Synthesis and characterization of succinylcholine-d18 and succinylmonocholine-d3 designed for simultaneous use as internal standards in mass spectrometric analyses

Article abstract of DOI:10.1002/jms.1230

Succinylcholine (SUX) is a routinely used yet potentially lethal depolarizing muscle relaxant, the detection of which poses severe problems to the clinical or forensic analyst: within a few minutes after its in vivo administration, SUX is broken down via succinylmonocholine (SMC) to yield the endogenous substances succinic acid and choline. For quantification of SUX and SMC in biological matrices using mass spectrometric detection, appropriate internal standards, i.e. deuterated analogs of the above substances, are indispensable but not commercially available. Internal standards for both substances were hence tailored to fit the analytical needs. The two-step synthesis and subsequent characterization of SUX-d₁₈ and SMC-d ₃ using a combination of nuclear magnetic resonance (NMR) spectroscopy, fast atom bombardment mass spectroscopy (FAB-MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) are described. SUX-d₁₈ was synthesized by reacting ethanolamine and iodomethane-d₃ in a first quaternization step to choline-d ₉, which in turn was esterified with succinyldichloride to yield the final product. SMC-d₃ was produced by esterification of succinic acid anhydride with dimethylaminoethanol, yielding desmethyl-SMC as intermediate product. The latter was then reacted with iodomethane-d₃ to obtain SMC-d₃. ¹H- and ¹³C-NMR data support the identity and purity as well as the designated deuteration of both preparations, findings which were further confirmed by FAB-MS as well as HPLC-MS/MS. Owing to a thoughtful design, the obtained substances SUX-d₁₈ and SMC-d ₃ feature different deuteration patterns at their trimethylamine moieties, and thus finally offer the possibility to simultaneously quantify SUX and SMC in clinical as well as forensic samples using isotope dilution mass spectrometry. Copyright

Full text of DOI:10.1002/jms.1230

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2007; 42: 929–939
Published online 7 June 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jms.1230
Synthesis and characterization of succinylcholine-d18
and succinylmonocholine-d3 designed for simultaneous
use as internal standards in mass spectrometric
analyses
Uta Kuepper,∗ Frank Musshoff and Burkhard Madea
Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany
Received 16 January 2007; Accepted 12 April 2007
Succinylcholine (SUX) is a routinely used yet potentially lethal depolarizing muscle relaxant, the detection
of which poses severe problems to the clinical or forensic analyst: within a few minutes after its
in vivo administration, SUX is broken down via succinylmonocholine (SMC) to yield the endogenous
substances succinic acid and choline. For quantification of SUX and SMC in biological matrices using mass
spectrometric detection, appropriate internal standards, i.e. deuterated analogs of the above substances, are
indispensable but not commercially available. Internal standards for both substances were hence tailored
to fit the analytical needs. The two-step synthesis and subsequent characterization of SUX-d18 and SMC-d3
using a combination of nuclear magnetic resonance (NMR) spectroscopy, fast atom bombardment mass
spectroscopy (FAB-MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC-
MS/MS) are described. SUX-d18 was synthesized by reacting ethanolamine and iodomethane-d3 in a first
quaternization step to choline-d9, which in turn was esterified with succinyldichloride to yield the final
product. SMC-d3 was produced by esterification of succinic acid anhydride with dimethylaminoethanol,
yielding desmethyl-SMC as intermediate product. The latter was then reacted with iodomethane-d3 to
obtain SMC-d3. 1H- and 13C-NMR data support the identity and purity as well as the designated deuteration
of both preparations, findings which were further confirmed by FAB-MS as well as HPLC-MS/MS. Owing
to a thoughtful design, the obtained substances SUX-d18 and SMC-d3 feature different deuteration patterns
at their trimethylamine moieties, and thus finally offer the possibility to simultaneously quantify SUX
and SMC in clinical as well as forensic samples using isotope dilution mass spectrometry. Copyright 
2007 John Wiley & Sons, Ltd.
KEYWORDS: succinylcholine; succinylmonocholine; synthesis; internal standard; isotope dilution; mass spectrometry
SUX is degraded by the unspecific plasma cholinesterase
(butyrylesterase; EC 3.1.1.8) to yield succinylmonocholine
(SMC), and finally, after complete hydrolyzation of ester
bonds, one molecule of succinic acid and two molecules of
choline.
The monitoring of SUX levels in a clinical setting and
especially the use of SUX as an asphyxiating agent in
(attempted) homicides or suicides confront the analyst with
several problems: firstly SUX is broken down within a few
minutes after its in vivo administration,1–7 and secondly the
postmortem detection of the more stable metabolite SMC
can no longer be regarded as a proof of SUX administration,
as it was recently found in SUX-negative control tissues.8,9
The analyte’s sensitivity to heat and alkaline conditions as
well as its pronounced hydrophilicity due to its precharged
character pose further problems, as these features complicate
SUX extraction.10,11
INTRODUCTION
The bis-quaternary amine succinylcholine (SUX) structurally
resembles the endogenous neurotransmitter acetylcholine
(ACh) and, as the latter, leads to an activation of post-
synaptic ACh-receptors. SUX is rapidly released by these
receptors, but compared to the native substrate ACh only
slowly degraded by the specific acetylcholinesterase (EC
3.1.1.7), thus resulting in a prolonged depolarization and
desensitization of the postsynaptic membrane. In the clinical
setting in which SUX is routinely employed as a mus-
cle relaxant during anesthetics, the concomitant respiratory
depression can be compensated by artificial respiration. If
respiratory assistance is not provided, a clinically effective
dose will cause hypoxic brain damage and finally lead to
death.
ŁCorrespondence to: Uta Kuepper, Institute of Forensic Medicine,
University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany.
E-mail: Uta.Kuepper@ukb.uni-bonn.de
Various techniques have been employed for the detec-
tion of SUX ranging from thin-layer chromatography12,13
Copyright  2007 John Wiley & Sons, Ltd.

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U. Kuepper, F. Musshoff and B. Madea
and gas chromatography of succinate esters12 to HPLC with
electrochemical1,3 or fluorescence detection,5 as well as to
several approaches using mass spectrometric detection such
as, for example, GC-MS of derivatized extracts,14,15 DI/EI-
MS16 and HPLC-ESI-MS.11 Lately, also tandem-MS tech-
niques like DI/ESI-MS/MS6,7,10 and HPLC-MS/MS(MS)9,17
have been reported. Only a minority of the latter authors
employing a mass spectrometric detection method did use
deuterated internal standards for quantification of SUX, i.e.
The present paper describes the successful synthesis
of SUX-d18 and SMC-d3 following modified methods of
Forney et al.14 and Phillips,22 respectively. For the first
time, deuterated internal standards for SUX and SMC
were designed for simultaneous use, and assessment of the
synthesis products was performed using elaborate analytical
techniques such as NMR, FAB-MS and HPLC-MS/MS.
EXPERIMENTAL
9
SUX-d6,6,7 SUX-d8 and SUX-d12.14,15
Chemicals
SMC has less often been the subject of research interest;
thus only a few authors have reported detection methods for
this metabolite, ranging from thin-layer chromatography13
to modern mass spectrometric methods like HPLC-ESI-
MS11 and HPLC-MS/MS(MS).8,9 Only in two studies a
deuterated internal standard, i.e. SMC-d4, was used for the
detection of SMC,8,9 but in both cases SMC-d4 was obtained
through hydrolysis of SUX-d8, thereby ruling out a parallel
quantification of both analytes. To the knowledge of the
authors, no simultaneous detection of SUX and SMC using
isotope dilution mass spectrometry has been achieved yet.
As suitable internal standards are not commercially
available, it was our immediate goal to synthesize deuterated
analogs of SUX and SMC and ascertain their identity as well
as their purity. Synthesis was designed in a way to ensure
different deuteration patterns of the trimethylamine groups
of both internal standards. Otherwise, owing to the above-
mentioned degradation of SUX to SMC, the parallel use
of both internal standards in a single sample must lead to
erroneous data: if the degradation product of the deuterated
SUX-analog is identical to the deuterated internal standard
for SMC, it will drastically interfere with the quantification
of the metabolite by causing an underestimation of SMC
concentration. The chosen derivatives SUX-d18 and SMC-
d3 ensure a correct determination of concentrations of the
metabolite as SMC-d9, which is the expected product of SUX-
d18 degradation, can easily be distinguished from the internal
standard SMC-d3 by mass spectrometric detection (Fig. 1).
Some authors have reported methods to synthesize
All chemicals used for synthesis were of analytical grade
and purchased from Sigma Aldrich, the only exception being
iodomethane-d3 which was obtained from Acros Organics.
Synthesis
SUX-d18 and SMC-d3 were prepared via 2-step syntheses as
detailed in the following sections.
SMC-d3
Step 1 – Preparation of desmethyl-SMC. One equivalent
(100 mmol, 10 g) of succinic acid anhydride and 1.12 equiv.
(112.2 mmol, 10 g, 11.29 ml) of dimethylaminoethanol in
100 ml of dry acetone were refluxed for 3 h in an oil bath
°
(75 C). After the reaction, a small amount of white precip-
itate was filtered off and discarded. The remaining acetone
filtrate was chilled on ice to crystallize the reaction prod-
uct, which was then filtered off and vacuum-dried. The
dry product was recrystallized from 5 ml of acetone. Before
employing the intermediate desmethyl-SMC in the following
reaction, its identity and purity were ascertained using NMR
spectroscopy and FAB-MS (data not shown).
Step 2 – Alkylation of desmethyl-SMC yielding SMC-d3. For the
subsequent reaction, 1 equiv. (10 mmol, 1.9 g) of desmethyl-
SMC and an excess of 3.2 equiv. (32 mmol, 4.65 g, 2 ml) of
iodomethane-d3 in 50 ml of dry acetone were refluxed for
°
24 h in an oil bath (75 C). After completion of the reaction
the solvent was distilled off, and the remaining residue was
dried under vacuum. The product was then recrystallized
from 10 ml of a 6 : 4 methanol : acetone (v/v) mixture and
collected by filtration. To the filtrate 10 ml of diethyl ether
were added to precipitate any solute product, which again
was collected by filtration.
SUX,18–20 SUX-d12 (via choline-d6),14 and possible (deuter-
21
ated) SUX precursors, as for example, choline-d9
or
desmethyl-SUX,15, as well as SMC22 and SMC-d4 via hydrol-
ysis of SUX-d8.8,9
Figure 1. Synthesis design featuring the differently deuterated trimethylamine groups of both internal standard substances. Possible
degradation of the internal standard SUX-d18 leads to SMC-d9, a metabolite that is clearly distinguishable from the deuterated
standard SMC-d3 using mass spectrometric detection.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

Synthesis of SUX-d18 and SMC-d3
931
Figure 2. Top: 1H-NMR spectrum of SUX-d18. The structural formula of the target compound is shown in the box. NMR signals are
assigned to distinct proton nuclei using matching letters. Signal multiplicity is described by s D singlet and m D multiplet. Bottom:
Extraction of the indicated region from the above standard 1H-NMR spectrum in superposition with the respective spectral region
from a 14N-decoupling experiment.
choline-d9 iodide was collected by filtration. Before employ-
ing choline-d9 in the following reaction, its identity and
purity were ascertained using NMR spectroscopy and FAB-
MS (data not shown).
SUX-d18
Step 1 – Preparation of choline-d9. Choline-d9 is commercially
available from various sources; for budget reasons, how-
ever, we chose to synthesize it ourselves. One equiv. of
ethanolamine (25 mmol, 1.53 g, 1.51 ml) and an excess of
3.2 equiv. (80 mmol, 11.6 g, 4.98 ml) of iodomethane-d3 in
10 ml of methanol were stirred for 24 h at room tempera-
ture. Sodium methoxide (2.1 equiv., 52.5 mmol, 2.84 g) was
dissolved in 25 ml of methanol and added in 7.5, 8.75 and
8.75 ml aliquots 1, 3.25 and 6.25 h after initiation of the reac-
tion, respectively, i.e. when the pH of the reaction mixture
had returned to neutral. Upon completion of the reaction,
the solvent was distilled off and the remaining product was
dried under vacuum. The residue was recrystallized twice
from 15 and 12 ml of ethanol, respectively, and purified
Step 2 – Reaction of succinyldichloride with choline-d9 yielding
SUX-d18. In a chilled reaction flask, 0.4 equiv. (1.68 mmol,
0.26 g, 185 µl) of succinyldichloride was added dropwise
to 1 equiv. (4.2 mmol, 1 g) of choline-d9 before addition of
10 ml of dry chloroform. The reaction mixture was slowly
°
heated in an oil bath and then refluxed for 12 h at 60 C.
For product cleanup, the solvent was removed using a
rotary evaporator and finally high vacuum. Together with
1 equiv. (4.2 mmol, 0.63 g) of NaI the dried product was
dissolved in 450 ml of 1 : 2 methanol : ethanol (v/v). SUX-
d18 iodide crystallized upon cooling of the reaction mixture,
Copyright  2007 John Wiley & Sons, Ltd.
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DOI: 10.1002/jms

932
U. Kuepper, F. Musshoff and B. Madea
Figure 3. 13C-NMR spectrum of SUX-d18. The structural formula of the target compound is shown in the box. NMR signals are
assigned to distinct carbon nuclei using matching letters. Signal multiplicity is described using s D singlet, t D triplet and
m D multiplet.
and was collected by filtration. A subsequent cleanup step
involved recrystallization of the product from 450 ml of a 1 : 2
methanol : ethanol (v/v) mixture and filtration of product
crystals.
(HPLC-)ESI-MS/MS
The data were acquired using a PE Sciex API 2000 mass
spectrometer in combination with an Agilent 1100 Series
HPLC. For ESI-MS/MS, a turbo ion spray source was used
°
in positive ion mode at a temperature of 350 C and an
ion spray voltage of 5000 V. Nitrogen was employed as the
collision gas.
NMR
Product ion spectra were recorded in the multiple
channel acquisition (MCA mode, 10 scans) using a 10 µg/ml
methanolic stock solution of each compound at collision
energies of 25 eV for both SMC and SMC-d3, as well as 18
and 16 eV for SUX and SUX-d18, respectively.
Subsequent mass spectrometric data were acquired in
the multiple reaction monitoring (MRM) mode with the
following transitions (quantifier in bold print, respective
collision energies in parentheses):
Spectra were recorded at room temperature using Bruker
DPX 300 and DPX 400 instruments (Bruker, Rheinstetten,
Germany). Chemical shifts are expressed in ppm relative to
the standard tetramethylsilane and were obtained through
referencing of solvent residual signals to the respective lit-
erature values.23 Data were analyzed using 1D-WinNMR
(Bruker Daltronic, Bremen, Germany). For NMR spec-
troscopy, SUX-d18 and SMC-d3 were dissolved in D2O and
MeOD to yield concentrations of approx. 35 and 65 mg/ml,
respectively. Both were measured on the DPX 300 instru-
ment.
SUX : 145.1 → 115.1 ꢀ15 eVꢁ; 145.2 → 79.2 ꢀ21 eVꢁ
SUX-d18 : 154.0 → 120.0 ꢀ16 eVꢁ; 154.0 → 98.0 ꢀ21 eVꢁ
SMC : 204.0 → 144.7 ꢀ20 eVꢁ; 204.0 → 98.6 ꢀ32 eVꢁ
SMC-d3 : 207.1 → 144.8 ꢀ22 eVꢁ; 207.1 → 98.8 ꢀ34 eVꢁ
NMR analysis on the intermediates was performed using
75 and 95 mg/ml samples in MeOD for desmethyl-SMC
and choline-d9, respectively. Choline-d9 and desmethyl-SMC
were measured on the DPX 300 and DPX 400, respectively.
For separation, a Phenomenex Synergi Hydro RP (150 ð
2 mm, 4 µm) column was used. Gradient elution using 5 mM
ammonium formate in H2O : acetonitrile 90 : 10 (v/v, solvent
A) and 5 mM ammonium formate in H2O : acetonitrile 10 : 90
(v/v, solvent B) was performed (0–3 min: 95% A/5% B;
3–5 min: 95% A →5% A/5% B →95% B; 5–8 min: 5% A/95%
FAB-MS
The spectra were acquired on a Concept 1H (Kratos,
Hofheim, Germany) instrument, using meta-nitrobenzyl
alcohol (mNBA) as matrix. The amount of product employed
for FAB-MS analysis was not exactly determined.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

Synthesis of SUX-d18 and SMC-d3
933
Figure 4. Top: 1H-NMR spectrum of SMC-d3. The structural formula of the target compound is shown in the box. NMR signals are
assigned to distinct proton nuclei using matching letters. Signal multiplicity is described using s D singlet, h D heptet, and
m D multiplet. Bottom: Extraction of the indicated region from above standard 1H-NMR spectrum in superposition with the
respective spectral region from a 14N-decoupling experiment.
B; 8–10 min: 5% A → 95%A/95%B ! 5% B; 10–13 min: 95%
A/5% B) at a flow rate of 200 µl/min.
For the characterization of the synthesized products a 5 µl
aliquot of a 1 µg/ml solution each of SUX-d18 and SMC-d3
was injected for analysis.
RESULTS
Recoveries
Final recovery for the intermediate desmethyl-SMC was
determined to be 36.4%. Combined SMC-d3 corresponded
to a recovery of 73.0%. For the intermediate choline-d9, a
recovery of 62.5% was determined. At 22.2%, the recovery
was lowest for synthesis of SUX-d18; however, it has to be
kept in mind that the methods were not optimized for high
recoveries but, on the contrary, care was taken to obtain the
cleanest possible material in each step.
Exemplary chromatograms were recorded by analyzing
spiked urine and serum sample extracts. Serum was esterase-
inactivated by addition of paraoxon. The aqueous sample
matrix was spiked with 1 µg/ml each of SUX and SMC
as well as the internal standard mix, yielding a final
concentration of 1 µg/ml of both SUX-d18 and SMC-d3. The
extraction procedure is based on ion pairing reactions and
employs a polymeric reversed phase solid-phase extraction
(SPE) cartridge. Quantitation was performed by calculating
the peak area (area under the curve, AUC) ratio of the
analyte’s to the respective internal standard’s signal, and
linear calibration curves were obtained for both biological
matrices. Method development, full validation and testing
of its applicability using biological samples are currently in
progress and will be described elsewhere.
NMR
SUX-d18 1H spectrum
For SUX-d18, the 1H spectrum comprises four distinct signals
(Fig. 2). Integration of these signals proves that signal (a) with
its odd integral value of 0.87 represents the solvent residual
signal, and the remaining three signals, each with an even
integral value of 4.00, can thus be attributed to the analyte. As
already indicated by the above-mentioned integral values,
four protons contribute to each of the latter signals. To
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

934
U. Kuepper, F. Musshoff and B. Madea
Figure 5. 13C-NMR spectrum of SMC-d3. The structural formula of the target compound is shown in the box. NMR signals are
assigned to distinct carbon nuclei using matching letters. Signal multiplicity is described using s D singlet, t D triplet and h D heptet.
Figure 6. FAB-MS spectrum of SMC-d3. The spectrum contains a main peak at m/z 207.1, corresponding to the isotopic molecular
mass of the [M]C ion. The minor peaks in this spectrum can be attributed to the complexes [2 ð M ꢀ H]C and [2 ð M C I]C. There is
no evidence of incomplete deuteration or contamination of the product.
further assign the 12 protons in question, the chemical shift
as well as the multiplicity data were used. The assignment
of the four protons contributing to the singlet in (d) is
unambiguous: only shielded protons, i.e. protons in a carbon-
rich environment, can give a comparable upfield signal.
All other protons are bound to carbons, which in turn are
bound to more electronegative heteroatoms, thus causing
a deshielding effect that leads to higher values for υ, and
the respective NMR signals can therefore be expected to be
more downfield shifted. The final assignment of the (b) and
(c) signals might seem surprising at first: the (c)-protons, in
the vicinity of an oxygen atom, are usually predicted to result
in more downfield signals as compared to the (b)-protons,
which are closest to a (less electronegative) nitrogen. The
presented assignment could however be confirmed using a
14N-decoupling experiment, showing that upon suppression
of 14N-coupling effects the multiplicity of the signal in
(b) resembles that in (c). It was thus proved that the protons
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

Synthesis of SUX-d18 and SMC-d3
935
Figure 7. FAB-MS spectrum of SUX-d18. The major mass peak at m/z 307.1 corresponds to [M ꢀ H]C, and the other two major
peaks can be attributed to [M C I]C at m/z 435.2 and [M-CD3]C at m/z 290.3. The minor peaks correspond to complexes between the
analyte and the matrix (m/z 461.3) or fragments of the matrix (m/z 354.3), as well as different fragments of the analyte or complexes
thereof (e.g. iodo-adduct to choline at m/z 239.2). There is no sign of incomplete deuteration or contamination of the substance.
SMC-d3 1H spectrum
(b) are the ones in the closest vicinity to the nitrogen atom.
The 1H spectrum of SMC-d3 contains six discernible
signals (Fig. 4). Integration data established that signals
(a) and (d) with their odd integral values of 0.96 and 0.52
are due to an H2O trace impurity and solvent residual
signal, respectively. Comparison of the chemical shifts with
reference data23 confirms this assignment. The remaining
four signals can all be attributed to the analyte. Integral
values were determined to be 2.0, 2.0, 6.0 and 4.0 for the
signals in (b), (c), (e) and (f), respectively. Together with
the chemical shift data, this leads to the conclusion that the
signal in (f) is derived from the most shielded protons in
the target compound, i.e. the protons of the succinic acid
moiety. The six methyl protons give rise to the singlet in (e),
this assignment being consistent with their expectedly lower
shielding, i.e. the higher chemical shift of the resulting signal,
due to the vicinal nitrogen. Two protons each contribute to
the signals in (b) and (c), respectively, and in this case the
same is true as for the 1H spectrum of SUX-d18: again it
could be proven via a 14N-decoupling experiment that the
protons denoted by (b) are the ones in the closest vicinity
to the nitrogen atom. The final assignment is summarized
in Fig. 4, which further shows that no excess resonances
indicative of organic impurities or incomplete deuteration
were detectable in the 1H-NMR spectrum.
The final assignment is summarized in Fig. 2, and it can be
stated that this assignment is consistent with the chemical
shift, integral as well as multiplicity data derived from the
acquired spectrum. The same figure emphasizes the fact
that no excess signals that would be indicative of organic
1
impurities or incomplete deuteration are visible in the H-
NMR spectrum of SUX-d18, and it can therefore be assumed
that the product is of high purity.
SUX-d18 13C spectrum
The 13C spectrum of SUX-d18 contains five discernible signals,
all of which can be assigned to the analyte (Fig. 3). The
signals in (a) and (e) can clearly be attributed to the carbons
in the carboxyl and succinic acid moieties of the molecule,
respectively: owing to the immediate vicinity of two oxygens,
the chemical shifts for the nuclei in (a) are at the downfield
end of the spectrum. In contrast, the carbon signals in (e),
not being bound to more electronegative heteroatoms, can
be found towards lower values for υ.
In 13C-NMR the signal intensity, usually small due to low
abundance and further signal splitting, is enhanced using
proton decoupling. With this in mind, the multiplet signal in
(d) can undoubtedly be assigned to the methyl carbons of the
analyte; the six respective carbons are each bound to three
deuterium atoms (nondecoupled) and one nitrogen atom
(nondecoupled), and the 13C signal is thus split multiple
times and consequently barely rises above the baseline. The
signals in (b) and (c) can be assigned using their respective
multiplicity: the fact that the carbon signal in (b) is a triplet
strongly suggests that the corresponding carbon nucleus, in
contrast to the one in (c) yielding a singlet signal, is the one
subjected to (13C, 14N) dipolar coupling, and therefore the
one in the closest vicinity to the nitrogen atom. The complete
assignment is depicted in Fig. 3. Also in the 13C spectrum of
SUX-d18 no excess signals indicative of organic impurities or
incomplete deuteration are visible.
SMC-d3 13C spectrum
In the 13C spectrum of SMC-d3, eight well-resolved res-
onances were detected (Fig. 5), one of which, the heptet,
represents the solvent residual signal. The remaining seven
signals were assigned as follows: resonances (a) and (b) are
derived from strongly deshielded carbon nuclei, i.e. the
carboxy- and ester-group carbons, respectively. In contrast,
the (g) and (h) signals can be attributed to the highly shielded
carbons in the backbone of the succinic acid moiety. The
remaining three signals can be assigned using a combination
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

936
U. Kuepper, F. Musshoff and B. Madea
Figure 8. Product ion spectra of SMC (a), SMC-d3 (b), SUX (c) and SUX-d18 (d). The figure emphasizes the differences in product ion
patterns between the deuterated and the respective nondeuterated compound in mass spectrometric analysis, and indicates their
applicability as internal standards in HPLC-MS/MS method development.
of multiplicity and integral data: The resonance in (d) is a
singlet and can therefore be attributed to the carbon next to
the ester bond. In contrast, the signals in (e) and (c) are split
into triplets, signifying close proximity to the nitrogen atom.
To further assign these resonances, the integral data was
taken into account: integration of 13C-NMR signals is not as
meaningful as the integral data in 1H spectra; it can, however,
be used as a clue for assignment. In this case, the integration
of the NMR signal in (e) yields a value that is about twice
that of signal (c), and it can therefore be assumed that the
two nondeuterated methyl carbons give rise to the former
resonance (the deuterated methyl carbon would be nonde-
coupled and therefore barely contribute to the signal), while
the latter is derived from the backbone carbon in the choline
moiety next to the quaternary amine group. The final assign-
ment is depicted in Fig. 5. The absence of excess signals in the
presented spectrum confirms that the preparation is correctly
deuterated and not contaminated with organic impurities.
The interpretation of the SMC-d3 FAB-MS spectrum is
summarized in Fig. 6.
SUX-d18
The FAB-MS spectrum of SUX-d18 is more complex: the major
mass peak in this spectrum is visible at an m/z of 307.1 which
corresponds to [M ꢀ H]C, and the other two major peaks can
be attributed to [M C I]C at m/z 435.2 and [M ꢀ CD3]C at
m/z 290.3. All other peaks are below 30% intensity: the ones
featuring a higher m/z correspond to complexes between the
target analyte and the matrix (m/z 461.3) or fragments of the
matrix (m/z 354.3), while peaks in the low-mass region of
the spectrum can be attributed to different fragments of the
analyte or complexes thereof, e.g. the choline moiety of the
product coupled to an iodine (m/z 239.2). The SUX-d18 FAB-
MS spectrum as well as its interpretation is depicted in Fig. 7.
No signal indicative of incomplete deuteration or signif-
icant amounts of impurities was found in any of the two
preparations.
FAB-MS
(HPLC-)ESI-MS/MS
SMC-d3
The product ion spectra of deuterated as well as nondeuter-
ated SUX and SMC are depicted in Fig. 8(a–d). Comparing
the respective signals at the approximate mass-to-charge
ratios of m/z 145, 99 and 101, it becomes visible that SMC
(Fig. 8(a)) and SMC-d3 (Fig. 8(b)) break down into identical
FAB-MS analysis of SMC-d3 yields a spectrum with one main
peak at m/z 207.1, which corresponds to the expected mass
of the [M]C ion. The minor peaks in this spectrum can be
attributed to the complexes [2 ð M ꢀ H]C and [2 ð M C I]C.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

Synthesis of SUX-d18 and SMC-d3
937
Figure 9. HPLC-MS/MS of an internal standard solution with 1 µg/ml of both SUX-d18 and SMC-d3. Eight transitions (1 quantifier
and 1 qualifier for each analyte) were monitored as detailed in ‘Experimental’. (a) Both deuterated analogs yield good signal
intensities for their respective quantifier as well as qualifier transitions. (b) In the same sample no nondeuterated SUX or SMC is
detectable above the background noise.
fragments. These fragments are, however, derived from dif-
ferent precursor ions, i.e. m/z 204.0 for SMC versus m/z 207.1
for SMC-d3, and distinguishable MRM transitions can there-
fore be defined. In contrast to the similar product ion spectra
of SMC and SMC-d3, the respective spectra of SUX (Fig. 8(c))
and SUX-d18 (Fig. 8(d)) are easily differentiated: the former,
with a precursor ion of m/z 145.1, gives rise to product ions
at m/z 115.1, 93.4 and 79.2, but the latter (m/z 154.0), in
contrast, produces fragments of m/z 120.0, 98.0 and 84.0. The
product ion spectra of the nondeuterated compounds and
their deuterated analogs were subsequently used to define a
set of unique MRM transitions for all substances.
As is visible from the diagram in Fig. 9(a), the synthesized
compounds yield good signal intensities for their respective
quantifier as well as the qualifier transition in HPLC-MS/MS.
No nondeuterated SUX or SMC was detectable above the
level of background noise (Fig. 9(b)).
Exemplary analyses of spiked serum (top) and urine
(bottom) sample extracts are depicted in Fig. 10. It is shown
that both nondeuterated analytes as well as their respective
deuterated analogs yield well-defined signals in HPLC-
MS/MS following sample extraction. The test samples were
correctly quantified upon consulting with each applicable
calibration curve (data not shown). Altogether, the presented
HPLC-MS/MS results demonstrate the applicability of both
SUX-d18 and SMC-d3 as reliable internal standards in mass
spectrometric analyses.
DISCUSSION
The synthesis and complete characterization of SMC-d3
and SUX-d18 is reported. Synthesis was performed using
two-step protocols and optimized for high purity of the
resulting preparations. The products were confirmed to be
chemically pure SMC-d3 and SUX-d18 using 1H- and 13C-
NMR spectroscopy as well as FAB-MS and HPLC-MS/MS:
In NMR spectroscopy, all resonances could undoubtedly
be assigned to defined nuclei or groups of nuclei. The
integration data, i.e. the number of protons or, in one case,
carbons giving rise to a certain signal, was always consistent
with the respective target structure. Multiplicity of signals
as well as 14N-decoupling experiments were also used for
assignment, and the results corresponded in all cases to the
respective structural formula. It was shown that in none of
the acquired spectra excess signals, i.e. evidence for organic
impurities or incomplete deuteration, could be detected,
findings that were confirmed by both mass spectrometric
methods.
Product ion scans of deuterated as compared to non-
deuterated compounds indicated the applicability of SUX-d18
and SMC-d3 as internal standards for mass spectrometric
analyses, and were used for the definition of unique MRM
transitions. The simultaneous detection of SUX and SMC
in biological media finally demonstrated the suitability of
both SUX-d18 and SMC-d3 as internal standards in the further
development of an HPLC-MS/MS method.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms

938
U. Kuepper, F. Musshoff and B. Madea
Figure 10. Exemplary HPLC-MS/MS chromatograms of spiked serum (top) and urine (bottom) sample extracts proving the
applicability of the synthesized compounds as internal standards enabling the simultaneous detection of SUX and SMC. Each
sample contained 1 µg/ml of both SUX and SMC. Internal standards were employed in concentrations of 1 µg/ml of each SUX-d18
as well as SMC-d3. For purposes of clarity only the quantifier transition is shown for each of the four compounds.
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able. Owing to thoughtful design, SUX-d18 and SMC-d3
feature different deuteration patterns at their trimethylamine
moieties and therefore offer the unique possibility to simul-
taneously quantify SUX and its metabolite SMC for clinical
and forensic purposes.
Acknowledgements
The authors would like to thank Prof. Dr M. Famulok and Dr S. Fusz
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Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 929–939
DOI: 10.1002/jms