Journal of Analytical Toxicology, Vol.

25, July/August 2001

Comparison of HPLC and GC-MS Methods for

Determination of Embutramide (A Component of
Tanax| or T-61| in Biological Specimens

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M. Giorgi 1, S. Bertini 2, G. Soldani1, and M. GiusianP,*
1Section of Pharmacology and Toxicology, Department of Veterinary Clinics, University of Pisa, Vie delle Piagge 2, 56124 Pisa,
Italy; 2Institute of Food Inspection, University of Parma, V. del Taglio 8, 43100, Parma, Italy; and 3Section of Forensic Medicine
and Toxicology, Department of Public Health, University of Pisa, V. Roma 55, 56100 Pisa, Italy

Abstract collapse because of paralysis of the intercostal muscles and

Tanax or T-61, a euthanasia solution commonly used in veterinary
medicine, has been often involved in suicide attempts (humans)
and malicious intoxications (animals). For forensic reasons, the
identification of one or more of the three components
(embutramide, mebenzonium iodide, and tetracaine hydrochloride)
of Tanax is needed to confirm the hypothesis of intoxication. This
study was performed with new high-performance liquid
chromatographic and gas chromatographic-mass spectrometric
methods to identify embutramide in biological matrices (blood,
liver, kidney) from different animal species. The good sensitivity
and specificity of both methods recommend their use in
toxicological analysis in both human and veterinary medicine.
diaphragm (6). Tetracaine hydrochloride belongs to the same
group as procaine, but it is 20 times more potent; it is added to
the solution because of its local ester anesthetic activity,which
reduces painful tissue reactions at the injection site (7). DMF is
an organic solvent commonly used in industry (e.g., in the
manufacture and processing of plastics) (8).
Tanax has often been involvedin deaths and suicide attempts
of human beings and may also be a factor to be considered in
cases of malicious death in apparently healthy animals such as
race horses or dogs (9). Analytical determination of Tanax in
human or animal tissues or biological fluids can support med-
ical diagnosis. Since 1983, several analytical methods have

r
Introduction

Tanax (T-61) is a euthanasia solution marketed since 1963 in

Germany and available in Canada and in the rest of Europe ~2" "OCH3
(1--4); it is commonly used in veterinary medicine for its nar- N-2-Ethyl-2-(3-methoxyphenyl)-butyl-4-hydroxybuta
namlde
cotic and curariformlike activity.This drug consists of a mixture Embutramide (EMB)
of three active compounds: N-2-ethyl-2-(3-methoxyphenyl)-
butyl-4-hydroxybutanamide (embutramide, EMB) (200 81 CH3 ~ ~ CH3 Ie
mg/mL), 4,4'-methylene bis-N,N,N-trimethylcyclohex-
anaminium di-iodide (mebenzonium iodide) (50 mg/mL), and
p-butylaminobenzoyl-dimethylamino-ethanol chloride (tetra-
4,4'-Methylenebis-N,N, N-trimet~ylcyclohexanaminlumdlodide
caine hydrochloride) (5 mg/mL) (Figure 1). These compounds
are dissolvedin a mixture ofN,N-dimethyl4ormamide(DMF) Mebenzonium iodide
and water (6:4, v/v).
O CH3
EMB is a general anesthetic derived from gamma-hydroxy-
butyrate that possesses a strong narcotic effect and induces a o/~N'-cH 3
deep anesthesia by paralyzing the brain centers that control
breathing in the central nervous system (CNS) (5). H3C-A,J"~
Mebenzonium iodide exerts a curariformlike action, para- p-Butylamlnobonzoyl-dlmethylamlno-ethanol chloride
lyzing the skeletal muscles and rapidly inducing a respiratory Tetracaine hydrochloride
Figure 1. Molecular formula of the three coformulants of Tanax.
"Authorto whomcorrespondenceshouldbe addressed.

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 Journal of Analytical Toxicology, Vol. 25, July/August 2001

been employed to detect Tanax components, despite some dif- v/v) adjusted to pH 4.7 with o-phosphoric acid. The volume of
ficulties: it is not easy to identify mebenzonium iodide in bio- the sample injected was 20 l~L.The detector was a Jasco model
logical materials because of the low solubility of quaternary 870 tuneable adsorbance detector, and the eluenl was moni-
ammonium compounds in most of the common organic sol- tored at 273 nm. Data acquisition, processing, and reporting
vents. Furthermore, tetracaine hydrochloride is quickly de- were handled using a Spectra PhysicData Jet Integrator (Darm-
graded by esterase enzymes. EMB is the most widely stadt-Kranicstein, Germany).
investigated coformulation constituent. Different analytical
methods to detect one or more constituents of Tanax have been
pointed out and described in the literature: thin-layer chro-
matography followedby a quantitative estimation by UV-spec-
IS
trophotometry (10), gas chromatography-mass spectrometry
(GC-MS) equipped with electron-ionization at 70 eV (11,12),
gas chromatography with nitrogen-phosphorus detection (13),
and GC-MS after derivatization (14).
The purpose of this study was to compare high-performance

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liquid chromatography (HPLC) and GC-MS methods for de-
termination of EMB, a component of Tanax or T-61 in biolog-
ical specimens.

Experimental

EMB and ambucetamide (AMB)(Figure 2) were kindly pro-

a~
vided by Hoechst Roussel Vet (Unterschleigheim, Germany)
and by Professor W. Lambert, University of Gent (Gent, Bel- ,..i ,-4

gium), respectively.Phenacetin (Figure 2) was purchased from

Sigma (St. Louis, MO).All the solvents were of HPLCgrade and
were obtained from commercial sources; water used was Milli
Q| filtered. Figure 3. HPLC chromatogram of drug-free whole blood (A). HPLC
chromatogram of a blood specimen (B).
Apparatus
HPLC instrumentation. The HPLCsystem used a Jasco model
880-PU pump to deliver mobile phase at the flow-rate of 0.9 1oo. 2 ~ 7

mL/min to a Waters Spherisorb ODS2 C18 5 t~m (150 mmx

4.6-ram i.d.) analyticalcolumn (Milford,]VIA).A Waters C18 No-
vapak Guard Pack precolumn was used to protect the analytical
column. The mobile phase consisted of CH3OH/H20 (65:35,

IZ7~ 1348B 1 , t ~

Time ( m i n i

HBCO~ ~/'~ CH3

~-(Dibutylamino)-4-methoxybenzeneacetamide
Ambucetamide

H~C---x F---x H
'o-4/ \x,_
~-CHB
0 ,o =~, , ~ i o
N-(4-Ethoxyphenyl) acetamide
Phenacetin .... " ~ " ~o~ " ~ " t ~ " I~ ~ ' " , : ~ o " ~:~";s~o" ~:~; '
Time (mini

Figure 2. Molecular formula of the analytical marker ambucetamide Figure 4. GC-MS chr0matogram of drug-free whole blood (A) and a
and of the internal standard phenacetin. blood specimen (B).

324
Journal of Analytical Toxicology, Vol. 25, July/August2001

GC-MS instrumentation. A Fison 8000 GC and a Fison MD Methods

800 MS (Milan, Italy) were used for the quantitative analysis of Samplepreparation. Quantitation of EMB concentration was
EMB. This compound was separated on a Mega| OV-1 fused- based on a six-point calibration curve. Standard curves for
silica capillary column (15 m x 0.18-mm i.d., 0.1-mm film EMB were prepared by adding EMB to pools of drug-free rat
thickness; Milan, Italy). The injector was operated in the split- blood, liver, and kidney at concentrations of 2.5, 5, 10, 50, 250,
less mode at 250~ helium was used as carrier gas at a column and 500 pg/mL These samples were then taken through the an-
head pressure of approximately 6 psi and a flow rate of 1 alytical procedure. For the HPLC analysis, the peak areas of
mL/min. Specimens (~1 mL) were injected at 100~ the split EMB relative to the internal standard (IS) were calculated and
valve was opened after 1 min, and the temperature was in- plotted versus the concentrations. An external standard proce-
creased to 295~ at a rate of 15~ When the temperature dure was followed for the extraction of the samples to assay by
reached 295~ the course was extended for 10 rain. The ion GC-MS analysis, and AMBwas used as an analytical marker for
source was operated at 180~ with an accelerating voltage of 70 the determination of the exact volume of the specimen in-
eV, 750 mA filament current, and 1 kV electron multiplier jected in the apparatus. The concentration of EMB in unknown
voltage (the conversion @nodes operated at 5 kV). Ion current samples was calculated from the calibration curves. Calibration
was acquired at the following mass-to-charge ratios: 121, 135, graphs for blood, liver, and kidney were constructed also for

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190 (EMB) and 121, 192, 248 (AMB). EMB determination in SIM mode (data not shown). In this
equation, x value was the concentration of EMB added to bio-
logical matrices in micrograms per milliliter, and y value was
Table I. Accuracy and Precision Data for EMB Analysis in the peak area of the base peak (m/z 121).
Whole Blood ~voohundred milligrams of NaHCO3 (in order to reach a pH
between 8.5 and 9.0) and 8 mL of dichloromethane (CH2C12)
Method Level n CV (%) containing 100 pg of phenacetin as IS (recovery 78 ___6%) were
added to a 1.0 mL volume of blood. Phenacetin was added only
HPLC Within.day for the HPLC sample preparations, whereas in extractions for
50 mg/mL 5 4.7 GC-MS analysis an external standard procedure was performed
500 mg/mL 5 4.6 (EMB recovery 92 _+4%). The extraction tubes were then placed
Day-to-day on an oscillatory mixer (10 rain) at the speed of 60 oscilla-
50 mg/mL 5 7.1 tions/min. After centrifugation for 10 rain at 2000 x g at room
500 mg/mL 5 6.2
temperature, the lower organic layer was transferred into a con-
ical tube. The solvent was evaporated to dryness under a gentle
GC-MS Within-day stream of nitrogen without heating. The residue was redissolved
50 mg/mL 5 4.3
500 mg/mL 5 4.0
in CHaOH (1 mL), vortex mixed, and transferred into 1.5 mL
Day-to-day screw-capped vial. A 20-tJL aliquot was injected onto the HPLC;
50 mg/mL 5 6.4 after the addition of the same volume of AMB solution (50
500 mg/mL 5 6.1 pg/mL), about 1 pL was injected onto the GC-MS system.
A pool of tissue samples was homogenized with an Ultra

AES-500 578 (11.637) Rf(1,10.000) Scan B+

121 135 190 3.2508
100] 98 I
t 89 91 I I 191
J43 , 8zl I 11TI /
i 161 177
r I 8.611 I'., 1o8"1I1.= h82 I
147 1491[ I
46 85 85 92 192 22 293
(.....~ ..... ,~ ~ ~6 292 ~4.276 1_295.3o8
.... ,...~...,.-.~1~..., ....100'.... ' ....120'.... ' ....1481
.... , ....180'.... ' ....180'.... ' "'1200'" ,l" 220"1"" ' 1 " ' 2 4 0 " 1. . . . i . . . . 2 5 0 1. . . . i . . . . 2 8 0 1. . . . i " " 3" 01 0" ' '1

mlz
,NVIBUA1 550(11.173) Rf(3,10.000) Scan B+
248 8.1506
100]

1 44 121 136 ._

"1 1 o:; 9194 lO,9 i1~11., e ,?

11,1 ,,I 8Se6l,, 7e I./ lO41 I ~111,3 11.49 111.6s 19ol 204 s217 2s3246 25O
oJill,... ~,11,.. ,g: ..... lit..,,,,. ~l,lL.,...,lk,I,.....,,lu,.,.,ll It...... ~E ......, I1( ............. h h....... I~'. ,/.................... 5
I'' '' I" "' I''''1 "''' I'''" I .... I''" '1'''' I'''' I'''' I''''1'' ''1'''' I'''' I" ' ' ' 1 .... I'''' I'''' I''''1''''1'''' I ', , , , i, ,, ,i,, ,,i,,,, i,,
40 60 80 100 120 140 100 180 200 220 240 250 250
m/z
Figure 5. Mass spectraof EMB and AMB.

325
 Journal of Analytical Toxicology, Vol. 25, July/August 2001

Turrax mixer after dilution with water (1:1, w/w), and 2-g
aliquots were extracted using the same protocol described for
blood samples.
The limit of detection (LOD) was defined as the smallest
amount of the analyte that could be detected with a signal-to-
noise ratio of 10:1. The limit of quantitation (LOQ) was esti-
mated as five times the LOD.
Reproducibility. Within-day reproducibility was evaluated by
analyzing five blood samples spiked with EMB to obtain two dif-
ferent levels (50 and 500 pg/mL). Day-to-day reproducibility was
determined by analysis of five replicate samples of the two
levels (50 and 500 pg/mL) on seven consecutive days. From the
data obtained, within-day and day-to-day correlation values
(CV) were calculated.
Drug administration. All the animals were administered by
cient were determined for the calibration curve by linear re-
gression (StatView). Comparison among mean values (blood,
liver, and kidney) was performed by one-way analysis of variance
for independent sample. When a significant overall difference
was detected, a Dunnett test was used. A value ofp _<0.05 was
considered to be significant.
Results
LOD and susceptibility to endogenous interferences are the
customary measures of the sensitivity and specificity respec-
tively, of an analytical method. There was no evidence of chro-
matographic or spectral interferences in either HPLC or

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intravenous, intracardiac, and intrapulmonary route with the GC-MS method used (Figures 3 and 4). In order to reduce the
minimum dose recommended by the producer. From the dead number of animals sacrificed, the present study hypothesizes
animals, blood, liver, and kidney were excised and stored at that the rat matrices utilized for the calibration curves do not
-20~ until assay. All the organs were sampled in different differ from those of the other species (horse, pig, etc) in the
parts before the analysis. contents of endogenous substances that can interfere with
Statistical analysis. Slope, intercepts, and correlation coeffi- EMB detection (12). The within-day and the day-to-day repro-
ducibility data of the EMB analysis in blood were tested (Table
I). In this study, GC-MS resulted the most specific analytical
Table II. Ion Ratios for EMB and AMB Added to Drug- method available for EMB detection and quantitation; its LOQ
Free Whole Blood at a Level of 5 pg/mL and Analyzed
was 250 ng/mL in SIM mode (ion acquired: EMB: 121, 135, 190;
with GC-MS in the SIM Mode
AMB: 121,192,248) (Figure 5), and in scan mode the LOQ was
Average ion ratio (%)* the same if compared with that of the HPLC method (2.5
m/z190/121 m/z135/121 pg/mL). To improve the qualitative identification of the SIM
mode, ion ratios for EMB and AMB were calculated (Table It).
EMB Calibration results are presented in Table III; they include the
Standard 73.4 (1.6) 63.1 (1.3) means of slope, y-intercepts, and correlation coefficients for
Fortified sample 72.2 (1.6) 61.9 (1.3) three separate calibrations by each method. Table IV shows the
detection of EMB by HPLC or GC-M$ in blood, liver, and kidney
m/z121/248 m/z192~248
from different animal species after intravenous, intracardiac,
AMB and intrapulmonary Tanax and three separate extractions by
Standard 75.0 (2.3) 47.2 (0.9) each method. No significant differences between the values
Fortified sample 73.6 (2.3) 45.5 (1.0) obtained with the two methods were observed.
* Values in parenthesesare the correspondingstandarddeviations.

Discussion
Table III. Calibration Curves Comparison for HPLC and
GC-MS Scan Mode* Analytical determination of Tanax in human or animal tissues
and biological fluids can assist medical diagnosis; in fact, a
Slope Intercept(m~/mL) r+ rapid evaluation of this poisoning is needed for the good out-
mean SD mean SD mean SD come of the patient from the acute intoxication due to the
three coformulants and to prevent the delayed toxicity caused
I-IPLC
by the solvent N,N-dimethylformamide (9). The presence of
Blood 2.170-+0.030 0.610-+0.710 0.998 _+0.006
EMB in biological matrices is an undoubtable sign of Tanax (T-
Liver 1.976 _+0.041 0.998 + 1.234 0.995 + 0.005
Kidney 2.245_+0.052 0.876_+ 0.654
61) intoxication; EMB is only present in this product, and it is
0.096 + 0.005
not available alone or in other pharmaceutical preparations.
GC-MS The comparison between the HPLC and GC-MS methods in-
Blood 1.302 + 0.046 8.002 + 10.654 0.997 __.0.005 dicates that liquid chromatography is a good alternative method
Liver 1.501 + 0.052 7.543 + 5.876 0.998 -+ 0.006 for EMB detection in biological matrices. In situations where
Kidney 1.145 _+0.062 10.230-+ 10.003 0.096 + 0.005
structural identity is required for legal purposes or for qualita-
* N = 3 calibrations.Curveswere constructedby plotting concentrations(x-axis) tive analysis of unknown compounds, MS is clearly superior to
versusthe ratio of the responseof the analyteto that of the internal standard the UV spectroscopic method of detection. However, in quanti-
(y-axis).Calibrationcurvesfor GC-MS are not shown (r = 0.998).
t Correlationcoefficient. tative applications where the need for spectral data is less in-
tense (e.g., analysis supported by a standard), a comparable

326
Journal of Analytical Toxicology, Vol. 25, July/August 2001

Table IV. Concentrations of EMB (pg/mL) in Blood, Liver, and Kidney from Different Animal Species Euthanized with Tanax
with Intravenous and/or Intrapulmonary and/or Intracardiac Injection and Detected with HPLC and GC-MS Methods*

Administration Dose Blood(mg/mL) Liver(mg/mL) Kidney(mg/mt)

Species route n (mL/kg) HPLC GC-MS HPLC GC-MS HPLC GC-MS
Rat intravenous 5 2 1352 + 407 1299 + 203 146 + 94 120 + 31t 321 + 110 399 _+52
Rat intrapulmonary 5 2 15.6 + 7.3 13.4 + 4.3 7.0 _+3.8 5.6 + 1.9 12.8 + 6.1 9.3 + 5.1
Rat intracardiac 5 2 1098 + 418 1158 + 305 36.6 + 9.2t 42.0 + 7.0 57.6 + 18.3 54.2 + 9.2
Cat intracardiac 2 1 517 _+82 489 + 65 18.3 + 9.1t 16.1 + 5.9t 45.3 + 17.8 48.6 + 9.5
Horse intravenous 1 0.1 17.6 + 9.3 23.1 + 8.9 n.a.* n.a. n.a. n.a.
Sheep intravenous 1 0.4 17.8 + 6.2 28.9 + 8.7 n.a. n.a. n.a. n.a.
Rabbit intrapulmonary 3 1 9.3 + 4.2 12.3 + 2.3 2.6 + 2.0 3.2 + 1 5.8 + 3.2 7.1 + 3.0
Pig intravenous 1 0.5 814 + 372 856 + 289 17.8 + 7.5 20.3 + 8.2t 7.85 + 6.1 6.2 + 3.1

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* Data are the mean + SD of 3-5 experiments, n = number of animals.
t p < 0.05 between liver and kidney concentrations.
* n.a., not assayed (no sample available).

sensitivity and precision can be achieved using the two appa-
ratus indifferently.In the HPLC method the UVdetector was se-
lected at 273 nm, whereas the maximum absorbance peak of the
EMB is at 210 nm. In these experimental conditions there were
no interference peaks deriving from biological matrices that
could make noise in the spectrum; however, the sensitivity of
the method decreased. The LOQ of 2.5 ]~g/mLallowed the de-
tection of EMB in all of the biological matrices deriving from
different animals euthanasized with the minimum recom-
mended dose of Tanax. In the present study, the highest level of
EMB was found in blood because of the rapid action of the drug
(10 s-3 rain is sufficient to cause death in all animal species em-
ployed in this study by different routes of administration).
Kidney showed a higher concentration of EMB when compared
to liver in all of the animal species except the pig. Although not
all of these data are significant (p < 0.05), this trend of concen-
trations is in accordance with previously reported data (12).
The higher EMB concentration in liver than in kidney of the pig
could be explainedby the fact that the animal used for the eval-
uation of EMB was previously employed in an experiment for
pulmonary transplant. It is possible that the previous adminis-
tration of anesthetic and analgesic drugs could have changed
the kinetics of the EMB; on the other hand, it is difficult to cor-
relate this result with a species difference.
Conclusions
Two selective and sensitive methods for EMB determination
were developed using HPLC and GC-MS apparatus. These sys-
tems may be used for forensic toxicological analysis in both
human and veterinary medicine.
Acknowledgments
The authors thank Hoechst Roussel Vet (Unterschleigheim,
Germany) and Professor W. Lambert (University of Gent, Bel-
gium), who kindly provided embutramide and ambucetamide,
respectively.
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Manuscript received March 20, 2000;
revision received August 8, 2000.

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