both help and hinder its detection as an electrophore, the structure of which is shown here:

The pyridine ring makes it possible to take advantage of purification by pH-controlled partitioning, as described above. However, this ring also might make this electrophore more susceptible to losses on acidic active sites in a GC column. An aliphatic hydroxyl is a problematic group for electrophore attachment. Unfortunately, the attachment of a pentafluorobenzyl moiety to such a hydroxyl, unlike an aromatic hydroxyl, should not yield a strong electrophore. The authors have demonstrated that pentafluorobenzoyl derivatization can be used, but pentafluorobenzoyl esters are susceptible to hydrolysis. It is therefore noteworthy that these workers were able to purify this compound at a trace level by reversed-phase HPLC. Nevertheless, the method was applied to relatively high levels of adduct in large amounts of DNA, as indicated in Table 1. The method was applied to biological samples even though contamination was present: a H2O blank was run for each set of samples, and the mean value for this blank (38 ( 16 fmol of analyte) was subtracted from the values observed for the samples. Potential sources of this contamination include the SpeedVac used for the evaporations, and the injector used for the HPLC separations (65). Trimethylamine is a gas at room temperature. The authors overcame this complication by starting with


trimethylamine hydrochloride, a solid, which was dissolved in aqueous sodium hydroxide, allowing trimethylamine to be extracted into hexane. After the latter solution was dried with anhydrous sodium sulfate, it was employed in the derivatization step with PFBCl. Perhaps triethylamine should be tested as a more convenient alternative to trimethylamine in such a derivatization reaction, since triethylamine is a liquid at room temperature, relatively volatile, and available as a highly purified reagent for trace derivatization reactions. Malondialdehyde (66, 67). The first procedure listed in Table 1 for the malondialdehyde-N1,N2-G adduct was tedious, and later the same group developed the improved, second procedure. Both procedures share the same initial steps. An aggressive phenol/chloroform extraction (15 min at room temperature, then 60 °C for 10 min; performed twice) was done on lysed cellular nuclei from liver after ribonuclease, protease, and adenosine deaminase digestion. The latter enzyme was included to convert deoxyadenosine to deoxyinosine, since deoxyadenosine eluted close to the analyte in the initial HPLC step. In the first procedure NaBH4 was used to reduce a CdN group in the deoxynucleoside form of the analyte prior to pentafluorobenzylation of the adduct, since the parent form of the deoxynucleoside adduct was unstable to the latter conditions. However, this also created an additional NH group that later needed to be derivatized, which was accomplished by tert-butyldimethylsilylation. During the PFBz reaction the sugar fell off since the PFBz group added at N7, creating a positive charge. These complications, including the additional HPLC purification required, were overcome in the second procedure by depurinating the deoxynucleoside adduct in 2.5% formic acid (60 °C, 45 min) prior to pentafluorobenzylation (PFBzBr, K2CO3, methanol, 1.5 h, room temperature), nylon filtration (K2CO3 removal), and GCEC-MS. Only the N7-PFBz derivative was monitored since an interfering peak coeluted with the corresponding N9-PFBz isomer. The structures of the two final derivatives that were detected in the two procedures are shown below.

Methyl N7-A, N1-A (68, 69). After urine (15 mL) was acidified with sulfuric acid, addition of silver nitrate gave a precipitate that contained the analytes, which, in turn, were extracted from the precipitate with 1 N HCl. The sample was neutralized with KOH, purified on C18-SiSPE, and derivatized with PFBzBr (acetonitrile/0.1 N KOH/95 °C/75 min). Strong conditions were used for the latter reaction to add two PFBz groups (sites unknown) to the analytes. Mild conditions, tested first, gave a product possessing a single PFBz group that could not be detected by GC-MS, apparently due to the residual active hydrogen. However, mono-PFBz derivatives of 6and 9-methyladenine, as standards, could be detected. While the reason for the overall low yield (0.25%) in the prior procedure was not defined, significant losses could have taken place during and after the PFBz reaction, due to the use of strong conditions followed by only evapora-


tion (giving a salt residue that could trap analyte), and extraction (CHCl3) prior to injection into the GC-MS. Fortunately rat urine contains a high concentration of these analytes (especially N1-methyl-A), enabling them to be detected by this method. The PFBz reaction was not successful for 3-methyladenine. Methyl N3-Tdn (34). As a standard, this deoxynucleoside analyte was pentafluorobenzylated under phase transfer conditions (aqueous KOH, tetrabutylammonium sulfate, CH2Cl2, CH3CN), and purified, after taking the organic layer, by preparative TLC. Methyl O2-C (29). As shown below, derivatization of 2 O -methylcytosine with PFBzBr/K2CO3 yields a product lacking the methyl group. Cytosine itself does not form this product; instead it appears to form N1-(pentafluorobenzyl)cytosine under these conditions. Apparently, the quite different pKa’s for the NH2 of O2-methylcytosine and cytosine (5.41 and 12.2, respectively) account for the contrasting behavior of these two compounds in this reaction.

Methyl O6-G (23, 29, 30, 56). This analyte was derivatized in the same way as 2′-(hydroxyethyl)-O6-G, which is discussed above, except that the last step, derivatization of the hydroxyethyl group, was not needed. 5-Methylcytosine (13, 17, 29). The structure of the electrophore formed from 5-methylcytosine is shown in Figure 2 (compound 1) as one of the examples in which an active hydrogen can be retained. As seen, this hydrogen, on N4, is sequestered since this site is converted into a pivalyl amide. In the method applied to human lymphocyte DNA, acid hydrolysis (3 h at 150 °C in HCO2H) was followed, after evaporation, by pentafluorobenzylation (K2CO3/acetonitrile, 60 °C, 3 h), CN-Si-SPE (discussed earlier in Sample Cleanup), pivalylation (pivalic anhydride, 4-(dimethylamino)pyridine, acetonitrile, 60 °C, 1 h), Si-SPE, HPLC, and then GC-ECD. The method illustrated the limitations of GC-ECD for this kind of methodology: in spite of the high level of 5-methylcytosine and the use of multiple sample cleanup steps, the GC-ECD chromatograms were busy with other peaks besides the analyte. Later this electrophore as a standard was tested by GC-EC-MS and found to give a typical spectrum for this type of derivative: essentially a single ion corresponding to M - PFBz (29). Methyl, Ethyl, Propyl, Butyl O6-G (42, 61, 70). The method for these adducts is equivalent to that cited above for 4-(hydroxybutyl) O6-G, except that one less trimethylsilyl group became attached to these electrophores. Methyl, Phenyl, Styrene Oxide N7-G (62). These adducts as standards at the 50 ng level were converted into electrophores using the same procedure described above for 2′-(hydroxyethyl) N7-G, except only in the case of styrene oxide was the second derivatization step (for the OH) necessary. Thus the above method appears to be general for small N7-G alkyl and aryl adducts with or without an attached hydroxy group. PhIP (39). Analogous to the above method for 4-aminobiphenyl C8-G (but with a lower hydrolysis temperature and more concentrated NaOH here), the DNA

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Figure 5. Detection limit by GC-EC-MS of O2-pivalyl-3′,5′- bis(trifluoromethyl)benzyl glycolate. Reprinted with permission (71).

sample (0.1 mL of 1 mg/mL) was subjected to alkaline hydrolysis (0.5 M NaOH, 100 °C, overnight) and the released adduct was extracted into ethyl acetate. After evaporation, the sample was pentafluorobenzylated (PFBzBr, ethyl acetate, diisopropylethylamine, 55 °C, 30 min) and evaporated. Since the electrophore could be protonated at low pH, it was cleaned up by partitioning as a function of pH (HCl/hexane then Na2CO3/ethyl acetate) prior to evaporation and detection by GC-ECMS. It is interesting that such high sensitivity was achieved in this method devoid of any antibody, solid phase extraction or HPLC sample cleanup steps. In part the relatively high molecular weight of the electrophore (564; structure proposed is shown below) in conjunction with the acid/base partitioning, may have helped. Interferences in GC-EC-MS tend to diminish at higher masses. The authors pointed out the need for a better method than A260 to quantify a small quantity of DNA (e.g., 50 µg), since results from replicate samples (adducts/108 nucleotides) could differ by as much as 2-fold, in spite of the use of an internal standard, which monitors the amount of adduct but not DNA.

2-Phosphoglycolate (71). In one type of oxidative damage to DNA, the C4′ hydrogen on the sugar is attacked, and this leads, with help from DNA repair enzymes, to strand cleavage and the release of 2-phosphoglycolate. Upon exposure to alkaline phosphatase, the latter compound is hydrolyzed to glycolate. Sequential treatment of standard glycolate with BTFMBzBr (acetone, 70 °C, 80 h) and pivalic anhydride (pyridine, 4-(dimethylamino)pyridine, 20 h, rt) gave the electrophore shown in Figure 5. Also shown in this figure is the detection as a standard of 87 zmol (1 zmol ) 10-21 mol) of this compound, which is the lowest detection limit observed so far by GC-EC-MS. In part this is due to the favorable detection properties of this electrophore. The result encourages the similar use of BTFMBzBr and pivalic anhydride as derivatization reagents for analo-

Invited Review


gous analytes, aside from the fact that this glycolate electrophore was only prepared at the milligram level. Thymidine (33). In addition to thymidine, which was the main focus, the electrophore methodology cited in Table 1 for this compound also was applied to deoxyguanosine, 5-(hydroxymethyl)deoxyuridine, and 8-hydroxydeoxyguanosine. Several combinations and reaction sequences were studied, involving acetylation, trimethylsilylation, trifluoroacetylation, and heptafluorobenzylation. Acetylation (of the alkyl hydroxyls) followed by pentafluorobenzylation (of the nucleobase) was selected as the best approach, and the preferred conditions for these reactions are better defined in the later work (discussed above for hydroxy C8-dG) by these workers. Uracil (26, 57). The DNA was purified from rat liver by the following sequence of steps: isolation and lysis of cellular nuclei, ribonuclease plus protease digestion, organic extraction, and ethanol precipitation. The organic extraction step was subdivided into a sequence of three substeps: phenol, phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol. Once the DNA was digested with uracil DNA glycosylase to release the uracil, the sample was simply dried and subjected directly to derivatization, as a suspension, with BTFMBzBr (acetonitrile, triethylamine, 30 °C, 25 min). Water was added and the electrophore was extracted into isooctane followed by GC-EC-MS. It is interesting that such high sensitivity (1 pg of uracil in 100 µg of DNA) was achieved by a relatively simple procedure, especially considering that the electrophore derivatization reaction was applied to the entire DNA/enzyme sample. This was certainly facilitated by the key step of specifically releasing the adduct from the DNA with an adduct-specific enzyme, and the associated steps were nicely optimized as well. Uracil also has been converted, as a standard, into an electrophore by a sequence of two derivatization reactions: pentafluorobenzoylation (PFBCl/N-methylmorpholine), followed by methylation (CH3I/Na2CO3/dimethylformamide), giving 3-PFB-1-Me uracil.

Future EC-MS vs Other MS Techniques. EC-MS will continue to be useful for the detection of DNA adducts, and it will certainly be joined more by other MS techniques. Electrospray MS, for example, is an important, more recent technique which can detect DNA adducts as underivatized nucleoside standards at the low picogram level (72-74). Versions of electrospray which provide “nanospray” (75) and “picospray” (76) have been introduced, at least for the detection of macromolecules, that consume tiny volumes of sample and greatly increase the fraction of total, sprayed sample that is introduced into the detection region of the mass spectrometer. Ordinary electrospray is very inefficient in this respect. The main weakness of EC-MS is the need to derivatize the analyte, which includes associated sample cleanup steps. Certainly one would avoid such derivatization if possible. In addition to the extra work it entails, structural details about the analyte can be lost. But the derivatization in EC-MS has three advantages as well. The first is that it can help to achieve sample cleanup by discriminating against interferences. Second, the analyte, once it becomes an electrophore, can be easier to recover during sample preparation due to its improved or more homogeneous solubility properties as a deriva-

tive. Third, gas chromatography, when applicable, is an ideal way to introduce trace samples into a mass spectrometer, since it both purifies the sample in general terms, and resolves many of its components. It can be easier to keep a mass spectrometer in a pristine and thereby ultrasensitive condition with sample introduction by GC as opposed to direct liquid introduction techniques. This is because gases can be much cleaner than liquids on a routine basis, and nonvolatiles do not pass through the GC column. Both electrospray-MS and EC-MS should have a significant future in the detection of DNA adducts. Laser desorption MS techniques also have potential to be useful in this area. Quantitative vs Qualitative MS Techniques. So far, EC-MS has been used only for the quantitative detection of DNA adducts. This is because the adduct is detected by EC-MS in a derivatized form as a single ion. This optimizes the sensitivity at the expense of qualitative structural information, which depends on fragmentation of the adduct and/or measurement of its elemental composition. The qualitative measurement by MS of DNA adducts has come from other MS techniques, some of which also have been used for quantitative purposes. This picture could change somewhat, since the expanding knowledge about electrophore derivatization, and the potential emergence of EC-MS equipment (see below) that can provide fragment and high resolution measurements, could create an opportunity for qualitative studies of DNA adducts by EC-MS. EC-MS Instrumentation. Quadrupole mass filter and sector instruments have been used so far to detect DNA adducts by EC-MS. Unfortunately the former instrument achieves maximum sensitivity only when it measures one ion signal at a time, and this is true for ordinary sector mass spectrometers as well (few in the field are fitted with an array detector). Potentially tandem quadrupole mass filter, quadrupole ion trap, time-of-flight, and Fourier transform ion cyclotron resonance mass spectrometers fitted with electron capture capability might emerge that provide high sensitivity. Overall, this could enhance our ability to measure multiple ions including spontaneous or induced fragment ions by EC-MS, broadening the usefulness of this technique for the measurement of DNA adducts. Sensitivity. EC-MS methods for the detection of DNA adducts will be most useful if they can be applied to a small amount of DNA, e.g., the amount of DNA (5-10 µg) in a small human sample such as a fingerprick of blood or a microbiopsy of tissue. Such very sensitive detection is important since it opens up practical epidemiology studies, and one good way to learn more about the meaning of DNA adducts is via epidemiology. The potential for GC-EC-MS to provide accurate and precise measurements of DNA adducts is important for such studies. Mechanistic studies, significant as well for the meaning of DNA adducts, would be enhanced too. Successful testing of small DNA samples by EC-MS has been very limited so far because of the limited performance (losses, interferences, and contamination) of many of the current procedures for sample preparation. Even for larger DNA samples, or samples where the “natural” background level of an adduct is relatively high, ultrasensitivity is needed because there is a need to test subpopulations of DNA adducts, e.g., those that are associated with a certain region of DNA or DNA sequence (77). There is probably no limit to the degree of sensitivity that we could use in the measurement of DNA


adducts. Further advances are needed in sample preparation for the detection of DNA adducts by EC-MS, and for the other MS techniques as well. Beyond the Teenage Years. EC-MS was said to have reached its “teenage years” at the outset of this review. The knowledge that is summarized here from the struggles and early successes of EC-MS can help it to grow further as a useful technique for measuring DNA adducts in biological samples. In part, its future depends on how effectively competing (and complementary) MS techniques like electrospray can achieve high sensitivity for such samples, and whether new forms of EC-MS will emerge.

Acknowledgment. I would like to acknowledge my colleagues, Ad de Jong and Paul Vouros, who critiqued my manuscript. This work was supported by NIH Grants CA 70056, CA 71993, and CA 65472, Grant OH02792 from the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control, Order No. 88-80536 from NIOSH, Robert A. Taft Labs, and a contract from the Health Effects Institute (HEI), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-816285) and certain motor vehicle and engine manufacturers. The specific contract was HEI Research Agreement 95-15. The contents of this article do not necessarily reflect the views of the HEI, or its sponsors, nor do they necessarily reflect the views and policies of the EPA or motor vehicle and engine manufacturers. Contribution No. 692 from the Barnett Institute.

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Detection of DNA Adducts by Electron Capture Mass Spectrometry

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