Liquid Chromatography–Tandem Mass Spectrometry Detection of ...

Case Report
Liquid Chromatography–Tandem Mass Spectrometry
Detection of the Quaternary Ammonium Compound
Mebezonium as an Active Ingredient in T61 ®
Katrin M. Kirschbaum 1 , Wolfgang Grellner 2 , Gertrud Rochholz 3 , Frank Musshoff 1, *, and Burkhard Madea 1
1 Institute of Forensic Medicine, University of Bonn, Bonn, Germany; 2 Institute of Forensic Medicine, University of Göttingen,
Göttingen, Germany; and 3 Institute of Forensic Medicine, University Hospital Schleswig-Holstein, Kiel, Germany
Abstract
Quaternary ammonium compounds pose an analytical challenge.
Mebezonium, a muscle-relaxing agent contained in veterinary
euthanasia solution T61, was analyzed in body fluids, organs, and
injection sites of a veterinarian by liquid chromatography–tandem
mass spectrometry (LC–MS–MS) method. Additionally,
embutramide and tetracaine, which are two other active
ingredients contained in T61, methadone, xylazine, and analgesics
were detected by LC–MS–MS and high-performance liquid
chromatography–ultraviolet detection methods. For detection of
mebezonium a solid-phase extraction (SPE) combined with ionpairing
reagent heptafluorobutyric acid was developed. Separation
was achieved on Phenomenex Synergi Hydro RP C 18 column
combined with ammonium formate buffer and acetonitrile
(pH 3.5). To enrich other drugs, liquid–liquid extraction procedures
were used. Most of these drugs were separated on a Restek Allure
PFP Propyl column using the mentioned mobile phase.
Mebezonium and embutramide were detected in femoral vein
serum in concentrations of 10.9 and 2.0 mg/L, respectively. The
concentration of xylazine and methadone in serum was 2.0 and
0.4 mg/L, respectively. The LC–MS–MS method with SPE combined
with an ion-pairing reagent allowed the quantitation of
mebezonium. Methadone was detected in toxic concentrations and
was, in combination with xylazine and T61, considered to be the
cause of death.
Introduction
Quaternary ammonium compounds that might be used as
muscle relaxants are analytical challenges. Because of a
missing chromophore, thermal instability, and hydrophilicity
* Author to whom correspondence should be addressed: Prof. Dr. Frank Musshoff,
Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany.
Email: F.Musshoff@uni-bonn.de.
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due to a permanent positive charge, they are difficult to extract
and to analyze (1,2).
Several methods for determination of quaternary ammonium
compounds are described in the literature. Solid-phase
extraction (SPE) with C 18- (3,4) or CBA-columns (5), partly in
combination with an ion pair reagent like heptafluoric acid
(HFBA), are used for screening or quantitative analysis. One
method describes a fluid–fluid extraction with addition of
potassium iodide (6) followed by liquid chromatography–mass
spectrometry (LC–MS) analysis.
The quaternary ammonium compound mebezonium iodide
(Figure 1) is a competitive inhibitor of acetylcholine at nicotinergic
acetylcholine receptors (7). It is an active substance in
T61, a euthanasia drug. Embutramide and tetracaine are also
active substances of the drug. Embutramide has narcotic properties,
it induces deep anesthesia and an inhibition of the respiratory
center located in the brainstem. Tetracaine is a local
anesthetic substance and decreases painful reactions at injection
sites (7).
In the literature, few cases of suicides or homicides involving
the use of T61 are described (7–17). In most cases, embutramide,
which is exclusively used in T61, was the only substance
analyzed because of the difficulties with the analysis of
mebezonium. Embutramide can be analyzed by thin-layer
liquid chromatography followed by ultraviolet (UV) detection
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Journal of Analytical Toxicology, Vol. 35, March 2011
Figure 1. The chemical structure of mebezonium iodide.

Journal of Analytical Toxicology, Vol. 35, March 2011
(8), by gas chromatography (GC)–MS (9,14,18,19) or by a nitrogen-phosphorus
detector (20), by high-performance liquid
chromatography (HPLC) followed by UV-spectroscopy (15,19),
or by MS (21). In only two cases of human intoxication in
which embutramide was analyzed were mebezonium (8) or
mebezonium and tetracaine (14) analyzed, too.
In one case, the quaternary ammonium compound was
quantified by thin-layer LC and UV detection (8). Kintz et al.
(14) used liquid–liquid extraction combined with potassium iodide
for extraction followed by LC–MS analysis.
In a case of suicide and fatal intoxication of a former veterinarian
(22), we analyzed each active ingredient of T61. The
man had suffered from metastatic intestine cancer, and shortly
before his death, a tumor at the base of his skull was diagnosed.
Several other veterinary drugs and analgesics were analyzed in
blood, urine, cerebrospinal fluid, brain, muscle, kidney, liver,
and injection sites as well as in a syringe and in drug bottles
found at the death scene. The former veterinarian not only injected
T61 but also xylazine, an α 2-adrenergic agonist used as
a sedative, analgesic, and muscle relaxant in animals (23), and
L-Polamivet ® , with levomethadone as active ingredient which
is used as analgesic.
Materials and Methods
Solutions and buffers were purchased in HPLC grade from
Merck (Darmstadt, Germany). Deuterated internal standards
were obtained from Cerilliant (Round Rock, TX). All other
substances were obtained from local pharmacies.
The quantitative analysis of most substances was performed
by LC–MS–MS systems. If available, deuterated internal standards
were used as analyte to internal standard concentration
for quantitation. Calibration was made by five or six concentrations
of spiked and extracted serum control samples. Concentrations
were in accordance with therapeutic and toxic
levels of each substance. The analgetics ibuprofen and paracetamol
were analyzed by routine methods using HPLC–DAD.
Detection of mebezonium
A recently published method for detection of succinylcholine
and succinylmonocholine (24) was modified and used for the
analysis of mebezonium. Samples of 0.5 and 1 mL were extracted
using 55-µm strata-C 18 cartridges (200 mg/3 mL, Phenomenex,
Aschaffenburg, Germany) with addition of HFBA as
an ion pair reagent and succinylcholine-d 18 as the internal
standard. The LC–MS–MS system includes an API 2000 triplequadrupole
MS and an Agilent 1100 HPLC-system. Analytes
were separated on a Synergi Hydro-RP C 18 column (150 × 2
mm, 4 µm, Phenomenex) using gradient elution with ammonium
formate buffer (5 mM, pH 3.5) and acetonitrile [eluent A
90:10 (v/v); eluent B 10:90 (v/v)]. Positive electrospray ionization
was used and analysis was performed by MRM, using the
precursor ion for mebezonium at m/z 148 and the product
ions at m/z 236 (target) and 60 (qualifier) and for succinylcholine-d
18 as internal standard using precursor ion at m/z 154
and product ion at m/z 120.
Detection of embutramide, xylazine, tramadol,
tilidine, nortilidine, bisnortilidine, 4-aminoantipyrine,
and 4-methylaminoantipyrine
To analyze embutramide, xylazine, tramadol, tilidine, bisnortilidine,
4-aminoantipyrine, and 4-methylaminoantipyrine,
we precipitated 100 µL of blood, urine, and cerebrospinal fluid
and 100 µL of tissue homogenized with water (1:10, m/m)
with 100 µL of acetonitrile as sample preparation. The supernatant
was injected directly. Chromatographic separation was
perfomed by an Agilent 1100 HPLC-system (Waldbronn, Germany)
with an Allure-PFP-Propyl column (50 × 2.1 mm, 5
µm, Restek, Bad Homburg, Germany) in combination with
ammonium formate buffer (5 mM, pH 3.5) and acetonitrile
[eluent A 90:10 (v/v); eluent B 10:90 (v/v)] was used with a total
run time of 15 min. For MS detection, API 2000 triplequadrupole
MS (Applied Biosystems, Darmstadt, Germany)
with ESI in positive mode was used. Analysis was performed by
multi-reaction monitoring (MRM) using a precursor ion and
two product ions for each substance: embutramide m/z 294 →
121 (T) and m/z 294 → 91 (Q), xylazine m/z 221 → 164 (T) and
m/z 221 → 90 (Q), tramadol m/z 264 → 58 (T) and m/z 264 →
115 (Q), tilidine m/z 274 → 155 (T) and m/z 274 → 77 (Q), nortilidine
m/z 260 → 155 (T) and m/z 260 → 77 (Q), bisnortilidine
m/z 246 → 155 (T) and m/z 246 → 229 (Q), 4-aminoantipyrine
m/z 204 → 56 (T) and m/z 204 → 94 (Q),
4-methylaminoantipyrine m/z 218 → 56 (T) and m/z 218 → 97
(Q).
Detection of codeine, methadone, and EDDP
Codeine, methadone, and EDDP were analyzed according to
a validated method by Kirschbaum et al. (25). After the addition
of 10 µL of an internal standard solution (codeine-d 3,
methadone-d 3, and EDDP-d 3) and 150 µL buffer pH 11 to 0.5
mL sample the mixture was extracted with 2 mL of n-butyl
chloride. Then the organic layer was evaporated to dryness
and reconstituted with 100 µL of eluent A. Chromatographic
separation was performed on an Allure-PFP-propyl column
(50 × 2.1 mm, 5 µm) at 40°C in combination with the eluents
described. The LC–MS–MS system includes an LC-20 prominence
HPLC system (Shimadzu) and an API 4000 MS (Applied
Biosystems). Positive electrospray ionization was used and
analysis was performed by MRM using the precursor ion and
two product ions for each substance: codeine m/z 300 → 152
(T) and m/z 300 → 115 (Q), methadone m/z 310 → 265 (T) and
m/z 310 → 105 (Q), and EDDP m/z 279 → 235 (T) and m/z 279
→ 250 (Q).
Results and Discussion
For the quantitative detection of the mebezonium ion an established
and validated method for succinylcholine and succinylmonocholine
using SPME with HFBA as an ion pair
reagent was suitable. The selectivity for mebezonium was tested
with blank serum and showed no interfering peaks. Precision
was 13.9% for 500 µg/L (n = 6) and 8.6% for 2500 µg/L (n = 6).
The accuracy was 101.2% and 99.3% (n = 6), respectively. Sam-
125

ples that were extracted using C 18 cartridges showed two peaks
with retention times of 2.8 and 3.1 min (Figure 2) when using
a gradient starting with 80% eluent A for the first 3 min decreasing
to 20% eluent A for the following 2 min. When starting
the gradient with 95% eluent A, the retention time changed to
1.2 min and only one peak was observed with an identical mass
spectrum of the product ion scan (Figure 3). The extraction
Figure 2. Product ion scan of an extracted serum sample spiked with 5 mg/L mebezonium and mass
spectra of the two peaks (RT 2.8 and 3.1 min) using extraction with C 18 cartridges and gradient starting
with 80% eluent A.
Figure 3. Product ion scan of an extracted serum sample spiked with 5 mg/L mebezonium and mass spectrum
of one peak (RT 1.2 min) using extraction with C 18 cartridges and gradient starting with 95% eluent
A.
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Journal of Analytical Toxicology, Vol. 35, March 2011
method with C 18 cartridges was applied to standard solutions,
extracted calibration samples and real samples. Only one peak
was detected after extraction with STRATA-XC cartridges using
a gradient starting with 80% eluent A. This peak was much
lower in its intensity. Mass spectra of the product ion scans
showed that there is the identical substance after extraction
with C 18 cartridges and a gradient starting with 80% eluent A in
both peaks (Figure 2). Therefore, the sum
of both peaks was used for quantitation.
Clustering of mebezonium with a variable
number of iodide ions and subsequent insource
fragmentation might be an explanation
for occurrence of two peaks. In
contrast to Kintz et al. (14), who quantitated
a specific ion of m/z 294, we used the
transition of m/z 148 → 236 of the double
charged molecule.
Additional substances could be quantitated
by few methods and simple extraction
procedures or precipitation. The especially
low sample volume needed for
each method enabled the quantitation of
all analytes. Extracted calibration curves
covered the range of the detected concentrations.
They were linear and revealed
correlation coefficients of r > 0.995. At
the lowest calibrator point, the signal-tonoise
ratio was always higher than 10 for
all target compounds.
Embutramide, mebezonium, tetracain,
xylazine, and methadone were detected in
fluids and tissues of the deceased veterinarian
(Table I). These substances were
also found in a syringe and drug-containing
bottles found at the crime scene,
as well as in high concentrations in injection
sites of a port located near the clavicula
and in the hypogastric region. Concentrations
of embutramide and
mebezonium of 2.0 and 10.9 mg/L, respectively,
in vein blood serum and 1.3
and 0.5 mg/L, respectively, in urine are
comparable to concentrations measured
in the case R.C. reported by Bertol et al.
(8). They detected concentrations of 3.0
mg/L embutramide and 4.5 mg/L mebezonium
iodide in blood and 2.0 mg/L embutramide
and 0.8 mg/L mebezonium iodide
in urine in an 80-year-old retired
veterinarian who had injected T61 intramuscularly.
Different fatal cases showed
much higher concentrations of embutramide
with 12.1 and 15.5 mg/L [cases
B.L. and D.C.S. (8)], 31.0 mg/L (9), 43.0
mg/L (14), and 90.0 mg/L (15). In those
cases the drug had been administered either
intravenously or orally.
The concentration of xylazine in serum

Journal of Analytical Toxicology, Vol. 35, March 2011
Table I. Concentrations of the Veterinary Drugs and Analgesics (µg/L) in Different Fluids and Tissues
Serum Serum Cerebrospinal
Analyte Urine Heart Left Vein* Fluid Brain Muscle Kidney Liver
Mebezonium 516 44,500 10,900 4660 442 3050 31,100 17,700
Embutramide 1280 6020 2020 1180 12,800 8140 34,600 48,100
Tetracaine 36.5 12.5 † n.p. ‡ n.p. n.p. n.p. n.p. 22.1
Xylazine 4630 1250 2030 582 4220 1360 12,200 9670
Tilidine 71.3 31.5 23.3 22.4 20.3 322 338 246
Nortilidine 73.0 15.5 11.1 11.9 8.29 88.1 129 194
Bisnortilidine 2180 30.1 27.7 22.2 162 89.4 1580 431
Codeine 90.9 < 30 < 30 < 30 < 30 < 30 < 30 < 30
Tramadol 144 1.83 1.48 2.17 n.p. n.p. 22.7 4.75
Methadone 601 171 442 54 777 442 3680 1430
EDDP < 10 < 10 < 10 < 10 < 10 < 10 < 10 12
4-Aminoantipyrine 6900 1540 1880 1060 2630 3570 3600 5160
4-Methylaminoantipyrine 3350 1970 1940 1360 2830 2920 2500 6650
Metamizole (sum) 10,250 3510 3820 2420 5460 6490 6100 11,810
Ibuprofen 3040 6680 n.t. n.t. n.p. n.p. n.p. n.p.
Paracetamol traces n.p. n.p. n.p. n.p. n.p. n.p. n.p.
* Femoral vein.
† Whole blood.
‡ Abbreviations: n.t., not tested and n.p., not present.
with 2.0 mg/L in the presented case was lower than in a case of
a non-fatal suicide attempt with 4.6 mg/L in serum (26).
Capraro et al. (27) reported a concentration of 0.5 mg/L in
blood 2 h after inhalation. In both cases, the injured persons received
treatment in an intensive care unit.
In the presented case serum concentration of methadone
was with 442 µg/L in the range of a potentially lethal dose and
can, in combination with xylazine and T61, be considered as
cause of death.
The analgesics tramadol, tilidine and its metabolites, metamizole
(detected as 4-aminoantipyrine and 4-methylaminoantipyrine),
and ibuprofen were found in concentrations below
their therapeutic ranges (Table I). Codeine and paracetamol
could also be detected in urine but not at injection sites. They
were probably taken as pain treatment for an existing tumor disease.
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Manuscript received May 17, 2010;
revision received July 14, 2010.