Derivatization of the mutagen MX (3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone) with butyl alcohols prior to GC-MS analysis

Article abstract of DOI:10.1016/S0043-1354(00)00456-5

An extremely potent mutagen, 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is commonly present in chlorinated drinking water. Due to its high mutagenic activity and according to WHO guidelines its concentration should be controlled in drinking waters. Determination of MX is difficult due to the low (ppt) levels at which the compound usually exists in drinking waters. Results obtained with butanols as MX derivatization agents are shown and derivatization with sec-butanol is presented as a method which significantly lowers GC/MS detection levels of MX. Copyright

Full text of DOI:10.1016/S0043-1354(00)00456-5

Wat. Res. Vol. 35, No. 8, pp. 1891–1896, 2001
2001 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
#
PII: S0043-1354(00)00456-5
0043-1354/01/$ - see front matter
DERIVATIZATION OF THE MUTAGEN MX (3-CHLORO-
4
(DICHLOROMETHYL)-5-HYDROXY-2(5H)-FURANONE)
WITH BUTYL ALCOHOLS PRIOR TO
GC-MS ANALYSIS
1
1
2
´
JACEK NAWROCKI *, PRZEMYSŁAW ANDRZEJEWSKI , HENRYK JELEN and
ERWIN WA˛SOWICZ
2
a
Department of Water Treatment Technology, Faculty of Chemistry, Adam Mickiewicz University,
Drzymały 24, 60-613 Poznan
´
Poznan
b
, Poland and Institute of Food Technology, Agricultural University of
´
, Wojska Polskiego 31, 60-624 Poznan, Poland
´
(
First received 6 March 2000; accepted in revised form 7 September 2000)
Abstract}An extremely potent mutagen, 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is
commonly present in chlorinated drinking water. Due to its high mutagenic activity and according to
WHO guidelines its concentration should be controlled in drinking waters. Determination of MX is
difficult due to the low (ppt) levels at which the compound usually exists in drinking waters. Results
obtained with butanols as MX derivatization agents are shown and derivatization with sec-butanol
is presented as a method which significantly lowers GC/MS detection levels of MX. # 2001 Elsevier
Science Ltd. All rights reserved
Key words}drinking water, derivatization methods, mutagenicity, MX
INTRODUCTION
characteristic triplet, has a low abundance compared
to the base peak of m/z=147 (Fig. 1). The base
Due to both technological and economical aspects,
chlorination is the most frequently used method for
water disinfection. For over 20 years chlorination has
been known to generate numerous by-products of
which some are proven dangerous to human health.
In 1986 3-chloro-4-(dichloromethyl)-5-hydroxy-
fragment ion forms a doublet together with m/
z=149, which reflects the cleavage of CHCl
2
group
(
Nawrocki et al., 1997). Despite its high intensity this
doublet is not characteristic enough to be used in the
MX identification by low resolution mass spectro-
metry. Electron capture detector (ECD) is usually
only suitable to detect MX present in clean matrices
while average chlorinated tap water cannot be
analyzed by ECD due to the presence of hundreds
of compounds at similar concentration levels.
2
(5H)-furanone (MX)}a compound of extremely
high mutagenic activity, comparable to that of
aflatoxins, was identified in potable water (Kronberg
et al., 1988; Meier et al., 1987) in many countries
including Poland (Kronberg et al., 1988; Backlund,
Different derivatization methods can improve the
detection of MX. Results achieved using propyl
alcohols have been described by the authors in
previous papers (Nawrocki et al., 1997, 1998). The
present work is aimed at the application of butyl
alcohols for the analysis of MX. The application of
sec-butanol is emphasized as the most useful
derivatization agent which improves MX detection
and lowers the MX detection limit. Furthermore, the
use of sec-butanol enantiomers allows a separation of
MX optical isomers. This can be important for
investigating the mechanism of mutagenesis of MX
as well as carcinogenic activity.
1
989; Suzuki and Nakanishi, 1990; Horth, 1990;
Smeds et al., 1997; Nawrocki et al., 1995). On the
basis of the available data it look like MX plays a
significant role in the total mutagenic activity of
water extracts.
In
a standard analytical procedure for MX
determination in potable water, sample derivatiza-
tion with methanol is used, followed by GC–MS. It
has some distinct advantages: simplicity, low-reac-
tion temperature and ease of derivative separation
from the substrate (mainly sulfuric acid excess).
However, the main drawback of this method is the
utilization of the [M-CH
tive analysis. This fragment, although forming a
3
O] fragment for quantita-
MATERIALS AND METHODS
*
Author to whom all correspondence should be addressed.
MX has been synthesized using the method of Padma-
Tel.: +48-61-848-55-11; fax: +48-61-848-66-87; e-mail: priya et al. (1985) at the Department of Organic Chemistry,
jaceknaw@amu.edu.pl ABO Akademii in Turku (Finland). A standard solution of
1
891

1892
Jacek Nawrocki et al.
À1
7
5 ng ml of MX was used in the experiments. The reaction ECD and peak intensities were compared. In case of a
of MX with butyl alcohols was the subject of interest and
the following alcohols were used: n-butanol, isobutanol (2-
methyl-1-propanol), sec-butanol (butanol-2), tert-butanol
positive reaction result, the fragmentation intensities of
m/z=199, 201, 203 [M–OR] were compared by GC/MS.
To minimize injection inconsistency and facilitate the
comparison of derivatization processes, equal volumes of
the extracts of MX derivatives were mixed together. The
mixture contained isopropyl–MX, n-butyl–MX, isobutyl–
(2-methylpropanol-2). All were purchased from Sigma-
Aldrich-Fluka. MX after methylation and isopropylation
was used for comparison in mass spectrometry evaluation.
MX standard solutions were derivatized separately in 3% MX, sec-butyl–MX and methyl-MX (v/v 1:1:1:1:1). A
alcoholic solutions of sulfuric acid. Samples were allowed to similar procedure was described previously Nawrocki
react for 1 h at a temperature close to the alcohol’s boiling et al. (1997, 1998).
point. These parameters were optimized if needed. The
optimized derivatization parameters for the alcohols tested
were as follows: methanol, 608C for 1 h (Kronberg et al.,
In a separate experiment enantiomers of sec-butyl alcohol
were used.
Apparatus. The following gas chromatographs were used
throughout the work:
1988); isopropanol, 858C for 1 h (Nawrocki et al., 1997); n-
butanol, isobutanol, sec-butanol and tert-butanol, 908C for
(1) Hewlett-Packard HP 5890II with quadrupole mass
spectrometer HPMSD 5971A. Separation was performed
1
0
h. The derivatized samples were extracted three times with
.3 ml of hexane. The extracts were analyzed using GC– on a Supelco MDN-5 column (30 m  0.25 mm  0.25 mm).
Fig. 1. The structure and full scan mass spectra of MX derivatized with methanol (MX+MeOH).
Table 1. (M–alkoxy group) fragment peak intensities and relative abundance ratios for MX derivatized with various alcohols
a
Sl. No.
Alcohol used
m=z fragments
Normalized intensity
Relative abundances of fragment ions m=z 199, 201 and 203 (%)
b
1
2
3
4
.
.
.
.
Methanol
199
5
9
5
57 Æ 2
100
2
2
01
03
61 Æ 2
c
Isopropanol
199
100
97
31
67
73
28
86
82
27
97
94
30
73
75
26
100
2
2
01
03
97 Æ 2
31 Æ 2
92 Æ 2
100
d
Isobutanol
199
2
2
01
03
38 Æ 2
e
Sec-butanol
199
01
203
99
201
03
199
100
2
95 Æ 2
31 Æ 1
100
96 Æ 2
31 Æ 1
98 Æ 2
100
Peak # 1
Peak # 2
n-butanol
1
2
5
.
2
2
01
03
34 Æ 1
a
b
c
The results are normalized to the intensity of m/z 199 ion resulting from fragmentation of pseudo-2-propyl ester of MX.
n=3.
n=5.
n=3.
n=3.
d
e

Derivatization of MX with butyl alcohols
1893
(2) Fisons 8000 with ECD detector in the GC–ECD constant. With these data it is easy to calculate the
evaluations. The following columns were used; RTX-5
30 m  0.32 mm  0.25 mm) and CP-Sil CP-8 (60 m Â
.25 mm  0.11 mm).
3) Hewlett-Packard HP 5890II coupled to AMD (AMD,
Germany) double focusing high resolution mass spectro-
abundance of isotopic ions which originated from
MX–isobutyl derivative and first peak of MX–sec-
butyl derivative.
(
0
(
The main obstacles of butyl alcohols application
meter. Compounds were separated on a DB-1 column for MX derivatization (comparing to the application
(
15 m  0.2 mm  0.25 mm).
of methanol) are caused by better solubility of butyl
alcohols in hexane (hexane is routinely used for
extraction of MX derivative from post-reaction
mixture) and the higher boiling points. Thus, this
RESULTS AND DISCUSSION
In the usual way of MX analysis in tap water procedure is slightly more complicated than deriva-
extracts, the hydroxyl group of the compound is tization with methanol since
methylated to yield a methoxy group. Identification
*
of the methyl pseudoester is done on the basis of the
mass spectrum. Unfortunately, the two isotopic ions
characterized by the highest intensities (m/z=147,
separation of products from the substrates is more
difficult (sulfuric acid excess),
higher temperature of the process.
*
149) cannot be used due to their low specificity in
complex water extracts. For qualitative and quanti-
Using high-temperature reaction vials solved the
tative purposes, the triplet of isotopic fragments problem of the higher process temperature.
M– OCH ] (m/z=199, 201, 203) resulting from the
[
3
presence of three chlorine atoms (Bruner, 1993) in the
MX molecule is used. The drawback of this cluster is
MS characterization of butylated MX
its low relative intensity, which causes difficulties Intensities of [M–alkoxy group] fragments. As
when MX is present in water at trace concentration. mentioned before, to facilitate a comparison of the
In the present investigation butanols were selected results obtained for particular alcohols, equal
for derivatization to give an isotopic cluster m/ volumes of alkoxy–MX hexane extracts were mixed
z=199, 201 and 203 of higher abundance than that together. Thus, both the derivatization efficiency and
resulting from methylation. The following criteria are the alkoxy–MX recovery were assessed together for
considered: formation of butyl–MX, intensity of the all alcohols. Table 1 and Fig. 2 give the relative
[M–alkoxyl group] fragment and application possi- intensities of the isotope peaks of MX derivatized
bilities of butyl derivatization.
with butyl alcohols [M–alkoxy group].
Table 1 reveals that the intensity of the triplet of m/
z=199, 201 and 203 of the isopropyl MX derivative
is the highest of all the alcohols used. The intensities
of the triplet from isobutanol derivatized MX are 13,
8 and 5 times higher than those from methylated
MX. Similar intensities are observed for the n-
butylated derivative. For sec-butyl alcohol, the
Generation of MX butyl derivatives
Positive derivatization results were observed for all
alcohols except tert-butanol. Therefore, for further
experiments the following alcohols have been chosen:
methanol, isopropanol, n-butanol, isobutanol and
sec-butanol (as racemate and separate enantiomers
R(À) and S(+)). Extracted ion chromatograms
(
EIC) for the ions of m/z=199, 201 and 203
confirmed the results obtained using GC/ECD,
indicating both the formation of MX butyl deriva-
tives and their separation by GC.
For sec-butyl alcohol two well-resolved peaks were
obtained. The presence of two peaks follows from the
fact that the C5 atom in the MX molecule is
asymmetric. Sec-butyl alcohol is also
a chiral
molecule. After derivatization, four diastereoisomers
are formed (RR, RS, SR, SS) of which two pairs have
been separated.
The retention times of butylated MX derivatives
are distinctively different with the exception of the
isobutanol derivative and the first of the two peaks of
the sec-butyl derivatives. Measurement of the abun-
dance of the second peak of MX–sec-butanol solved
the problem of the quantitation of the overlapping
peaks, because ratios of abundance of adequate
isotopic ion (m/z=199, 201 and 203) from first and
second peaks of MX derivatized with sec-butanol are
Fig. 2. Relative abundance of fragments [M.–OR] (m/
z=199, 201 and 203) for MX derivatized with butyl
alcohols.

1894
Jacek Nawrocki et al.
intensities of the ion triplets are comparable to those 201, 202, 203 and 204). The even mass ions are also
of isopropyl derivatives. Thus, the derivatization of present for ethyl, propyl and sec-butyl derivatives,
MX with isopropyl alcohol as well as with butyl but their intensities are much smaller.
alcohols is much more favorable for the detection of
MX in complex water extracts than methylation.
According to the data of elemental analysis of the
isotopic ions (shown in Table 2) it can be concluded
Full range mass spectra of butyl–MX are shown in that the presence of the even mass ions at m/z=200,
Figs 3–5 (the mass spectra for the second peak of 202 and 204 is related to the protonated fragment
MX–sec-butanol derivative, with higher retention [M+H – OR].
time, are practically identical to that with lower
retention time}as in Fig. 5). The intensity ratios of Separation of MX enantiomers. The formation of
isotopic ions m/z=199, 201 and 203 generated from diastereoisomers in the reaction of MX with sec-butyl
sec-butyl pseudoester and that of isopropyl one are alcohol can be utilized for the separation of MX
similar to the theoretical values (for fragment ions enantiomers. When racemic sec-butanol is used,
containing three chlorine atoms, Bruner (1993)). In two pairs of diastereoisomers are separated.
the case of n-butyl and isobutyl derivatives, the Derivatization with R(À) or S(+) sec-butanol
intensity ratios are different. The dominating ion is of allows a separation of the MX enantiomers. The
m/z=201, and instead of the triplet m/z=199, 201 mass spectra of the resolved diastereoisomers do not
and 203 we observe a sextet cluster (m/z=199, 200, show any differences in the fragments nor in their
Fig. 3. The full scan mass spectra of MX derivatized with n-butanol.
Fig. 4. The full scan mass spectra of MX derivatized with iso-butanol.

Derivatization of MX with butyl alcohols
1895
Fig. 5. The full scan mass spectra of MX derivatized with sec-butanol, peak #1 (lower RT).
a
Table 2. Identification of fragments (M. –OR) of the MX derivatized with butyl alcohols by HR mass spectrometry
b
No
Alcohol used
m/z received
m/z calculated
Mass deviation (ppm)
Sum formula
Identified fragment
3
3
3
3
5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
.
.
.
.
.
.
.
.
Sec-butanol Peak # 1
Sec-butanol Peak # 1
Sec-butanol Peak # 1
Sec-butanol Peak # 1
Sec-butanol Peak # 1
Sec-butanol Peak # 2
Sec-butanol Peak # 2
Sec-butanol Peak # 2
Sec-butanol Peak # 2
Iso-butanol
198.91350
199.91981
200.90842
201.91648
202.90671
198.9189
199.91815
200.90956
202.90677
198.91197
199.92162
200.91084
201.91620
202.90767
203.91379
198.91206
199.91816
200.90749
201.91550
202.90577
198.91203
199.91986
200.90909
201.91692
202.90614
198.91203
199.91986
200.90909
202.90614
198.91203
199.91986
200.90909
201.91692
202.90614
203.91396
198.91203
199.91986
200.90909
201.91692
202.90614
À7.4
0.3
3.3
C
C
5
H
H
2
O
O
2
Cl
Cl
[M – OR]
[M+H –OR]
[M – OR]
[M+H – OR
[M –OR]
[M –OR]
[M.+H –OR]
[M –OR]
[M –OR]
[M.–OR]
[M.+H –OR]
[M.–OR]
[M+H –OR]
[M –OR]
[M+H –OR]
[M –OR]
[M.+H –OR]
[M –OR]
[M.+H –OR]
[M –OR]
5
5
3
2
5
3
5
3
3
2
2
5
37
37
C
C
C
5
H
5
H
5
H
2
2
2
O
O
O
2
Cl
Cl
Cl
Cl
2.2
À2.8
2
2
37
2
Cl Cl
3
3
3
3
5
0.7
8.6
C
C
H
5 2
O
O
2
Cl
Cl
5
H
5 3
2
3
2
3
5
37
À2.3
À3.1
0.3
À8.8
À8.7
3.5
C
C
5
H
H
2
O
2
Cl
Cl
5
37
2
.
5
2
O
2
Cl Cl
3
3
3
3
5
0.
1.
2.
3.
4.
5.
6.
7.
8.
9.
0.
C
C
H
5 2
O
O
2
Cl
Cl
5
Iso-butanol
Iso-butanol
Iso-butanol
Iso-butanol
Iso-butanol
n-butanol
n-butanol
n-butanol
H
5 3
2
35
2
35
2
35
37
37
C
C
C
C
5
H
5
H
5
H
5
H
2
3
2
3
O
2
Cl
Cl
Cl
Cl
Cl Cl
O
O
O
2
2
2
37
2
37
2
À7.5
35
0.8
Cl Cl
3
3
3
3
5
À0.1
8.5
8.0
7.0
1.8
C
C
5
5
H
2
O
O
2
Cl
Cl
5
H
3
2
35
2
35
2
35
37
37
C
C
C
5
H
5
H
5
H
2
3
2
O
O
O
2
Cl
Cl
Cl
Cl
Cl Cl
n-butanol
n-butanol
2
2
37
2
a
ATT: Accepted mass error Æ 10.0 ppm; Isotopic ions of fragmentation [M.–alkoxy group] which appear on mass spectra but did not fulfill
the acceptable mass error criteria (i.e. Æ 10.0 ppm) are not included in the table.
Mass difference between columns 3 and 4.
b
intensities (see Fig. 5). The practical importance of The unique group of ions used for identification and
the separation of MX enantiomers has not been fully quantitation of MX is an isotope cluster of m/z 199,
established yet.
201 and 203. Identification of MX (using LR/MS) is
performed by a comparison of two parameters:
Potential applications of butylated MX
*
retention time of a standard and the MX peak in a
sample and,
the relative intensities of the cluster peaks m/z 199,
201 and 203 (56 : 100 : 63 for methanol and
100 : 97 : 32 for MX derivatized with isopropanol
or other alcohols).
Butyl alcohols react easily with MX forming
alkoxy derivatives. This opens up the possibility of
using these derivatives for the analysis of MX by
GC–MS.
For the determination of MX in potable water,
high-resolution mass spectrometry is routinely used.
*
Improved detectability of the triplet ions would allow The most abundant ion in a cluster (m/z=201 for
us to use cheaper analytical method with low- methylated MX and m/z=199 for other derivatives)
resolution mass spectrometry. GC–LRMS is used is selected as a target ion, whereas the others serve as
mainly in the selected ion monitoring (SIM) mode. qualifiers.

1896
Jacek Nawrocki et al.
Abundance of isotopic ion depends, at least, on 2. Butyl alcohols yield a high intensity triplet at
two parameters:
m/z=199, 201 and 203, which can be used for
the quantitative and qualitative analysis of MX.
. Derivatization of MX with sec-alcohol can facil-
itate the analysis of MX by low-resolution mass
spectrometry in the cases of crude, impure
matrices. In this case, the detection limit is lower
than that obtained using isopropanol as derivati-
zation agent.
*
first is derivatization efficiency,
second is relative intensity of chosen isotopic ions
3
4
*
compared to the total intensity of all ions resulting
from the fragmentation of the compound.
Thus, the limit of detection (LOD) for MX deriva-
tized with alcohols depends on intensity of the cluster
(m/z 199, 201 and 203 resulting from a cleavage of the
alcoxy group). For derivatization with isopropyl
alcohol the m/z=199, 201 and 203 cluster is the
most abundant, the LOD of derivatized MX is
estimated at 220 pg inj . The limit of detection for Acknowledgements}Prof. dr Leif Kronberg from Abo
methylated MX is tenfold higher at 2000 pg inj
Thus, the real samples, due to low MX concentra-
tions in tap water (ppt level), have to be preconcen-
. The separation of MX enantiomers can shed new
light on the mutagenic activity of MX enantio-
mers.
À1
À1
Akademii Turku (Finland) is acknowledged for supplying
us with MX standard. Financial support from the Polish
Committee for Scientific Research (grant No. 3T09A17308)
.
and
a joint grant of A. Mickiewicz University and
trated 15,000–100,000 times prior to GC/MS Agricultural University (PU-9) are acknowledged.
analysis.
For all tested alcohols except sec-butanol, one
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strong mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-
2
the cluster determines the limit of detection (quali-
fiers m/z=199 for methanol and m/z=203 for other
alcohols). In the case of sec-butyl alcohols used for
(5 h)-furanone (MX) in chlorinated raw and drinking
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82.
2
MX derivatization, two well-resolved peaks of two Bruner F. (1993) Gas Chromatographic Environmental
diastereoisomers are obtained. Although the inten-
sities of ion fragments are slightly lower than the
corresponding ones for isopropyl derivative (see
Table 1), there are two additional parameters for
identification of MX. The first one is the difference in
retention times of the enantiomers. In this case it is
possible to identify MX without monitoring the
lowest intensity ion. The other one is the presence of
Analysis. VCH, New York.
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(
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abundance of fragment m/z=199 of the peak #1 to
the abundance of fragment m/z=199 of the peak #2
ratio, should be the same as in sec-butylated MX Nawrocki J., Andrzejewski P., Jelen
´
H. and Kronberg L.
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(5H)-furanone) in water. Chem. Anality. 43, 687–693.
Nawrocki J., Andrzejewski P., Kronberg L. and Jelen H.
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tion of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-fur-
anone in water. J.Chromatogr. A 790, 242–250.
(
standard).
As a result, the detection limit is lowered as it is
determined by the abundance of the qualifier ion m/
z=201 which is relatively more abundant compared
to m/z=203. Therefore, the detection limit is
approximately three times lower (ratio of intensities
2
´
Nawrocki J., Kronberg L. and Andrzejewski P. (1995) MX
}zwiazek o silnej aktywnosci mutagennej w wodzie do
´
picia. (MX}the strong mutagenic activity compound in
of m/z=201 and 203, see Fig. 2.) and equals
À1
75 pg inj .
The resolution of MX enantiomers can contribute
´
tap water). Ochrona Srodowiska 3(58), 19–22.
Padmapriya A. A., Just G. and Lewis N. G. (1985)
Synthesis of 3-chloro-4-(dichloromethyl)-5-hydroxy-
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isomers of hydroxyfuranones.
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2(5H)-furanone in drinking water in Japan. Chemosphere
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CONCLUSIONS
1
. Butyl alcohols (except tert-butanol) react easily
with MX. From the point of view of GC/MS
analysis, each of the butyl alcohols is better than
methanol for MX derivatization.