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Quantitative Analysis of the DNA Adduct w,3Ethenoguanine Using Liquid Chromatography/
Electrospray Ionization Mass Spectrometry
Ten-Yang Yen,? Nadia I. Christova-Gueoguieva, Nova Scheller, Sharon Holt, James A. Swenberg and
M. Judith Charlest
Department of Environmental Sciences and Engineering and Curriculum in Toxicology, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599-7400, USA
The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS
methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring
its own sets of problems, including the production of artifacts, interferences and sample to sample variability in
derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric
(LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (eGua), a promutagenic DNA adduct of vinyl
chloride exposure. The response of eGua to isotopically labelled internal standard [ "CC,]eGua was linear
(us = 0.999) and reproducible from 0.027 to 0.538 pmol PI-'. We obtained an accuracy of 86 f 14% by analyzing
chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic eGua. The analysis of CEO-treated
calf thymus DNA samples not enriched with authentic eGua provided a precision of 15%. The detection limits with
a signal-to-noise ratio (S/N) 2.5:l were obtained in the determination of authentic eGua at 5 fmol per injection.
The detection limit obtained in the routine analysis of the biological samples was 50 fmol eGua with S/N = 3: 1.
The applicability of the method was established by determining ~ G u ain rats treated with CEO by portal vein
injection and an unexposed human liver. It was observed that the concentration of EGua in the rat livers increased
with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of
the adduct in rat livers. In the human liver DNA sample, sGua was quantitated at 0.06 f 0.01 pmol mg-' DNA.
KEYWORDS: liquid chromatography/electrospray ionization mass spectrometry; DNA adduct; N2,3-ethenoguanine; vinyl chloride
The formation of DNA adducts due to the electrophilic
interactions between chemical carcinogens or their
metabolites and DNA can be the initial step of chemical
carcinogenesis. The molecular dose of such adducts represents a more accurate determinant of potential risk of
exposure to carcinogens than those of external exposure
such as air, water and diet. Several analytical techniques, such as high-performance liquid chromatography (HPLC) combined with fluorescence or
electrochemical detection, 32P post-labeling and gas
chromatography/mass spectrometry (GC/MS), have
been employed to quantitate DNA adducts.' The
greater need for specificity and sensitivity has led to the
development of GC/MS method^.^-'^ Such methods
require chemical derivatization (i.e. silylation, electrophore labelling), which introduces its own sets of problems. Different reagents have different derivatizing
efficiencies and affinities for targeted analytes. For DNA
adducts that are very polar, the derivatization is often
difficult and less successful. Also, interferences and artifacts can arise from the derivatization process that can
lead to difficulties in identifying and quantifying the
Direct analysis of DNA adducts by using liquid chromatography combined with electrospray ionization
mass spectrometry (LC/ESI-MS) can obviate those
problems arising from the employment of chemical deriv a t i z a t i ~ n . ~ ' -In
~ ~ this paper, we present data to
demonstrate that DNA adducts in biological samples
can be quantified precisely and accurately by using LC/
ESI-MS. N2,3-Ethenoguanine (EGua), a highly promutagenic adduct generated by vinyl chloride (a known
human carcinogen) exposure,21was selected as a model
for DNA adducts because the procedure for its isolation
was well characterized in our l a b ~ r a t o r y Our
. ~ findings
are relevant to the analysis of other DNA adducts that
are generated exogenously or endogenously.
t Author to whom correspondence should be addressed
$ Present address: Department of Environmental Toxicology, University of California, Davis, CA 95616, USA.
CCC 0030-493X/96/111271-06
0 1996 by John Wiley & Sons, Ltd.
HPLC-grade water and methanol were obtained from
Fisher Scientific (Fair Lawn, NJ, USA) and Mallinckrodt (Paris, KY, USA), respectively. Calf thymus DNA
Received 30 June 1996
Accepted 23 July 1996
was purchased from Sigma (St Louis, MO, USA). Other
chemicals utilized were of analytical grade and were
obtained from Fisher Scientific. The Amberlite IR 120
utilized in strong cation-exchange columns was
acquired from Serva Feinbiochemica (Westbury, NJ,
USA). Octadecyl (C,,, 40 pm particle size) used for
solid-phase extraction columns was obtained from J. T.
Baker (Phillipsburg, NJ, USA). All glassware used in
sample preparation was silanized.
N2,3-Ethenoguanine (EGua) and ['3C4]-N2,3ethenoguanine (['3C4]~Gua) were synthesized according to protocol described elsewhere.3722
We determined
the residue of unlabeled EGua in the ['3C4]~Guato be
< 2% by conducting an LC/ESI-MS experiment in
which the [M -tH I + ion of EGua and [ ' 3 C 4 ] ~ G ~were
selectively ion monitored.
Preparation of in vitro and in vivo samples
Caution: Chloroethylene oxide (CEO) is hazardous and
should be handled with protective clothing in a well
ventilated hood.
CEO-treated calf thymus DNA was prepared according to the protocol developed by G u e n g e r i ~ hBriefly,
25 mg of calf thymus DNA (2.5 mg ml-'), was incubated with 210 pmol of CEO in 0.1 M potassium phosphate buffer (pH 7.4) at 25°C for 20 min. The treated
calf thymus DNA was then precipitated with 50 ml of
cold ethanol, centrifuged at 3000 g for 10 min, dried
under a stream of nitrogen, dissolved in HPLC-grade
water and stored at 4°C. Aliquots (20-50 pg) of this
CEO-treated calf-thymus DNA were taken for mild
acid hydrolysis.
Male Sprague-Dawley rats (0.4-0.8 kg) from Charles
River Laboratories (Raleigh, NC, USA) were employed
as control and exposed animals. The exposed animals
were killed at 2, 4 or 6 h after exposure to 0.07 or 0.29
pmol of chloroethylene oxide per gram body mass by
portal vein injection. Control rats were killed at the
same time invervals during the experiment. Livers were
removed, frozen and stored at - 80 "C. The human liver
sample (H 116) was obtained from the Tennessee Donor
Service and stored at - 80 "C. DNA was extracted from
the livers by using an ABI 304A nucleic acid extractor
(Applied Biosystems, Foster City, CA, USA). After two
phenol-chloroform extractions and one chloroform
extraction, DNA was precipitated by using sodium
acetate and propan-2-01.~ The yields and purity of
DNA were determined by measuring the UV absorption at 230, 260 and 280 nm. Aliquots of the rat liver
DNA (1.88-4.42 mg) and the human liver DNA (7.4 mg)
were prepared for mild acid hydrolysis.
Extraction, isolation and enrichment of
N2,3-et henoguanine
Prior to the DNA mild acid hydrolysis, 4 pmol of
['3C4]eGua were added to each sample. The DNA was
hydrolyzed in 0.2 M HC1 for 90 min at 80°C. EGua and
['3C,]~Gua, was isolated by applying each of the
hydrolyzates to individual strong cation-exchange
columns packed with Amberlite IR 120 AS 3545 resin.3
NaCl (0.25 M) was used as mobile phase. The fraction
from 5 to 10 ml of the eluate was collected, applied to a
C,, solid-phase extraction column, followed by 5 ml of
water to remove NaCl (all materials for these lowpressure chromatographic separations were prepared in
disposable glass Pasteur pipets (5.75 in) plugged with
glass-wool). EGua and [13C4]~Guawere eluted from the
C1, column with 4 ml of methanol. The solvent was
evaporated and the dry residue was dissolved in 30 p1 of
water for LC/ESI-MS analyses.
LC/ESI-MS analyses
LC,ESI-MS analyses were conducted using a Beckman
Gold liquid chromatographic system (Beckman Instruments, Arlington Heights, IL, USA) coupled to a Finnigan 4000 quadrupole mass spectrometer. The mass
spectrometer was retrofitted with a pneumatic electrospray source (Analytica of Branford, Branford, CT,
USA). The solvent (water and/or methanol) was passed
through a mixing tee. One end of the tee was connected
to a pressure-balance column C,,, 150 x 4.6 mm id.),
while the other end was connected to a Cl, capillary
column (150 x 0.3 or 0.8 mm id.; Hypersil, 3 pm particles size) (LC Packings, San Francisco, CA, USA). The
flow rate was 4 p1 min-' for the 0.3 mm i.d. column and
25 pl min-' for the 0.8 mm id. column. A piece of
fused-silica capillary (30 cm x 50 pm i.d. x 375 pm 0.d.)
directed the eluent of the capillary column to the electrospray needle. The sample injection volume was controlled by a Rheodyne Model 77253 injector (20 pl
external loop) or a Valco CI4WS injector (0.5 pl internal loop). Chromatographic separations were accomplished by increasing the mobile phase from 100%
water to 25% water-75% methanol in 18 min, altering
the phase to 5% water-95% methanol in 4 min and
passing 100% methanol through the column for 1 min.
A voltage of 3.6 kV was applied to the electrospray
needle and 70 psi of nebulizer gas (nitrogen) was
employed to stabilize the spray. The voltage difference
between the exit of the glass capillary and the first
skimmer in the differential pumping region was optimized at 130 V for detection of the [M + H]+ ion of
EGua (m/z = 176). Data were acquired and processed by
a Technivent Vector data system (Teknivent Maryland
Heights, MO, USA). Full-scan data were obtained by
scanning from m/z 20 to 350 in 1 s. For selected-ion
monitoring (SIM) analyses, two ions (m/z 176 and 180)
were monitored with a dwell time of 0.7 s.
Quantification of EGua was accomplished by measuring the peak areas of the [M + H I + ion of EGua (m/z
176) and the [M + H]+ ion of [13C4]eGua (m/z 180) in
standard solutions containing 27, 54, 135, 269 or 538
fmol pl-1 of eGua and 500 fmol pl-1 of [13C4]~Gua.
The calibration curve was constructed by plotting the
response of the ratio of EGua to [13C4]sGua vs. the
concentration of eGua. The equation of the calibration
curve was determined by linear regressions (leastsquares method) of the data. During sample analyses,
the reproducibility of the data was verified by analyzing
a standard solution after every five samples. The relative
difference throughout the day among these standards
was less than 15%. For every eight samples, one
rapid decrease in the ion signal with a small fraction of
fragment ions [see Fig. l(c)].
In previous work, we investigated the factors that
affect the electrospray response of nucleobases to determine how to optimize ESI-MS methods for the analysis
of DNA ad duct^.^^ We found that for compounds with
pK, d 4, the sample solution acidified with weak acid
to promote the formation of protonated ions in solution
can increase the ion signal less than threefold. However,
the LC/ESI-MS ion signal for eGua in a watermethanol solution acidified with 0.1% formic acid was
three times weaker than the response in watermethanol that was not acidified. Therefore, we did not
utilize any modifiers in the mobile phase to separate
eGua from other components in the sample extract.
Because the electrospray response is dependent on
the analyte concentration,2s*26it is desirable to employ
small internal diameters of liquid chromatographic
method blank containing 4 pmol of [13C,]~Gua was
performed to assure the accuracy of measurement. The
EGua signal was not detectable in the method blank.
Optimization of LC/ESI-MS of EGua
The base peak of the LC/ESI mass spectrum for EGua is
the [M + H I + ion at m/z 176 with a retention time of
10.5 min [see Fig. l(a) and l(b)]. We hoped to dissociate
these ions collisionally to gain structural information
about the adduct, but attempts to do so by increasing
the voltage between the skimmer and the exit of the
glass capillary regions from 130 to 200 V resulted in a
1 ooO0ooo
1 4
1 7 6 t3U+-
(M+H)'-a-1= 135
(M+H)'-a-b= 94
d z
Figure 1. Determination of EGua (3.8 pmol): (a) reconstructed ion chromatogram at m/z 176; (b) background-subtracted mass spectrum
of EGua; (c) collision-induced dissociation mass spectrum of EGua. The relative intensity of the ion signal is normalized to the response of
the analyte shown in (A).
columns with low flow rates to increase the analyte
peak concentration and the analyte electrospray
r e s p o n ~ e . ~We
~ - ~compared
the response of EGua
obtained by using C I Bcapillary columns (150 x 0.3 mm
i.d. and 150 x 0.8 mm id.). With 25 fmol of EGua
injected, S/N = 20: 1 was obtained by using the 0.3 mm
id. column compared with S/N = 4 : 1 by using the 0.8
mm i.d. column. Although, as expected, we achieved
greater sensitivity when using the 0.3 mm i d . column,
we discovered two disadvantages. First, we observed
5-50 fmol of EGua in a solvent blank analyzed after the
analysis of standards containing > 3 pmol of EGua. This
carry-over of EGua was eliminated by washing the
column with 95% MeOH-5% H,O for 1 h or more.
Second, we observed a decrease in signal when we
injected 3 4 pl of a sample solution extract from CEOtreated calf thymus DNA. This decrease, probably due
to the presence of other electrolytes in biological
samples, did not occur when we injected 6 pl of the
sample solution on to the 0.8 mm i.d. column. Moreover, EGua was not detected in solvent blanks analyzed
after the determination of < 5 pmol of EGua. Therefore,
the 0.8 mm i.d. column was considered to be more
appropriate for the analysis of biological samples.
Evaluation of the method by quantification of EGua in
CEO-treated calf-thymus DNA
We investigated the accuracy, precision and detection
limits of the LC/ESI-MS method by analyzing the
response of EGua to [13C,]~G~afrom standard solutions and samples. We present the response factors for
EGua (Table 1) from the analysis of 27-538 fmol pl-'
standards with 0.5 p1 on-column injection to construct a
five-point calibration curve on five different days. The
reproducibility of the analyses varied less than 15%
(relative standard deviation), and the response of EGua
to [13C4]sGua was linear with a mean r2 = 0.999 from
linear regression analysis. We further evaluated this
response by analyzing blank samples spiked with 8 p1 of
27-538 fmol pl-1 EGua standards that were carried
through the sample preparation procedures. The data
are summarized in Table 1. These results are similar to
those obtained by direct analysis.
We evaluated the precision of the method by analyzing replicate samples of CEO-treated calf-thymus DNA
(n = 14), and we determined the accuracy of the method
by analyzing matrix spikes (sample contained 20 pg of
CEO-treated calf-thymus DNA enriched with 3 pmol of
authentic EGua). A mean f standard deviation of
55.3 f 8.3 pmol EGua mg-' CEO-treated calf-thymus
DNA was obtained for the analysis of 14 samples of
CEO-treated calf-thymus DNA that provided a precision of 15% among the samples. A mean f standard
deviation of 2.57 k 0.35 pmol (n = 3) was obtained for
the average increase in EGua concentration for CEOtreated calf-thymus DNA enriched with 3 pmol of
authentic EGua samples which provided an accuracy of
86 & 14%.
We also conducted a more stringent test of the accuracy of the method by determining EGua from unexposed rat liver DNA (4 mg) spiked with 8 p1 of 27-538
fmol p1-l EGua. In Fig. 2, we present the data as the
response factor of EGua to [13C4]eGua for these
samples and for the data presented in Table 1. The data
acquired from the analysis of rat liver DNA (open
squares) are close to the calibration curve, demonstrating high accuracy for the analysis of spiked control rat
liver DNA samples. The limit of detection for an
authentic standard of EGua was 5 fmol using the 0.3
mm capillary column with S/N = 2.5: 1, and 15 fmol
using the 0.8 mm capillary column with S/N = 3 : 1.
Application of method to quantify EGua in CEO-treated
rat livers and in an unexposed human liver
Rats were treated with CEO via portal vein injection
(0.07 or 0.29 pmol CEO per gram rat body mass). Two,
four or six hours after exposure, the concentration of
EGua was determined in the exposed rat liver DNA.
The results are summarized in Table 2. The lowest concentration of EGua, 0.15 f 0.05 pmol mg-' DNA, was
found in rat 4 treated with 0.07 pmol of CEO 6 h after
exposure. The highest concentration of EGua
Table 1. Response factors of EGua to I''CC,J&Guaby
r2 = 0.999
Response factorb
Direct analysis of
standard solutions
EGua (pmol PI-')'
(n 5 )
0.07f 0.01
0.1 3 *0.01
0.29 0.01
0.55f 0.02
1.06f 0.03
Standard solutions processed
through sample preparation
procedures (n 3)
0.08f 0.01
0.15 f 0.01
0.32f 0.02
0.57f 0.03
1.08f 0.03
Each standard solution contains the same concentration of
''C.+Gua (0.5pmol PI-').
bThe response factor is the ratio of the m/z 176 area to the
m/z 180 area, and the value given is the mean f the standard
deviation of the number of replicates indicated.
[email protected])
Figure 2. Plot of the response factor of cGua in control rat liver
DNA (4 mg) spiked with the standard solutions (open squares) vs.
the concentration of cGua; the solid line is the calibration curve
obtained from the direct analysis of standard solutions (open
circles; data presented in Table 1 ), and the open triangles are for
the analysis of standard solutions processed through the sample
preparation procedures.
Table 2. Concentrations of N2,3-ethenoguanine in biological samples
Oose of CEO
(pmol g- ')'
Time after
Rat liver DNA
Human liver DNA
Mean f SD
(pmol mg-' DNA)
1.49*0.19( n = 8 )
0.35k0.04 (n "4)
0.30f0.04 (n = 3)
0.15 f 0.05(n = 5)
0.06f 0.01
Detection limit
(S/N- 3 : 1 )
by extrapolation
51 *18
'Values are the amount for chloroethylene oxide per gram rat body mass administered
by portal vein injection.
bValue is the mean *standard deviation for three measurements of the sample extract.
(1.49 f 0.19 pmol mg-' DNA) was found in rat 1
treated with 0.29 pmol of CEO 2 h after exposure. We
also applied LC/ESI-MS to measure endogenous EGua
present in human liver. The chromatograms obtained
with SIM at m/z 176 (EGua) and 180 (['3C,]eGuaj are
shown in Fig. 3. The concentration of ~ G u ain the
human liver DNA sample was 0.06 f 0.01 pmol mg-'
DNA (we employed 7.4 mg of DNA and obtained S/
N = 5: 1 on 70 fmol). If we extrapolate the data to
obtain S/N = 3 : 1 on these measurements, we obtain a
detection limit of about 50 fmol in biological samples.
This limit of detection is about 50 times greater than the
detection limit that can be routinely obtained by using
Tima (mill)
ml7A 80
Figure 3. Selected-ion monitoring chromatograms of human liver
DNA analyzed for cGua and [lSC,]cGua.
derivatization with trifluoromethyltetrafluorobenzyl
bromide and gas chromatography/electron-capture
negative chemical ionization high-resolution mass spectrometry (GC/ECNI-HRMS), the technique routinely
employed in our laboratory to determine ~ G u a . ~ '
The isolation and detection of DNA adducts (modified
bases) from unmodified bases in biological samples is a
great challenge. The concentration of EGua is about
0.1-2.5 per lo6 of guanine for in viuo samples; therefore,
it is impossible to analyze DNA hydrolyzates directly
by using capillary LC/ESI-MS without employing
analyte isolation and enrichment procedures. The
employment of a cation-exchange resin followed by C,
solid-phase extraction method is able to separate the
analyte and remove most of guanine and adenine from
the DNA hydrolyzates. However, the sample solutions
are still complex mixtures. This is evident by the suppression of the ion signal when > 4 p1 of solutions of
extracts were injected on to a 0.3 mm capillary column.
Because of this sample loading limitation and carryover problems associated with the usage of the 0.3 mm
capillary column, we employed a 0.8 mm capillary
column for routine sample analyses, even though the
determination of cGua using the 0.3 mm capillary
column provided greater sensitivity.
Using eGua as a model compound, we demonstrated
that LC/ESI-MS provides for accurate and precise
quantification of the DNA adduct. The response of
EGua to [13C,]~Gua is linear (r2 = 0.999) and reproducible from 27 to 538 fmol PI-'. We obtained an accuracy of 86 f 14% by analyzing CEO-treated calf
thymus DNA enriched with EGua. The analysis of
CEO-treated calf thymus DNA samples not enriched
with EGua provided a precision of 15%.
We also demonstrated the applicability of the LC/
ESI-MS method by determining EGua in livers of rats
treated with CEO by portal vein injection. Using a
small number of samples, we observed that the concentration of EGua in DNA from CEO-treated SpragueDawley rat livers increases with increasing dose of
CEO, and decreases with time after exposure. Such a
reduction in the concentration of EGua suggests rapid
repair of EGua in rat livers. Additional support for this
conclusion comes from evidence for urinary excretion of
the related adduct 1,N6-ethenoadenine and the demonstration of 1,N6-ethenoadenine and EGua repair by glyc ~ s y l a s e . We
~ ' ~also
~ ~ succeeded in the quantification of
endogenous EGua from a human liver DNA (7.4 mg)
The detection limit of this method for EGua in biological samples is about 50 fmol with S/N = 3 : 1. This is
about 50 times less sensitive than the GC/ECNI-HRMS
method employed in our laboratory. Further work is
needed to increase the sensitivity. This may be accomplished by using immunoaffinity chromatography for
sample enrichment to increase analyte recovery and
reduce other complex mixtures and by sharpening the
electrospray needle to enhance ionization efficiency.33934
Also, the amount of DNA required for the analysis
could be reduced by using the column switching
(LC/LC) method with injecting a greater proportion of
the sample.35 The method as presented can be
employed to determine EGua in vitro and to investigate
dose-response relationships in animals at levels above
the detection limit of our method. In our laboratory, we
have used the method to investigate artifactual formation of EGua during chemical derivatization. While at
this time the GC/ECNI-HRMS method is capable of
detecting attomolar quantities of EGua, in cases where
the sample size is not a limitation or the analyte is very
polar, the LC/ESI-MS method may be the method of
choice. Whichever method is chosen, good-quality
assurance measures should be employed (e.g. the
analysis of procedural blanks and matrix spikes, and
confirmation of the identity of the compound by
another method when one ion is monitored) to ensure
accurate quantification of low levels of DNA adducts.
This research was supported by a grant from NIEHS, P42 ES05948.
We thank Dr R.Sangaiah for the synthesis of chemical standards. We
also thank Dr Craig Whitehouse for providing the electrospray ionization source.
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