A Fatality Related to the Veterinary Anesthetic Telazol - Journal of ...

[Case Report
Journal of Analytical Toxicology, Vol. 23, October 1999
A Fatality Related to the Veterinary Anesthetic Telazol
Christopher J. Cording*, Robert Detuca, Thomas Camporese, and Elizabeth Spratt
Westchester County Department of Labs and Research, 2 Dana Road, Valhalla, New York, 10595
[Abstract }
A 45-year-old male veterinarian was found dead in bed. Police
investigation showed no evidence of trauma or other suspicious
circumstances. Autopsy was unremarkable except for cardiomegaly
and hepatosplenomegaly. Toxicological analysis revealed the presence
of Telazol and ketamine. Telazol is a veterinary anesthetic agent that
is composed of equal parts of tiletamine and zolazepam. Tiletamine
is a disassociative anesthetic similar to ketamine and phencyclidine,
and zolazepam is a diazepine derivative tranquilizer used to minimize
the muscle hypertonicity and seizures associated with tiletamine.
Quantitation of tiletamine and zolazepam was performed using gas
chromatography-mass spectrometry in the selected ion monitoring
mode following a solid-phase extraction. Postmortem blood, urine,
and liver concentrations of tiletamine were 295 ng/mL, 682 ng/mL,
and 196 ng/g, respectively, whereas postmortem concentrations of
zolazepam for the same tissues were 1.71 pg/mL, 1.33 pg/mL, and
15.5 pg/g, respectively. Blood and urine ketamine levels were 37
ng/ml, and 381 ng/mL, respectively. The cause of death was
ruled an acute mixed drug intoxication of tilelamine, zolazepam,
and ketamine with the manner of death ruled as unclassified.
Introduction
Telazol is a non-narcotic, nonbarbiturate, injectable veterinary
anesthetic agent that is equal parts by weight of tiletamine and
zolazepam. Tiletamine (2-[ethylamino]-2-[2-thienyl]-cyclohex-
anone hydrochloride) is an arylcyclohexylamine structurally
related to phencyclidine and ketamine. Like ketamine and phen-
cyclidine, it produces dissociative anesthesia primarily by selec-
tively interrupting the association pathways to the brain before
producing a somesthetic sensory blockade (1). Tiletamine pro-
duces a spectrum of central nervous system (CNS) effects
ranging from excitement and ataxia at low doses to catalepsy and
anesthesia at higher doses. These CNS effects are highly species
specific, but catalepsy prevails in all species at moderate doses
(2). The anesthesia produced by tiletamine resembles a catalep-
toid state in which the eyes remain open with a slow nystagmus
gaze (3).
Zolazepam (4-[o-fluorophenyl]-6,8 dihydro-l,3,8-trimethyl
pyrazolo [3,4-e][1,4] diazepin-7[1H]-l-hydrochloride)is a non-
phenothiazine diazepine tranquilizer used to reduce the muscle
hypertonicity and seizures associated with tiletamine (1).
Telazol produces anesthesia within 5-]2 rain of administration
* Author to whom correspondence should be addressed.
and optimum muscle relaxation within 20-25 min (1). Telazol is
approved for use in cats and dogs, although it has also been
proven to be effective for restraint and induction of anesthesia in
a wide variety of animals ranging in size from rats to polar bears
(4). Telazol is supplied as 500 mg of active drug. With the addi-
tion of 5 mL of supplied diluent, it is equivalent to a solution of
50 mg/mL of base for each compound.
Case History
A 45-year-old male veterinarian, (104 kg, 1.8 m) was found in an
unresponsive state by a home-care worker who was tending to
the deceased's 79-year-old mother. The deceased was found
kneeling by his mother's bed with his torso draped over his
mother's legs. The home-care worker called the deceased's sister
who arrived on the scene and called the police. Police arrived and
determined that there was no trauma or other suspicious cir-
cumstances. Found at the scene were two syringes, two empty
vials of Telazol, an empty vial of ketamine, and a bottle con-
taining 49 Centrine pills. Centrine is the trade name for
aminopentamide, which is an anticholinergic (5). The deceased
had a history of ketamine use.
The mother was transported to a hospital where she sub-
sequently died. She suffered from Alzheimer's disease and was
unable to recount any details of her son's death. Fears of a
murder-suicide were dispelled when the mother's toxicology
report proved to be negative. Her autopsy showed that death was
due to natural causes.
An autopsy was conducted on the deceased on the day of death.
The findings were unremarkable except for cardiomegaly and
hepatosplenomegaly. The body was slightly decomposed. There
were no injection sites noted. A general toxicological screen for
drugs and ethanol was performed on blood and urine specimens
collected at autopsy using immunoassay, headspace gas chro-
matography, and high-performance liquid chromatography-
mass spectrometry (HPLC-MS).
Experimental
Materials
Tiletamine and zolazepam were supplied individually by Wyeth-
Ayerst Labs (St. Davids, PA) in association with Fort Dodge
Animal Health (Fort Dodge, IA). Phencyclidine (PCP)-ds was
552 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

Journal of Analytical Toxicology, Vol. 23, October 1999
purchased from Radian (Austin, TX) as a methanolic solution at
a concentration of 100 lJg/mL. SKF-525A was purchased from
Sigma Chemical Co. (St. Louis, MO), and 5-ethyl-5-p-tolyl-
barbituric acid was purchased from Aldrich Chemical Co.
(Milwaukee, WI). The chemicals used in the extraction were
analytical grade. The solid-phase extraction columns employed
were United Chemical Technologies (Bristol, PA) Clean Screen
ZSDAU020 extraction columns.
Methanolic stock solutions were made for the tiletamine and
zolazepam at a concentration of 1 mg/mL. This stock was fur-
ther diluted to a combined working standard of I IJg/mL. This
working standard was used to prepare calibration standards in
blood of 12, 25, 50, 100, and 300 ng/mL for the tiletamine and
zolazepam. An independent blood-based control was also pre-
pared. A PCP-d5 stock solution of 3.33 IJg/mL was prepared as an
internal standard from the 100-1Jg/mL Radian standard.
Instrumentation
A Hewlett-Packard (HP) HPLC-MS system was used for the
initial screening. It consists of an HP 1050 series HPLC pump,
autoinjector, and UV detector with an HP 59980B particle beam
interfaced with an HP 5989B MS. It used an Altima (Deerfield,
IL) C18, 5-1Jm Direct Connect Guard cartridge (#88058)
Abundance #145: Tiletamine rrt=0.159 (*)
116
8000
6000
4000 II0123
2000 51 69 83 ,Jl,,..,,fl ..,,,~38,i,,,,
0 LH,J.., , .l,l.h. hd ,.
9 /z--> '"6'o"'~'o'"L66"L~6"L~6"L~
195
Figure 1. Structure and particle beam LC-MS El mass spectrum of tiletamine.
Abundance
m/z-->
8000
6000
4000
2000.
0
attached to a 5-1Jm Altima C18 (20 mm x 2.1 mm) column. The
HPLC was run with a gradient that began with 100% of a mobile
phase A (deionized water/acetonitrile, 90:10, 0.05% trifluo-
roacetic acid) for 5 min and ended with 100% of a mobile phase
B (deionized water/acetonitrile, 20:80, 0.05% trifluoroacetic
acid) after 55 min at a flow of 0.25 mL/min with a column tem-
perature of 40~ Total run time was 70 min which allows for re-
equilibration of the column. The UV detector scanned between
205 and 290 nm. The MS was set in the electron impact (EI)
mode to scan for ions in the range of 50-500 ainu. The struc-
tures and LC-MS EI spectra for tiletamine and zolazepam are
shown in Figures I and 2, respectively.
A Hewlett-Packard (Palo Alto, CA) model 5890 series II+ gas
chromatograph (GC) with a 5972 series mass selective detector
was employed for the quantitation. The column was an HP-
Ultra-1 crosslinked methyl siloxane column (12 m x 0.2-ram
i.d., 0.3-1~m film thickness). The injector temperature was set to
250~ and the detector was set to 280~ The initial oven tem-
perature was 135~ The temperature was then ramped to 220~
at a rate of 25~ After being held at 220~ for 1 min, the
temperature was ramped to 320~ at a rate of 30~ for a
total run time of 8.73 min. The MS was run in the EI mode with
selected ion monitoring using a target ion and two qualifying
iL I1206 223 240 268 284 319
#146: Zolazepam rrt-0.238 (*)
2~:72 5
69
145
51 i09 134k 180 199 230
i'; .... 160 1go '' '260 .... 2g, ~360'
Figure 2. Structure and particle beam LC-MS El mass spectrum of zolazepam.
32~ 3s~68 394 42o
'' '3~0 .... 460'''
S-
~
NH~Hs
OHc I
~H3 CH3
CH2 ,____~N
~ F HCl
553

ions for each compound. The target ion for tiletamine was 166
with qualifiers of 195 and 110; the target ion for zolazepam was
285 with qualifiers of 267 and 257; and the target ion for PCP-ds
was 205 with 247 and 248 as its qualifying ions. Retention times
for tiletamine, PCP-ds, and zolazepam were 3.74, 4.84, and 6.87
min, respectively (Figure 3).
Procedure
The LC-MS screening procedure in which the compounds
were detected was a liquid-liquid extraction at both neutral and
alkaline pHs. Buffer (1 mL, pH 7) was added to a 5-mL sample of
blood or urine. Both I lag of SKF-525 A and 6 lag of 5-ethyl-5-p-
tolybarbituric acid were added as internal standards. After the
addition of 7 mL of hexane/toluene/isoamyl alcohol (90:5:5), the
samples were vortex mixed and centrifuged. The organic layer
was removed to a clean test tube. Buffer (1 mL, pH 9.5) was
added to the remaining aqueous layer. The alkaline fraction was
then extracted using 6 mL of hexane/isoamyl alcohol (99:1). The
two extracts were combined and dried down under nitrogen. The
samples were reconstituted with 250 laL of deionized water/ace-
tonitrile (55:45), 0.05% trifluoroacetic acid. The samples were
then dried down to approximately half their volume to remove
the acetonitrile. The injection volume was 50 laL.
The tiletamine and zolazepam confirmation was performed
using the solid-phase method for phencyclidine as described by
United Chemical Technologies (6). Two milliliters of 0.1M phos-
phate buffer pH 6.0 was added to 3 mL of sample. To all samples
75 laL of PCP-ds was added as an internal standard
for an equivalent of 250 ng. Three grams of a 1:1
liver homogenate was measured into a test tube
resulting in an actual weight of 1.5 g for the liver
extract. The same amount of internal standard
and buffer were added to the liver sample. In- Sample
house controls were run at a concentration of 25 Blood
ng/mL and 100 ng/mL for tiletamine and Urine
zolazepam. Liver
The columns were prepared with 3 mL of
methanol (MeOH) followed by 3 mL of deionized
Results and Discussion
Journal of Analytical Toxicology, Vol. 23, October 1999
The screening panel for drugs of abuse by enzyme multiplied
immunoassay technique (EMIT) was found to be negative for
both urine and blood. Telazol did not show any cross-reactivity
with the Behring Diagnostics Inc. (Cupertino, CA) benzodi-
azepine assay. Although the phencyclidine assays were also neg-
ative, the urine showed an elevated negative value 16 units below
the cutoff value of 25 ng/mL. It is not known if this cross-reac-
tivity was due to the presence of ketamine or tiletamine or both.
Headspace analysis for ethanol and other volatiles was also neg-
ative. The blood was also screened for aminopentamide by HPLC
and was found to be negative.
Tiletamine and ketamine were detected by the HPLC-MS
screening. The EI spectra were searched by the Pfleger mass
spectra library, which was supplied by Hewlett-Packard, and
matched tiletamine as norketamine. Tiletarnine resembles nor-
ketamine in that it shares a 166 ion. After the addition of tile-
tamine to an in-house mass spectra library, the peak was
properly labeled. The HPLC-MS also detected the presence of
ketamine. Zolazepam was not extracted by the HPLC-MS
screening method.
Although no recovery studies were done for the solid-phase
extraction of tiletarnine and zolazepam, there appeared to be no
difficulty in combining the analyses of these two compounds into
one method. The United Chemical Technologies extraction pro-
cedures for phencyclidine and benzodiazepines were nearly iden-
tical. In both extractions, sample preparation and column
Table I. Concentrations of Tiletamine, Zolazepam, and Ketamine in Blood,
Urine, and Liver
Tiletamine Zolazepam Ketamine
295 ng/mL 1.71 ns/mL 37 n~mL
682 ng/mL 1.33 nglmL 381 ng/mL
196 ng/g 15.5 pg Analysis not performed
water and finally I mL of 0.1M phosphate buffer
pH 6.0. After vortex mixing and centrifuging, the
Abundance
TIC: 0701006.D
samples were loaded onto the prepared solidphase
extraction columns. The columns were
then washed with 3 mL of deionized water
250000
Tile amine
Zolazepam
followed by 1 mL of 0.1M acetic acid and finally
3 mL of MeOH. The columns were dried for 5 rain
200000
under vacuum, and the samples were then eluted
with 3 mL of methylene chloridelisopropyl alco-
150000
PCP D- 5
hol/ammonium hydroxide (78:20:2). The eluent
was dried down under nitrogen and reconstituted
100000
with 100 IJL of ethyl acetate for injection onto the
GC-MS. The injection volume was I laL.
soooo
The ketamine confirmation was performed separately
using the same extraction method and
0
Time-->
.... ~ , .... k_______._~ , .... , .... I .... , .... , .... ,,,,,,,,,,,,,,,,
3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
GC-MS parameters as described for tiletamine
and zolazepam. Ketamine had a retention time of
4.49 min.
Figure 3. GC-MS total ion chromatogram of 300 ng/mL extracted tiletamine, PCP-ds, and
zolazepam.
554

Journal of Analytical Toxicology, Vol. 23, October 1999
preparation were the same. The methods differ only in their
respective column washes and elution solvents. Zolazepam
showed a good response at the lowest calibration level, so the
differences in column wash and elution solvent did not appear to
be significant.
Limit of detection, limit of quantitation, and linearity studies
were not done. The calibration curves were linear with coeffi-
cients of correlation (r 2) of 1.00 and 0.999 for tiletamine and
zolazepam, respectively. The samples were run at dilutions that
fell within the upper and lower calibrators. The sole exception
was the liver zolazepam results.
Postmortem blood, urine, and liver concentrations of tile-
tamine, zolazepam, and ketamine are listed in Table I. Data
regarding toxic human dosages of Telazol are nonexistent.
Although there is a preponderance of veterinary data regarding
dosages required for chemical restraint and surgical anesthesia
of various animal species, it is difficult to correlate these data to
humans. Among the nonhuman primates most closely related to
humans, chimpanzee, orangutan, and gorilla, dosages required
for chemical restraint were 3.63 mg/kg, 2.72 mg/kg, and 1.35
mg/kg, respectively. (7) According to an article by Eads (7), larger
species of primates required smaller doses of Telazol on a weight
basis to produce equivalent results.
Tiletamine belongs to the same class of drugs as ketamine and
phencyclidine. In most respects the pharmacokinetics of these
drugs are the same. In terms of potency, tiletamine is between
ketamine and phencyclidine with ketamine being the weakest
and phencyclidine the most potent. (3) The usual intravenous
dose of ketamine required for induction of anesthesia ranges
from 1 to 4.5 mg/kg. (8) A dose of 2.5 mg/kg resulted in an
average plasma concentration of I IJg/mL after 12 rain, which fell
to 0.5 IJg/mL after 30 rain. (9) Phencyclidine intoxication is
associated with blood levels of 0.007-0.240 IJg/mL. (10) The
usual intravenous dose of PCP was 1-3 rag. (11) Toxic concen-
trations of phencyclidine range from 0.3 to 25 ~g/mL in the
plasma. (8) The deceased had 1000 mg of tiletamine available to
him, which would result in a dose of 9.6 mg/kg if taken all at
once. A dose of 8.25 mg/kg was fatal to a gorilla after 5 h with
death due to respiratory depression. (7) The blood concentra-
tions of tiletamine and zolazepam suggest that it is probable the
decedent did not take the full dose available at one time.
The deceased also took ketamine. Although the postmortem
blood concentration of ketamine was low, 37 ng/mL, ketamine
and tiletamine are the same class of compound, and at least an
additive effect can be assumed.
Assessing the role of zolazepam as a causative agent in this
death is much more difficult. According to an article by Lin et al.
(3), zolazepam was the least likely to cause depression when
compared with chlordiazepoxide and diazepam, which is why it
was chosen in the development of Telazol. Chlordiazepoxide and
diazepam are the two most commonly used benzodiazepines in
veterinary medicine. The postmortem blood concentration of
zolazepam was 1.71 I~g/mL. It is very possible that the zolazepam
played a role in enhancing the respiratory depression induced by
the tiletamine and ketamine; however, it is not possible to
describe the extent of that enhancement.
The tremendous difference in the blood concentrations of the
two drugs at first glance appears to be difficult to explain consid-
ering that Telazol is equal parts of tiletamine and zolazepam. The
most likely explanation for the disparity in blood concentrations
of tiletamine and zolazepam would be differences in the distri-
bution, metabolism, and elimination of these two compounds.
The cause of death in this case was ruled an acute mixed drug
intoxication of tiletamine, zolazepam, and ketamine, and the
manner of death was ruled unclassified.
References
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Veterinary Medicine Publishing Group, Lenexa, KS 1997,
pp 747-748.
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and respiratory effects of tiletamine-zolazepam. Pharmacol.
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a review of its pharmacology and use in veterinary medicine. J. Vet.
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Columns Applications Manual, Revision 691070, Bristol, PA.
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8. R.C. Baselt and R.H. Cravey. Disposition of Toxic Drugs and
Chemicals in Man, 4th ed. Chemical Toxicology Institute, Foster
City, CA, 1995.
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Pharmacokinetics of ketamine in man. Anaesthesia 24:260--263
(1975).
10. D.S. Pearce. Detection and quantitation of PCP in blood by use of
2H s PCP and selective ion monitoring applied to non-fatal cases of
PCP intoxication. Clin. Chem. 22:1623-1626 (1976).
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Manuscript received March 1, 1999;
revision received May 26, 1999.
555