JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 34, 421È426 (1999)

Quantitative Determination of DNA Adducts

Using Liquid Chromatography/Electrospray
Ionization Mass Spectrometry and Liquid
Chromatography/High-resolution Inductively
Coupled Plasma Mass Spectrometry¤

Christoph Sietho†,” Ingo Feldmann, Norbert Jakubowski and Michael Linscheid*°

ISAS Institute of Spectrochemistry and Applied Spectroscopy, P.O. Box 101352, D-44013 Dortmund Germany

The quantitative determination of nucleotides from DNA modiÐed by styrene oxide is described using a com-
bination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ioniza-
tion mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography
(HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O‘ and 14N16OH‘) was employed to
determine quantitatively the content of modiÐed nucleotides in standard solutions based on the signal of phosphor-
us ; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of
the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data sug-
gested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative
determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene
oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modiÐed in 108 unmodiÐed nucleotides in a
5 lg DNA sample, which comes close to the best methods available for the detection of chemical modiÐcations in
DNA Copyright ( 1999 John Wiley & Sons, Ltd.
KEYWORDS : modiÐed nucleotides ; liquid chromatography/electrospray ionization mass spectrometry ; liquid
chromatography/inductively coupled mass spectrometry ; inductively coupled plasma high-resolution mass spectrometry ;
quantitative determination

INTRODUCTION major and minor DNA modiÐcation (for a review, see

ModiÐcation of the common nucleobases in DNA by
metabolites of xenobiotic compounds is considered to
be a major step in the genesis of cancer.1 It is still
unknown which type of modiÐcation is most efficient
for the induction of the series of events eventually
leading to cancer, since most carcinogens react with
DNA at several di†erent sites and with the formation of
more than one product. This results in a general uncer-
tainty about the role of speciÐc adducts, even though
some reports have been published about the e†ect of
* Correspondence to : M. Linscheid, Department of Chemistry,
Humboldt University Berlin, Hessische Strasse 1È2, D-10115 Berlin,
Germany. E-mail : michael.linscheid=chemie.hu-berlin.de
¤ This contribution is dedicated to the memory of Professor
Wilhelm Richter.
” Present address : Fa. Spectronex, Basle, Switzerland
° Present address : Department of Chemistry, Humboldt University
Berlin, Hessische Strasse 1È2, D-10115 Berlin, Germany.
Contract/grant sponsor : Bundesminister fuŽr Forschung und Tech-
nologie (BMBF).
Contract/grant sponsor : Ministerium fuŽr Wissenschaft und For-
schung des Landes Nordrhein-Westfalen (MWF).
Contract/grant sponsor : Fonds der Chemischen Industrie (VCI).
Ref. 2). A few studies on the dosimetry of adducts have
been published with regard to DNA3h7 and proteins in
relation to the occurrence of cancer,2,8h12 although the
picture is far from being understood.
One of the major obstacles is the difficulty of eluci-
dating the great number of unknown structures at very
low levels and to determine the adducts quantitatively.
The most successful method up to now to establish the
presence of adducts is post-labelling.13,14 The main lim-
itation of this approach is the inability to identify
unknowns without synthetic reference compounds. Fur-
thermore, the efficiency of the labelling reaction varies
depending on the nature of the adducts.15
Recently, accelerator mass spectrometry (AMS) was
employed in this Ðeld owing to its exceptional inherent
detection capabilities for 14C, allowing a search for very
low levels of radioactive markers.6,16 Even experiments
with humans may become possible, since the level of
radioactive label is well below the tolerated amount for
humans. The limitation here is again that only known
compounds can be studied.
The introduction of capillary electrophoresis and
micro-high-performance liquid chromatography
(HPLC) interfaced with tandem mass spectrometry
(MS)N, especially using electrospray ionization. ESI,

CCC 1076È5174/99/040421È06 $17.50 Received 15 October 1998

Copyright ( 1999 John Wiley & Sons, Ltd. Accepted 31 December 1998
422 C. SIETHOFF ET AL .
indicated a new way to elucidate the structures of
unknown DNA adducts in very minute amounts ; even
at the oligomer level, modiÐcations were tractable with
ESI-MS.17 However, reliable quantitative determi-
nation remained an unsolved problem, since standards
are not available. Furthermore, it was unclear whether
the response of di†erent adducts to ESI was similar
enough to allow a precise determination of the most rel-
evant adducts based on a single internal standard.
A solution to this problem could be found only if a
common feature in all nucleotides regardless of their
structure could be used (equivalent to radioactive phos-
phorous in post-labelling), which would allow a sensi-
tive and quantitative determination based on an
internal standard. Such a feature could be the natural
phosphorus in nucleotides with a detector speciÐc for
that element. The technique of choice is inductively
coupled plasma (ICP) MS, since its detection capability
for elements is considered to be independent of the spe-
ciation of the element, in our case phosphorous, because
the high-temperature plasma atomizes any molecule
completely.18
EXPERIMENTAL
Instrumentation
The HPLC system consisted of a low-pressure gradient
pump (Model M480 ; Gynkotek, Germering, Germany),
an injection value (Model 7125 ; Rheodyne, Cotali, CA,
USA) with a 50 ll sample loop and a reversed-phase
column (Nucleosil, 25 cm ] 4 mm i.d.). A second pump
was connected via an additional valve after the column
used for ICP-HRMS only to maintain a liquid Ñow to
the nebulizer unit during a regeneration cycle (100%
methanol).
The HPLC system was interfaced to the following
mass spectrometric detectors. (1) A MAT90 double-
focusing sector Ðeld mass spectrometer (Finnigan MAT,
Bremen, Germany) with an ESI II (Finnigan MAT)
electrospray ion source was used. The eluent was split
in a ratio of 10 : 1, allowing a D2 ll min~1 Ñow to enter
the ESI sprayer. The instrument was set to a resolution
of about 1500. [M [ H]~ ions were always monitored
to ensure the best detection capability. The electron
scavenger gas SF (Messer-Griesheim, Germany) was
6
used to suppress electrical discharge. (2) An ICP-HRMS
system (prototype ELEMENT mass spectrometer, Fin-
nigan MAT) was used, using a hydraulic high-pressure
nebulizer (HHPN), which is described in more detail
elsewhere,19 for high efficiency sample introduction. A
resolution of about 1500 was sufficient for baseline
separation.18,20 The spectrometer was scanned electri-
cally over the mass window relevant for phosphorus.
The instrument was tuned using a solution of phos-
phoric acid with a concentration of 1 lg P ml~1 in 25%
methanol. To avoid carbon deposition at the skimmer
and sampler oriÐces, oxygen at a Ñow-rate of 20 ml
min~1 was added to the carrier gas of the HHP nebu-
lizer.
In addition, a UV detector (UVIS 200, Linear
ScientiÐc) was employed at a wavelength of 254 nm.
Copyright ( 1999 John Wiley & Sons, Ltd.
Chemicals
The nucleotides and calf thymus DNA (Boehringer
Mannhein, Mannheim, Germany) were used without
further puriÐcation. The solvent were high-purity grade
methanol and propan-2-ol (Merck, Darmstadt,
Germany). Water was puriÐed in-house with a Seralpur
DeltaUV system (Seral Reinstwasser Systeme,
Ransbach-Baumbach, Germany).
For in vitro reactions of styrene oxide and DNA, 5
mg of lyophilized DNA were dissolved in 1 ml of 30 mM
ammonium acetate bu†er at 37 ¡C. Duplex DNA was
formed by heating the solution to 96 ¡C and then for 60
min at 60 ¡C. For the reaction the DNA was brought to
37 ¡C. Then, 10 mg of styrene oxide (Merck) in 1 ml of
DMSO were added. The samples were incubated at
37¡C and after 24 or 48 h extracted twice with diethyl
ether. Digestion to 5-monophosphates followed by
means of nuclease P1 (Boehringer Mannheim) (6 U
mg~1 DNA) for 3 h at 37 ¡C. The concentrations used
for the quantitative determinations were calculated with
an average molecular mass for the necleotides of 307.7 g
mol.~1
For ICP-MS, 10 mg of each mononucleotide in 1 ml
of ammonium acetate (30 mM, pH 7) were allowed to
react with 25 mg of styrene oxide at 37 ¡C for 24 h. The
solutions were divided in aliquots and stored at
[40 ¡C. For the measurements, the solutions were
diluted again (1 : 5) with 30 mM ammonium acetate.
RESULTS
The reaction of styrene oxide with DNA components
results in a large number of di†erent adducts, the
majority of which have been described already.21h24 A
few more were found in the course of this study and a
comprehensive discussion is given in a separate pub-
lication.25 Typically, styrene oxide reacts mainly with
the N-7 nitrogen of guanine, much less with N-3 of
guanine and adenine and to some extent with the exo-
cylic heteroatoms of all nucleobases. The endocyclic
adducts induce the cleavage of the glycosidic bond and
subsequently the Ðssion of the DNA. To complicate
matters, not only are isomers formed owing to di†erent
attachment sites in the nucleobases and the styrene
oxide moiety (a or b), but also steroisomers, yielding a
highly complex product mixture. This mixture can be
separated using reversed phase HPLC and a few typical
structures of stable adducts are shown in Fig. 1 to indi-
cate the possible adduct sites in bases and in the styrene
moiety.
The HPLC system with ICP-MS detection was
employed to monitor the phosphorus signal at m/z
30.97. To avoid interference with the plasma molecules
15N16O` and 14N16OH`, a resolution P1500 is
required, which makes a high-resolution mass spectro-
meter mandatory. Since we had to use a gradient Ñow
with methanolÈwater, inÑuences of the solvent system
and the bu†ers could impose problems during the chro-
matography. Methanol is known to alter the plasma
characteristics by changing the desolvation properties of
the aerosol and by deposition of carbon from incom-
J. Mass Spectrom. 34, 421È426 (1999)
 QUANTITATIVE LC/MS OF NUCLEOTIDES 423

Figure 1. Structures of styrene oxide DNA adducts. The selection represents typical attachment sites : (A) b-SO -N-3 adduct to adenosine
5¾-monophosphate ; (B) a-SO -N-2 adduct to guanosine 5¾-monophosphate ; (C) b-SO -N-3 adduct to cytidine-5¾-monophosphate ; (D)
b-SO -N-3 adduct to uridine-5¾-monophosphate, a deamination modification formed during the reaction. a,b-SO refers to the attachment
site in the styrene oxide moiety.

plete oxidation on the sampler and skimmer surfaces. served as a control for the reproducibility of the mea-
Generally the signals decrease with increasing propor- surements, since they should remain the same in all
tion of methanol, which could result in variations of the measurements. The results of these experiments are
ion source sensitivity. shown in Fig. 4 ; very small signals and signals of com-
The Ðrst step here was to optimize the system based pounds with short half-lives (such as the N-7 adducts of
on the signal-to-noise ratio using an eluent containing guanosine) were omitted from the calculation.
25% methanol because this is the important range for We explain the variations in the slopes [Fig. 4(b)] by
the elution of the modiÐed nucleotides. To determine small errors made during the preparation of the stan-
the inÑuence of the methanol in the eluent correctly, in dard solutions ; since the standard addition is based on
the next experiment we added phosphoric acid 1 ppm the dilution of a solution, small errors may cause signiÐ-
phosphorous) to both solvents at a constant concentra- cant deviations. A comparison of the ICP-MS traces
tion during the analysis time. The signal trace for phos- (m/z 30.97, single ion monitoring), ESI-MS traces
phorus is shown in Fig. 2(a). These data were taken to
calculate the best-Ðtting function, which was used as
correction function for the subsequent experiment. It is
important to note that the signal-to-noise ratio remains
constant since the noise level also decreases.
The validity of this approach was tested in the next
experiment. Repetitive injections of phosphoric acid
(2 ppm P) were made and the correction function was
applied [Fig. 2(b)]. The stability is between 10 and 20%,
which is sufficient for our purposes. This experiment
was included in the analysis routine after every three
runs to compensate for variations and to establish the
long-term stability of the correction function.
To enhance the precision of the measurements
further, at the end of each run 5 ll of a 2 ppm P solu-
tion were injected and the concentrations of adducts
were calculated based on the area of that signal as inter-
nal standard. Typical chromatograms of the [M [ H]~
ions of the four nucleotides are shown in Fig. 3.
For the next step, solutions were prepared with
styrene oxide adducts of each of the three nucleotides
5@-adenosine monophosphate, 5-cytidine mono-
phosphate and 5-guanosine monophosphate separately
and the content of the adducts based on the phosphorus
was determined using ICP-MS. These solutions were
used as standard solutions later. Then a DNA sample
was reacted with styrene oxide and digested to mono-
nucleotides. Aliquots of this mixture were spiked with Figure 2. (A) Data used for the construction of the correction
function. The decrease in signal intensity is accompanied by a
standard solutions and, by means of the standard addi- decrease in baseline noise, resulting in a constant signal-to-noise
tion protocol, the sensitivity of ESI-MS for each adduct ratio. (B) Repetitive injections of phosphoric acid with application
was determined. The signals of the thymine adducts of the correction function.

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 421È426 (1999)
424 C. SIETHOFF ET AL .

Figure 3. Separation of the four product mixtures of the reaction of styrene oxide with the nucleotides (A) 5¾-cytidine, (B) 5¾-thymidine,
(C) 5¾-adenosine and (D) 5¾-guanosine. The peaks which are quantified based on the internal standard (last peak, 2 ppm) are indicated in
each case. Sources of errors leading to variations in the sensitivity are unresolved peaks, e.g. in the adenine and guanine traces.

Figure 4. Results of the standard addition experiment for the nucleotide adenosine 5¾-monophosphate as an example. (A) ÍM É HËÉ trace
of the mono-adducts. The unresolved doublets 2/3 and 6/7 were jointly quantified, hence the content is higher than the others. (B) Results
of standard addition experiments. The intercepts with the y -axis reflect the concentration of the adducts in DNA incubated with styrene
oxide. The result of the calculation is given in (C). The numbered adducts refer to (1) b-N-1 adduct, (2,3) b-O -phosphates, (4,5) a-O -
phosphates, (6,7) a-N-6 adduct and (8) b-N-6 adduct. A comprehensive account of the structure elucidation will be given elsewhere.25

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 421È426 (1999)
 QUANTITATIVE LC/MS OF NUCLEOTIDES 425

([M [ H]~, single ion monitoring) and UV (254 nm)
data for adduct mixture separations supports that
assumption, since the traces are nearly identical (Fig. 5).
This was also true for all the other nucleotides, regard-
less of the concentrations.
DISCUSSION
The main obstacle for the quantitative determination of
adducts of DNA components was the impossibility of
combining quantitative determinations and the eluci-
dation of unknown structures. Known components can
be determined precisely (using AMS) or newly formed
unknown adducts detected (with post-labelling), but to
detect and quantify unknown adducts without synthetic
standards was not possible. With ESI-MS only, the
structures of unknown adducts could be elucidated, but
without pure synthetic standards quantitative results
are difficult to obtain. Only by employing both LC/ESI-
MS and LC/ICP-MS can we elucidate the structure and
determine the concentration of an adduct in the same
sample. The strength of the approach is that we do not
have to rely on a synthetic (radioactive) label as in post-
labelling ; rather, the naturally common feature of all
nucleotides, the phosphate group, is employed. ICP-MS
detection is generally not a†ected by the speciation of
an element,26 which allows one to detect any phosphate
with the same efficiency ; thus even inorganic phosphate
is a suitable internal standard. It seems inevitable,
though, that high-resolution mass spectrometry has to
be used to avoid interferences.
As mentioned already, an increasing content of meth-
anol in the eluent (see Fig. 2) changes the signal inten-
sity during the separation, but we observed no decrease
in instrumental sensitivity. As we have shown for
ESI-MS, the sensitivity for the detection of all adducts
here is the same within the error limits obtained in the
measurements. This is due to the fact that all adducts
are chemically very similar and the charge for negative
ions is located in the phosphate group, rendering the
modiÐcation site less important. The comparison shown
in Fig. 5 supports this assumption since the traces given
for ICP-MS, ESI-MS and UV detection are identical.
Only single signals typical for the detectors indicate the
speciÐcity. In the UV trace a small amount of styrene
oxide is visible, but this signal is not observed with ESI
and ICP-MS. On the other hand, signals show up in the
phosphorus trace which are not visible in the ESI-MS
signal ion [M [ H]~ trace, because those adducts obvi-
ously have a di†erent mass. As we have shown, loss of
H O with the formation of cyclic structures is a
2
common reaction for styrene oxide ; the details will be
discussed elsewhere (C. Sietho† and M. Linscheid, to be
published). A few minor signals are shown in the traces
without structure assignment, since the small amount
did not allow structure elucidation. Thus all adducts
can be quantiÐed by means of electrospray data, which
have been calibrated based on the ICP-MS data.
For the styrene oxide adducts determined using LC/
ESI-MS, detection limits between 0.01 and 0.03 lg ml~1
were obtained. With a DNA concentration of 5 mg

Figure 5. Comparisons of the HPLC separations of reaction mixtures of 5¾-monophosphates of cytosine (C), thymidine (T), adenosine (A)
and guanosine (G) using UV detection (254 nm, upper traces), phosphorus detection at m /z 30.97 with ICP-HRMS (middle traces) and
monitoring ÍM É HËÉ ions (ESI-MS ; m /z values are given on the traces). Styrene oxide is visible only in the UV trace ; adducts with
molecular masses other than the adducts appear in the phosphorus traces.

Copyright ( 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 421È426 (1999)
426 C. SIETHOFF ET AL .
ml~1 this represents a molar concentration of 16.2
mmol l~1, calculated with a mean molecular mass of
307.7 g mol~1 for a nucleotide. Since 20 ll were used
for injection on to the regular HPLC column (4 mm
i.d.), a detectable concentration of 4.6 ] 10~8 M (or 920
fmol absolute) results if the average adduct detection
limit is 0.02 lg ml~1. The mean molecular mass of the
adducts is 427.7 g mol~1. This means that for a concen-
tration of 5 mg ml~1 DNA we can detect one modiÐ-
cation in 3.5 ] 105 unmodiÐed nucleobases. On
replacement of the regular HPLC column with a micro-
column of 50 mm ] 320 lm i.d., the detection capabil-
ities improve dramatically. A 1 ll injection volume
yields the same ion abundance owing to the much
higher peak concentration. This results in a detection
limit of seven modiÐcations in 106 unmodiÐed nucleo-
bases using single ion monitoring. With on-column pre-
concentration of a 50 ll injection volume, we could
demonstrate that 14 modiÐcations in 108 nucleotides
can be detected, which comes close to results obtained
with the post-labelling technique.
CONCLUSION
The reaction of styrene oxide with DNA served as an
instructive example to demonstrate that the LC/ICP-
MS signal of phosphorus can be used to determine
modiÐed DNA components. It is important to realize
that it is not necessary at this stage to know the struc-
tures of the adducts, since the measurement is based on
the atomic signal of phosphorus. The only assumption
is that the molecular masses of the adducts in a study
are almost the same to allow the calculation of molar
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J. Mass Spectrom. 34, 421È426 (1999)