Mass spectrometric behavior of thiazide-based diuretics after electrospray ionization and collision-induced dissociation.

Article abstract of DOI:10.1021/ac020020e

The mass spectrometric behavior of 21 thiazide-based compounds after electrospray ionization in the negative ion mode and collision-induced dissociation was investigated on a triple-stage quadrupole mass spectrometer. The mass spectra show individual and common fragmentation patterns, the generations of which are discussed based on comparable molecular structures of commercially available substances and the synthesis of unlabeled, deuterated, and 15N-labeled analogues. The synthesis of deuterated thiazides is perfomed by condensation of 4-amino-6-chloro-1,3-benzenedisulfonamide with appropriately labeled aldehydes, while the introduction of 15N into the sulfonamide groups of thiazides was achieved by the synthesis of 4-amino-6-chloro-1,3-benzenedisulfonamide(15N2) from 3-chloroaniline via 4-amino-6-chloro-1,3-benzenedisulfonyl chloride. The most common fragments determined are m/z 269, 205, and 126 for 6-chloro-7-sulfamoyl-3-alkyl-3,4-dihydro-1,2,4-benzothiadiazine-1,1-dioxides and m/z 303, 239, and 160 for 6-trifluoromethyl-7-sulfamoyl-3-alkyl-3,4-dihydro-1,2,4-benzothiadiazine-1,1-dioxides. Individual fragmentation behaviors were found that mainly depended on the C-3-linked side chain.

Full text of DOI:10.1021/ac020020e

Anal. Chem. 2002, 74, 3802-3808
Mass Spectrometric Behavior of Thiazide-Based
Diuretics after Electrospray Ionization and
Collision-Induced Dissociation
Mario Thevis,*,†,‡ Hans Schmickler,§ and Wilhelm Scha1nzer†
Institute of Biochemistry, German Sport University Cologne, Carl-Diem-Weg 6, 50933 Cologne, Germany, and Institute of
Organic Chemistry, University of Cologne, Greinstrasse 4, 50939 Cologne, Germany
clinical, forensic, and antidoping analysts require informative and
unambiguous data about administered pharmaceuticals. Therefore,
different analytical techniques, such as high-performance liquid
chromatography (HPLC),8-12 gas chromatography (GC),13-15 and
capillary electrophoresis (CE)16 coupled to ultraviolet (UV) detec-
tors or mass spectrometers (MS), were developed. Especially the
use of LC/ MS/ MS has proven to be an appropriate means to
provide detailed information about thiazide-based diuretics and
other compounds belonging to this class of remedies. In particular,
the doping analysis in human and equine sports profits from
avoiding time-consuming sample preparation steps and the pos-
sibility of sensitive and selective measurements of thiazides.17,18
Due to the acidic character of thiazides and sulfonamides in
general, the use of negative ion electrospray ionization proved to
be reasonable. Some gas-phase reactions of organic compounds
after hydrogen abstraction were reviewed by Bowie,19 showing
possible rearrangements, elimination, and fragmentation routes
as a result of collision-induced dissociation. The knowledge of gas-
phase reactions of negatively charged ions after collisional
activation is of analytical significance and important for the
determination and identification of compounds by mass spectrom-
etry. The goal of the present study is the elucidation of the mass
spectrometric behavior of thiazides after negative ion electrospray
ionization in correlation to their individual structures by means
of a triple-stage quadrupole mass spectrometer. The mass spectra
of 21 compounds with a 7-sulfamoyl-1,2,4-benzothiadiazine 1,1-
The mass spectrometric behavior of 2 1 thiazide-based
compounds after electrospray ionization in the negative
ion mode and collision-induced dissociation was investi-
gated on a triple-stage quadrupole mass spectrometer.
The mass spectra show individual and common fragmen-
tation patterns, the generations of which are discussed
based on comparable molecular structures of commer-
cially available substances and the synthesis of unlabeled,
deuterated, and 1 5 N-labeled analogues. The synthesis of
deuterated thiazides is perfomed by condensation of
4 -amino-6 -chloro-1 ,3 -benzenedisulfonamide with appro-
priately labeled aldehydes, while the introduction of 1 5
N
into the sulfonamide groups of thiazides was achieved by
the synthesis of 4 -amino-6 -chloro-1 ,3 -benzenedisulfon-
amide(1 5 N2 ) from 3 -chloroaniline via 4 -amino-6 -chloro-
1 ,3 -benzenedisulfonyl chloride. The most common frag-
ments determined are m / z 2 6 9 , 2 0 5 , and 1 2 6 for
6-chloro-7-sulfamoyl-3-alkyl-3,4-dihydro-1,2,4-benzothia-
diazine-1 ,1 -dioxides and m / z 3 0 3 , 2 3 9 , and 1 6 0 for
6 -trifluoromethyl-7 -sulfamoyl-3 -alkyl-3 ,4 -dihydro-1 ,2 ,4 -
benzothiadiazine-1 ,1 -dioxides. Individual fragmentation
behaviors were found that mainly depended on the C-3 -
linked side chain.
The analysis of products based on the structure of 6-chloro-
7-sulfamoyl-3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide (hydro-
chlorothiazide, Figure 1) has gained attention ever since the
diuretic effect of thiazides was observed with chlorothiazide by
Novello and Sprague in 1957.1 Owing to the wide variety of
analogous products developed in the following years,2-7 the
(7) Whitehead, C. W.; Traverso, J. J.; Sullivan, H. R.; Marshall, F. J. J. Org. Chem.
1 9 6 1 , 26, 2814-2818.
(8) Rosado-Maria, A.; Gasco-Lopez, A. I.; Santos-Montes, A.; Izquierdo-Hornillos,
R. J. Chromatogr., B 2 0 0 0 , 748, 415-424.
(9) Smith, R. M.; Murilla, G. A.; Hurdley, T. G. J. Chromatogr. 1 9 8 7 , 384, 259-
278.
† German Sport University Cologne.
‡ Current address. Department of Chemistry and Biochemistry, University
of California at Los Angeles, 3021 Young Hall, 607 Charles E. Young Drive E.,
Los Angeles, CA 90095. Phone: 310-794-7308. Fax: 310-206-7286. e-mail:
mthevis@chem.ucla.edu..
(10) Leahey, W. J.; McMeekin, J. R. J. Chromatogr. 1 9 8 7 , 421, 401-406.
(11) Cooper, S. F.; Masse´, R.; Dugal, R. J. Chromatogr. 1 9 8 9 , 489, 65-88.
(12) Tisdall, P. A.; Moyer, T. P.; Anhalt, J. P. Clin. Chem. 1 9 8 0 , 26, 702-706.
(13) Lisi, A. M.; Trout, G. J.; Kazlauskas, R. J. Chromatogr. 1 9 9 1 , 563, 257-
270.
§ University of Cologne.
(1) Novello, F. C.; Sprague J. M. J. Am. Chem. Soc. 1 9 5 7 , 79, 2028-2029.
(2) Werner, L. H.; Halamandaris A.; Ricca S., Jr.; Dorfman L.; DeStevens G. J.
Am. Chem. Soc. 1 9 6 0 , 82, 1161-1166.
(14) Ahnoff, M.; Ervik, M.; Lagerstro¨ m, P. O.; Person, B. A.; Vessman, J. J.
Chromatogr. 1 9 8 5 , 340, 73-138.
(15) Maurer, H. H. J. Chromatogr., B 1 9 9 9 , 733, 3-25.
(16) Jumppanen, J.; Sire´n, H.; Riekkola, M. L. J. Chromatogr., A 1 9 9 3 , 652, 441-
450.
(3) DeStevens, G.; Werner, L. H.; Barret, W. E.; Chart, J. J.; Renzi, A. H.
Experientia 1 9 6 0 , 16, 113-114.
(4) Novello, F. C.; Bell, S. C.; Abrams, E. L. A.; Ziegler, C.; Sprague, J. M. J.
Org. Chem. 1 9 6 0 , 25, 965-970.
(17) Thieme, D.; Grosse, J.; Lang, R.; Mueller, R. K.; Wahl, A. J. Chromatogr., B
2 0 0 1 , 757, 49-57.
(5) Sherlock, M. H.; Sperber, N.; Topliss, J. Experientia 1 9 6 0 , 16, 184-185.
(6) DeStevens, G.; Werner, L. H.; Halamandaris, A.; Ricca, S., Jr. Experientia
1 9 5 8 , 14, 463.
(18) Garcia, P.; Popot, M. A.; Bonnaire, Y.; Tabet, J. C. Proc. 11th Int. Conf. Racing
Analysts Veterinarians 1 9 9 6 , 498-502.
(19) Bowie, J. H. Mass Spectrom. Rev. 1 9 9 0 , 9, 349-379.
3802 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
10.1021/ac020020e CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/22/2002

Figure 1. (A) Structures of commercially available and synthesized thiazide-based compounds. Structure names, INN; if others, in quotes. (B)
Structure of methiazide-d4. (C) Structure of [15N2]-ethiazide.
dioxide structure (Figure 1) are discussed, and elimination steps
and rearrangements for the generation of common and individual
fragment ions are proposed, based on the synthesis and analysis
of different alkylated thiazides in addition to deuterated and 15N-
labeled analogues.
ether (distilled before use) was from KMF (St. Augustin, Ger-
many), and acetonitrile was from J. T. Baker (Deventer, Nether-
lands).
Thiazides. Chlorothiazide was purchased from Sigma (Dei-
sendorf, Germany), benzthiazide from Knoll AG (Ludwigshafen,
Germany), bendroflumethiazide from ICI Pharma (Plankstadt,
Germany), trichlormethiazide from Merck, polythiazide from
Pfizer (Karlsruhe, Germany), methyl chlothiazide from Abbott
Laboratories Ltd. (Queensborough, England), butizide from Boeh-
ringer (Mannheim, Germany), and bemetizide from Schwarz
Pharma GmbH (Monheim, Germany). The reference materials
althiazide, ethiazide, epithiazide, cyclothiazide, cyclopenthiazide,
and hydroflumethiazide were kindly provided by Simon Biddle
from HFL, UK. Methiazide, methiazide-d4, prothiazide, n-buthi-
EXPERIMENTAL SECTION
Chemicals. 3-Chloroaniline, chlorosulfonic acid, 4-amino-6-
chloro-1,3-benzenedisulfonamide, acetaldehyde, propionaldehyde,
butyraldehyde, valeraldehyde, capronaldehyde, 3-methylmercapto-
propionaldehyde, acetaldehyde-d4, D2O, DCl (6 N in D2O), ethanol-
d, ammonium deuteride (12 N in D2O), and [15N]-ammonium
hydroxide were purchased from Aldrich. Ethanol, toluene (dried,
over molecular sieve), n-heptane, and sodium chloride were
obtained from Merck (Darmstadt, Germany), tert-butyl methyl
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3803

azide, n-penthiazide, and [15N2]-ethiazide were synthesized in our
laboratory.
Table 1. 1H and 13C Chemical Shifts of Synthesized
Methiazide-d4
Synthesis of 6 -Chloro-7 -sulfamoyl-3 -alkyl-3 ,4 -dihydro-
1 ,2 ,4 -benzothiadiazine 1 ,1 -Dioxides. The preparation of not
commercially available alkylated analogues of hydrochlorothiazide
(e.g., methiazide, methiazide-d4, [15N2]-ethiazide, prothiazide, n-
buthiazide, and n-penthiazide) was performed according to the
method of Whitehead et al.7 The synthesis of methiazide is
described in the following: In a volume of 40 mL of warm ethanol
and 6 N aqueous HCl (50:50, v/ v) 2.85 g of 4-amino-6-chloroben-
zene-1,3-disulfonamide (I) was suspended. A total of 1.5 equiv of
acetaldehyde (0.66 g, 840 µL) was added, and the mixture was
stirred for 1 h. After storage at 4 °C for 10 h, the precipitate was
collected on a medium-porosity frit and washed with bidistilled
water until the residue of mineral acid was removed. The resulting
methiazide (1.6 g, 52%) was dried in a desiccator over phosphorus
pentoxide under reduced pressure. Smaller amounts of synthe-
sized diuretics were extracted from the reaction mixture by
dilution with a 5-fold volume of bidistilled water and subsequent
solid-phase extraction of the thiazide. To do this, the mixture was
transferred onto a PAD-I column (bed height 3 cm, inner diameter
1 cm), washed with distilled water, and the product was eluted
with methanol.
carbons
δ
hydrogens
δ
C-3
C-5
C-6
C-7
C-8
C-9
C-10
C-11
63.19
118.31
136.65
129.45
127.34
119.81
148.41
19.25
N-2
N-4
C-5
C-8
7.65
8.00
6.90
8.08
7.44
SO2NH2
and variable parameters of orifice, ring electrode, and inter-
quadrupole lenses were optimized for maximum abundance of
the ions of interest (M - H)-. The collision gas was nitrogen
obtained from a Whatman 75-72 K727 nitrogen generator, and the
collision offset voltage was 25 eV for all product ion scans.
Nuclear Magnetic Resonance Spectroscopy. Structure
confirmation of the synthesized methiazide-d4 was performed on
a Bruker DRX 500 instrument with a solution of the analyte in
CD3OD or CD3OH. The one- and two-dimensional experiments
1
were 1H, H Watergate (at 20, -10, and - 44 °C), 13C, HMBC
(CD3OH, -44 °C), and ROESY (CD3OH, -44 °C).
For the preparation of deuterated methiazide-d4 (Figure 1B)
the acetaldehyde was replaced by its deuterated analogue and the
condensation with I was performed in ethanol-d and DCl in D2O.
Synthesis of [1 5 N2 ]-Ethiazide. To introduce selectively two
15N-atoms into the thiazide structure, the intermediate product of
I, 4-amino-6-chlorobenzene-1,3-disulfonyl chloride, was prepared
in accordance to the method described by Novello et al.4
3-Chloroaniline (6.4 g, 0.05 mol) was added dropwise to 67 g of
chlorosulfonic acid (0.6 mol) placed in an ice-cooled three-necked
flask under constant stirring. Further, 35 g of sodium chloride
(0.6 mol) was added portionwise. The mixture was heated slowly
to 90 °C and maintained at this temperature for 1 h. After cooling
to ambient temperature, the product was extracted with 500 mL
of tert-butyl methyl ether, the organic layer was evaporated to
dryness by means of a rotary evaporator under reduced pressure,
and the residue was dried in a desiccator over phosphorus
pentoxide in vacuo for 12 h. The crude crystals were finally
purified by recrystallization from toluene/ n-heptane (yield: 2.4
g, 15% of the theory).
RESULTS AND DISCUSSION
Nuclear Magnetic Resonance Spectroscopy. The NMR
experiments provided unambiguous information about the struc-
ture of the synthesized methiazide-d4. The H and 13C chemical
shifts are shown in Table 1.
Mass Spectrometry. 3-Hydrogenated and 3-Alkylated 7-Sulfa-
moyl-3,4-dihydro-1,2,4-benzothiadiazine 1,1-Dioxides. On the basis
of the mass spectrum of the synthesized methiazide (Figure 2),
fundamental fragmentation steps after collision-induced dissocia-
tion of the quasimolecular ion are discussed.
1
In all product ion scan spectra of (M - H)- of hydrochlorothi-
azide and 3-alkylated 7-sulfamoyl-3,4-dihydro-1,2,4-benzothiadiazine
1,1-dioxides, the fragments m/ z 269, 205, 126, and 78 are present
(Table 2, Figure 3A), which are missing in atmospheric pressure
chemical ionization (APCI) mass spectra as reported in the
literature.20 The generation of m/ z 269 is proposed to be initiated
by the fission of the C-3-N-4 bond and a subsequent elimination
of RsCHdNH. Here, the presence of a single bond between
atoms 3 and 4 and the following cleavage of the linkage seems to
be essential for the existence of m/ z 269. Two of the investigated
compounds, chlorothiazide and benzthiazide, do contain double
bonds at this particular position and do not generate the common
fragment m/ z 269 or any other corresponding ion (Table 2). The
proposal that hydrogens are shifted within the suggested frag-
mentation is supported by the appearance of m/ z 272 and 271 in
the spectrum of methiazide-d4 (Table 2). Thus, two or three
deuteriums of C-3 or its neighboring methyl group have to migrate
from the leaving group to the anion. Other studies dealing with
stably labeled compounds report a comparable “scrambling” of
hydrogens in a negatively charged gas-phase ion during its
dissociation.21 The position of charge could not be determined,
but it is known from the literature19,21 that, with chemical and
electrospray ionization in the negative mode, generally the most
A total of 500 mg of the resulting 4-amino-6-chlorobenzene-
1,3-disulfonyl chloride (II) was treated with 6 N [15N]-ammonium
hydroxide at 80 °C under argon for 1 h. After being cooled to
ambient temperature, the mixture was stored at 4 °C for 10 h,
the precipitate was collected on a medium-porosity frit and washed
with bidistilled water (yield: 380 mg, 85% of the theory). The
doubly nitrogen-labeled 4-amino-6-chlorobenzene-1,3-disulfona-
mide was then condensed to the corresponding [15N2]-ethiazide
with propionaldehyde as described above.
Mass Spectrometry. All analyses were performed on a PE
Sciex API2000 triple quadrupole mass spectrometer (PE Bio-
systems, Foster City, CA) equipped with an electrospray interface.
The flow rate of the solution containing the analyte was 10 µL/
min, introduced by a syringe pump into the ESI interface. All
analytes were solved in a mixture of acetonitrile/ water (50:50, v/ v)
at a concentration of 5 µg/ mL. The ionization mode was negative
(20) Garbis, S. D.; Hanley, L.; Kalita, S. J. AOAC Int. 1 9 9 8 , 81, 948-957.
(21) Bowie, J. H. Mass Spectrom. Rev. 1 9 8 4 , 3, 161-207.
3804 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

Figure 2. ESI product ion scan of m/z 310 of synthesized methiazide.
acidic hydrogen is abstracted. Therefore, the charge is typically
located at the sulfonamide group. The following elimination of
sulfur dioxide (-64 amu) from m/ z 269 generates m/ z 205. This
fragment was also observed in the mass spectrum of 4-amino-6-
chlorobenzene-1,3-disulfonamide (Table 2), which can initially lose
SO2NH (-79 amu) under hydrogen rearrangement, producing the
same ion m/ z 205. The exchange of all nitrogen-bonded hydrogens
by deuteriums led to the expected corresponding fragment m/ z
209. A further loss of SO2NH produces the common ion m/ z 126
with the structure of deprotonated 3-chloroaniline, which was
demonstrated in an earlier study.18 In case of 6-trifluoromethylated
thiazides, such as hydroflumethiazide and bendroflumethiazide,
the counterparts to m/ z 269, 205, and 126 are detected with m/ z
303, 239, and 160, respectively. Obviously, the kind of substitution
of C-6 (Cl or CF3) does not essentially influence the presented
fragmentation process. The composition of m/ z 78 is proposed
to be SO2N- since the fragment shifts to m/ z 79 with 15N-labeling
but is constant under the conditions of deuterium labeling.
In Figure 3B, the generation of ions with a comparatively
decreased abundance is proposed, e.g., for ethiazide. All mass
spectra of 3-n-alkylated thiazides contain a fragment of (M - H
- 64)-, due to the loss of SO2 from the quasimolecular ion and a
possible formation of a five-membered ring structure. Subse-
quently, the elimination of HCN is observed, which includes
exclusively the nitrogen N-2. This was proven by the synthesis of
15N-labeled ethiazide, in which only the nitrogens of the sulfona-
mide group and N-2 were isotopically substituted. The analysis
of [15N2]-ethiazide showed the fragment m/ z 262 (corresponding
to m/ z 260) after removal of SO2 from (M - H)- and the following
loss of 28 mass units producing the fragment m/ z 234, owing to
15N. Further, the hydrogen involved in the
the elimination of HC
rearrangement of the HCN elimination definitely originates from
C-3 and not from any nitrogen. Evidence to this effect was obtained
by the analysis of methiazide-d4, which also expels 28 mass units
with DCN (Table 2). Thus, the removal of HCN of 3-alkylated
thiazides includes the migration of the alkyl group from C-3,
possibly to N-4, as demonstrated with ethiazide and its fragment
ion m/ z 233 in Figure 3B.
In general, the 3-alkylated and 6-chlorinated thiazides such as
methiazide, ethiazide, prothiazide, n-buthiazide, butizide, n-penthi-
azide, cyclopenthiazide, bemetizide, and cyclothiazide eliminate
HCl (Table 2), the hydrogen of which obviously originates from
different sources. With methiazide-d4, the loss of HCl and DCl is
observed in equal intensities, and also 4-amino-6-chlorobenzene-
1,3-disulfonamide, without the typical thiazide cyclization and
alkylation at C-3, eliminates HCl. A comparable phenomenon
(elimination of HCF3 or HF) is not present for those thiazides
bearing a trifluoromethyl group at C-6.
Another common fragment of 3-hydrogenated or 3-alkylated
and 6-chlorinated thiazides is m/ z 190 (Table 2). Its proposed
structure is shown in Figure 4A. The fact that N-4 remains in the
fragment was proven by [15N2]-ethiazide, which does not generate
an ion m/ z 191 owing to the absence of the amine group of the
sulfonamide unit. In the mass spectra of the 6-trifluoromethylated
thiazides, bendroflumethiazide and hydroflumethiazide, the frag-
ment m/ z 224, corresponding to m/ z 190, is also observed.
A distinction between 3-alkylated thiazides with identical
masses but different side-chain constituents such as butizide and
n-buthiazide or an open-chain structure compared to a cyclic
substitute appears to be difficult by means of mass spectrometry.
(22) Martinez-Alvarez, R.; Martin, N.; Seoane, C.; Suarez, M.; Perez, R.; Nour
Kayali, H. R. Rapid Commun. Mass Spectrom. 2 0 0 1 , 15, 758-762.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3805

Figure 3. (A) Proposed generation of the fragment ions m/z 269,
205, and 126 of 6-chloro-7-sulfamoyl-3-alkyl-3,4-dihydro-1,2,4-ben-
zothiadiazine 1,1-dioxides and hydrochlorothiazide. (B) Postulated
successive elimination of SO2 and HCN from ethiazide.
However, the influence on the fragmentation process of a double
bond or a complete functional unit such as a phenolic ring (e.g.,
bemetizide, Figure 1) is intense and informative with respect to
its structure. So, cyclothiazide (Figure 1) generates an ion m/ z
322 (Table 2) from the quasimolecular ion by a classical retro-
Diels-Alder rearrangement (Figure 4B), in addition to the
common fragments of 3-alkylated thiazides. In contrast, bemetizide
produces an ion (M - H - 106)- by the elimination of ethylben-
zene at the expense of the common fragmentation pattern. So,
m/ z 269, 205, and 126 appear with decreased intensities and
further individual ions are generated (Table 2). The mass
spectrum of bendroflumethiazide, which bears a benzyl group at
C-3, contains all common ions (with respect to the shift owing to
the substitution of Cl by CF3) and the ion m/ z 328, the origin of
which is the elimination of toluene from the quasimolecular ion.
3-Chloroalkylated 6-Chloro-7-sulfamoyl-3,4-dihydro-1,2,4-benzothia-
diazine 1,1-Dioxides. The halogenation of the C-3 alkyl side chain
results in a fragmentation pattern different from the one described
for 3-hydrogenated and 3-alkylated 7-sulfamoyl-3,4-dihydro-1,2,4-
benzothiadiazine 1,1-dioxides. Typical representatives of this class
of thiazides are methylclothiazide, trichlormethiazide, and teclothiaz-
ide (Figure 1). The mass spectra of those compounds are
dominantly characterized by the elimination of HCl and the
subsequent loss of SO2 (Table 3). In contrast to those thiazides
3806 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

Figure 4. (A) Proposed fragment ion structure of m/z 190 of
6-chloro-7-sulfamoyl-3-alkyl-3,4-dihydro-1,2,4-benzothiadiazine 1,1-
dioxides. (B) Possible retro-Diels-Alder rearrangement of the quasi-
molecular ion of cyclothiazide (m/z 388) to m/z 322.
without chlorination of the 3-methyl group (methiazide) or with
an alkyl side chain in general, the base peak is not (M - H)-.
Also, the fragmentation to m/ z 269, 205, and 126 is not observed,
probably due to the initial formation of double bonds into the
thiazide structure by neutral loss of hydrochloric acid. The lack
of those common fragment ions was also reported for chlorothi-
azide and benzthiazide, both of which contain a 3-4-double bond.
3-Alkylthiomethyl-6-chloro-7-sulfamoyl-1,2,4-benzothiadiazine 1,1-
Dioxides. The mass spectrum of epithiazide is shown as an
example for the substances of this class (Figure 5). All compounds
contain a side chain including a thioether functionality. Except
those compounds bearing a 3-4-double bond (benzthiazide) or a
2-N-methyl group (polythiazide), the diuretics of this section
(Table 3) generate the common fragments m/ z 269, 205, 126, and
78. The thioether linkage turned out to be of great interest since
it obviously represents a preferred position of dissociation. With
epithiazide, benzthiazide, and polythiazide, fragmentation occurs
on both sides of the sulfur atom, generating characteristic
fragments for this particular thiazide structure. The dissociation
of the side chain, including the elimination of the sulfur atom,
involves the migration of a hydrogen from the leaving group to
the remaining ion (e.g., m/ z 310 of epithiazide, Figure 5). The
ion m/ z 341 of althiazide and epithiazide (Table 3) obviously
results from a homologous fission of the S-C bond remote from
the thiazide nucleus. The corresponding fragments of benzthiazide
(m/ z 339, owing to its 3-4-double bond) and polythiazide (m/ z
355, owing to its 2-N-methyl group) are present, too. Such a
fragmentation requires the generation of two radicals from an
even-electron anion. Although the “even-electron rule” is com-
monly accepted23 and appropriate for many cases of mass
spectrometry, the literature reports several exemptions,24 the
number of which increases proportional to published fragmenta-
(23) McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra, 4th ed.;
University Science Books: Mill Valley, CA, 1993; Chapter 8.
(24) Karni, M.; Mandelbaum, A. Org. Mass Spectrom. 1 9 8 0 , 15, 53-64.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3807

Figure 5. ESI product ion scan of m/z 424 of epithiazide.
tion studies.25-29 In the case of althiazide, only the fission of the
S-C-bond remote from the thiazide nucleus is observed, and not
a neutral loss of the side chain including the sulfur atom. This
may be explained by the formation of a leaving group, which is
able to stabilize the generated radical by an allylic double bond.
In contrast, the mass spectrum of methyl thioethiazide does not
contain an ion resulting from the elimination of a methyl radical,
which would correspond to the homologous fragmentation de-
scribed above. Here, only the neutral loss of methylene sulfide
from (M - H)- is found (-48 Da, Table 3), generating m/ z 322.
The presence of a trifluoroethyl group in the side chain of
epithiazide and polythiazide intensively influences the fragmenta-
tion process. Besides the ions resulting from eliminations includ-
ing or excluding the sulfur atom, the multiple loss of HF (-20
Da) is observed from (M - H)- producing m/ z 404, 384, and
364, respectively (Figure 5). After removal of three HF molecules,
the subsequent elimination of SO2 generates m/ z 300. The
hydrogens expelled from the thiazides as HF obviously do not
originate from the nitrogens N-2 and N-4 since the product ion
spectrum of epithiazide after exchange of nitrogen-bonded hy-
drogens by deuteriums still presents the loss of three HF, but no
DF molecules.
that contain common and individual fragment ions, the generation
of which is dominantly influenced by the N-4-linked side chain.
This consists of n-, iso-, or cyclic alkyl groups, phenolic groups,
mono-, bis-, or trischlorinated methyl groups, or thioether chains.
The alkylated thiazides mainly generate the common fragments
m/ z 269, 205, and 126 besides their quasimolecular ion, except
those bearing a 3-4-double bond in the thiazide nucleus. The
exchange of the C-6-linked chlorine atom by a trifluoromethyl
group results in a shift of 34 mass units to the fragments m/ z
303, 239, and 160, respectively, in accordance to the Biemann shift
rule. The introduction of double bonds or heteroatoms such as
sulfur or halogen atoms into the side chain generates fragmenta-
tion patterns that show individual ions, especially those originating
from a homologous fission of linkages from an even-electron ion
or those generated by the elimination of hydrogen halides.
The possibility of differentiation of thiazide-based compounds
by mass spectrometry enables the characterization and classifica-
tion of compounds belonging to this class of drugs. The individual
and diagnostic fragment ions support the identification of sub-
stances, e.g., in biological matrixes of doping control samples, and
the knowledge of fragmentation behavior should facilitate the
identification of derivatives of these particular remedies.
CONCLUSION
ACKNOWLEDGMENT
The collision-induced dissociation of thiazides after electrospray
We thank Simon Biddle from HFL, UK, for supplying different
reference compounds and the Bundesinstitut fu¨ r Sportwissen-
schaft, Bonn, for financial support.
ionization in the negative ion mode gives rise to mass spectra
(25) Gioacchini, A. M.; Czarnocki, Z.; Arazny, Z.; Munari, I.; Traldi, P. Rapid
Commun. Mass Spectrom. 2 0 0 0 , 14, 1592-1599.
(26) Smyth, W. F.; McClean, S.; Ramachandran, V. N. Rapid Commun. Mass
Spectrom. 2 0 0 0 , 14, 2061-2069.
(27) Domingues, M. R. M.; Domingues, P.; Gajewski, M.; Czuchajowski, L.;
Ferrer-Correia, A. J. Rapid Commun. Mass Spectrom. 2 0 0 1 , 15, 908-914.
(28) Justesen, U. J. Mass Spectrom. 2 0 0 1 , 36, 169-178.
Received for review January 9, 2002. Accepted April 5,
2002.
(29) Qin, X. Z. J. Mass Spectrom. 2 0 0 1 , 36, 911-917.
AC020020E
3808 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002