Journal of Analytical Toxicology 2012;36:647 –656
doi:10.1093/jat/bks078 Advance Access publication September 20, 2012

Case Report

Analysis of Clothing and Urine from Moscow Theatre Siege Casualties Reveals
Carfentanil and Remifentanil Use
James R. Riches, Robert W. Read, Robin M. Black, Nicholas J. Cooper and Christopher M. Timperley*
Detection Department, Defence Science and Technology Laboratory (Dstl), Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK
*Author to whom correspondence should be addressed. E-mail: cmtimperley@dstl.gov.uk

On October 26, 2002, Russian Special Forces deployed a chemical
aerosol against Chechen terrorists to rescue hostages in the
Dubrovka theatre. Its use confirmed Russian military interest in
chemicals with effects on personnel and caused 125 deaths
through a combination of the aerosol and inadequate medical care.
This study provides evidence from liquid chromatography –tandem
mass spectrometry analysis of extracts of clothing from two British
survivors, and urine from a third survivor, that the aerosol comprised a mixture of two anaesthetics—carfentanil and remifentanil—whose relative proportions this study was unable to identify.
Carfentanil and remifentanil were found on a shirt sample and a
metabolite called norcarfentanil was found in a urine sample. This
metabolite probably originated from carfentanil.

Introduction
On October 23, 2002, Chechen terrorists seized the Melnikov
Street Theatre in Moscow during a sell-out performance of the
musical “Nord-Ost,” taking over 800 hostages and demanding
immediate and unconditional withdrawal of Russian troops
from Chechnya. The siege ended in the early morning of
October 26 after a Special Forces unit belonging to the Russian
Federal Security Service (FSB) pumped a chemical aerosol into
the building and stormed it. At least 33 terrorists and 129 hostages died during or shortly after the raid. The terrorists were
shot dead after falling unconscious to the effects of the
aerosol, explosives strapped to them were removed, and a
bomb in the auditorium was deactivated. Two hostages were
shot by the terrorists, while 125 died through a combination of
the aerosol and inadequate medical treatment following the
rescue. Medical treatment of the casualties was complicated
because the Russian government did not disclose the composition of the aerosol. The head of the Moscow Public Health
Department announced that all but one of the hostages killed
in the raid had died of the effects of the gas, which was surmised to comprise an anaesthetic or chemical warfare agent.
Foreign embassies in Moscow issued official requests for more
information on the aerosol to aid treatment, but were ignored.
Armed guards were posted at Moscow hospitals and doctors
were ordered not to release any of the casualties. Refusing to
disclose the content of the aerosol used, the Russian government informed the United States Embassy on October 28 about
some of its effects. Based on this information and examination
of some of the casualties, doctors concluded that the aerosol
had contained a morphine derivative. On October 30, Russia
responded to increasing domestic and international pressure
with a statement by its Health Minister, Yuri Shevchenko (1),
who identified the aerosol as that of a fentanyl derivative,
although the precise composition was not disclosed. Shevchenko
# Crown copyright 2012.

stated that fentanyl was an anaesthetic that fell into the category of non-lethal medical preparations and that the troops
had not used any substances prohibited by the Chemical
Weapons Convention.
After the siege, various hypotheses were proposed to
account for the Russian explanation that an aerosolised form
of fentanyl had been used: a mixture of fentanyl and the anaesthetic gas halothane (2), or fentanyl alone, for example,
but these ideas were soon discredited based on the insufficient potency of these chemicals (2 –4). A recent European
Court of Human Rights report (5) of a legal case against
Russia, lodged by 64 Russians that survived or lost relatives in
the siege, provides an authoritative account of events. Therein
the Russian Government revealed that the aerosol was a
“mixture based on derivatives of fentanyl” and “a composite
chemical compound of general narcotic action,” suggesting
more than one component. However, the composition was
not disclosed to the court. This case report provides evidence
from the analysis of clothing from two British survivors, and
urine from a third survivor with a Russian name, that the
aerosol comprised a mixture of two fentanyls, carfentanil and
remifentanil.

Case History
Clothing and blood samples from British survivors held in the
theatre throughout the siege were received at the Defence
Science and Technology Laboratory (Dstl) Porton Down on the
afternoon of October 28, 2002, for analysis. A jumper and
leather jacket from Casualty 1 (male), a shirt from Casualty 2
(female), and two blood samples from each casualty, were provided. The blood samples had been taken approximately 19 h
(Casualty 1) and 12 h (Casualty 2) after the chemical aerosol
had been released into the theatre. The casualties had been
near an exit door, which appears to have been a major factor
in their survival. They were among the first casualties evacuated from the theatre after it was assaulted.
Both casualties were interviewed separately by British police
on November 12 and 13, 2002. The following information is
taken from these interviews (6): During the morning of
October 26, the hostage-takers pointed to a white aerosol that
appeared to be emerging silently from the balcony wall. The
aerosol—dense, white and cloudy—spread evenly and descended slowly. Casualty 1 claimed it had an indescribable
smell, Casualty 2 remembered it as odourless; people were not
coughing or spluttering, even on the balcony. Both casualties
knelt on the floor and covered their faces with their clothing.
From first spotting the aerosol to being overcome by it took
“10–30 seconds” for Casualty 1 and “at least 30 seconds” for

 Casualty 2. Neither casualty saw the assault team enter the
theatre.
Upon awakening in the hospital, Casualty 1 recalled vomiting
and seeing blood, which concerned him, but strangely, he felt
no pain in his stomach. Nurses told him not to sleep, but eventually he dozed off. Two bottles of fluid were administered by
intravenous drip; he was told they contained sugar. He received
at least two injections. One was supposed to make him go
to the toilet. He did not know what the others were for.
He was given pyjamas to wear because his clothes were wet.
He noticed them drying on a radiator and put them on to
go home.
Casualty 2 was led down steps and placed in a vehicle. On
the way to the hospital, she drifted in and out of consciousness, and in the hospital a drip was inserted into her arm: she
was surprised to feel no pain. Doctors appeared worried by the
blood in her hair. She thought it must have been blood from a
terrorist. Later, she found blood on her clothes and realized
she had probably been dragged from the theatre.
A single urine specimen was received from a 56-year-old
man (Casualty 3) who had survived the siege. The specimen
was provided on October 31, 2002, five days after the man had
inhaled the chemical aerosol, and arrived at Dstl Porton Down
one day later. According to the accompanying medical notes,
the man had been exposed to the aerosol for “maybe 1 hour”
and had fallen unconscious for “at least 2-3 hours.” After being
rescued, he had been moved to a hospital in Moscow, where
he had received: a mucolytic, corticosteroids, frusemide (a diuretic), amikacin and ofloxacin (both antibiotics), ipratropium
(an anticholinergic drug) and propranolol (a beta-blocker). He
was later discharged and had not received any medication the
day before he donated the urine sample.
Other eyewitness accounts suggest that the Russian Special
Forces pumped the chemical aerosol through the ventilation
system (5) at approximately 5:00 a.m. Other hostages inside
the theatre also saw and smelt the aerosol, were rapidly
overwhelmed by it, and after 30 –60 min were evacuated to
hospitals in Moscow. Unconscious, they had inhibited tendon,
pupil and corneal reflexes, respiratory depression and cyanosis.
The Russian Government (5) commented that hospital care
for those most intoxicated involved oxygen administration,
mechanical ventilation and injection of naloxone (a standard
antidote for treating opioid overdose) (7). Less affected individuals were torpid, disorientated and vomiting, had pinpoint
pupils, bradycardia and hypotension, and received symptomatic
treatment.

Experimental
Materials and methods
Fentanyl and its analogues are highly physiologically active: analytical standards should be handled with care, wearing gloves
and eye protection, and working in an efficient fume cupboard.
Sample storage
Separately packaged clothing items that arrived at Dstl Porton
Down were stored in a sealed container in a cold room (48C).
Two blood samples were obtained from each survivor (Casualties
1 and 2). They were supplied in ethylenediaminetetraacetic acid
648 Riches et al.

(EDTA)-treated (4 mL) and Z-serum sep-clot activator (8 mL)
Vacutainer tubes (BD, Franklin Lakes, NJ). EDTA-treated tubes
were centrifuged for 5 min at 5,000 rpm and stored at 48C.
The Z-serum clot-activated samples were similarly stored.

Standards
Fentanyl hydrochloride, cis-3-methylfentanyl free base, carfentanil oxalate, sufentanil citrate, lofentanil oxalate, remifentanil
hydrochloride, norcarfentanil and remifentanil acid were
synthesised in-house and were .98% pure by 1H and 13C
nuclear magnetic resonance (NMR) spectroscopy. Stock solutions of the standards were prepared in Milli-Q water (0.5 mg/
mL), except for cis-3-methylfentanyl free base, which was dissolved in acetonitrile (Distol grade, BDH Ltd., Poole, Dorset,
UK), and subsequently diluted.

Clothing samples
Cutting and placing fabric swatches into sample bottles was
conducted with new equipment in a room separate from the
laboratory in which the solvent extraction and analysis was performed. Three swab samples using methanol-soaked 10 cm2
sections of lint cloth (RS Components Ltd., Oxford, UK) that
had been pre-cleaned with dichloromethane (CH2Cl2) were
taken from different areas of the leather jacket of Casualty 1:
front left-hand side (including the sleeve), front right-hand side
(including the sleeve) and the back (excluding the sleeves).
The surface area of each swabbed section was approximately
2,500 cm2. The samples were extracted with water (10 mL) for
30 min with occasional shaking. Aliquots of each extract
(3 mL) were then treated with 1 M aqueous NaOH (200 mL)
and extracted with CH2Cl2. The samples were vortexed and
centrifuged, and the CH2Cl2 layer was separated, concentrated
to dryness (nitrogen stream at 408C) and redissolved in CH2Cl2
(100 mL) for gas chromatography –mass spectrometry (GC –
MS) analysis.
Samples of the front sections of the jumper and shirt from
Casualties 1 and 2, respectively, of area 10   10 cm, were
extracted with CH2Cl2 (200 mL). Extracts were decanted into
clean sample bottles. Excess solvent was removed under a
nitrogen stream and the residues were extracted with water
(200 mL). This procedure allowed fentanyls to be extracted as
free base (organic extract) or salt form (aqueous extract). The
dichloromethane extracts were filtered. Aliquots of the filtered
extracts were evaporated to dryness and redissolved in CH2Cl2
(100 mL) for analysis by GC –MS. Aliquots (10 mL) of the
aqueous extract of each item of clothing were treated with
1 M aqueous NaOH (500 mL) and extracted with CH2Cl2
(1 mL), as described for the leather jacket extracts. Aliquots of
the organic extracts were extracted with water (10 mL). The
water extracts were treated with 1 M aqueous NaOH (500 mL)
and extracted with CH2Cl2, as already described. Fresh portions
of the jumper and shirt were also extracted with water only
and aliquots of these samples (10 mL) were basified and
extracted with CH2Cl2. To investigate the detection limit, two
samples of the shirt were spiked with 1 mg of the fentanyl salt
mixture and 9 mg of sufentanil (internal standard). Samples
were extracted with CH2Cl2, then water, and with water only,
as already described.

 Phase extraction
Solid-phase extraction (SPE) was used to clean samples before
analysis by liquid chromatography –tandem mass spectrometry
(LC –MS-MS) based on the method of Shou (8), but scaled up
to reflect the larger sorbent bed volume of the SPE cartridges
used (130 mg versus 25 mg) instead of the SPE plates described
in the published method. Plasma (1 mL) or aqueous extracts
(up to 5 mL) were loaded onto 130 mg Bond Elut Certify SPE
cartridges (Varian Ltd., Oxfordshire, UK) preconditioned with
2 mL of methanol–water –5% acetic acid solution. Cartridges
were then washed with 5% aqueous acetic acid (1 mL) and
methanol (1 mL) and dried under gentle vacuum. Cartridges
were eluted with two 0.75 mL aliquots of 2% aqueous NH4OH
in 4:1 v/v chloroform–isopropanol. Samples were concentrated
to dryness under a stream of nitrogen and dissolved in 100 mL
of 95:5:0.05 v/v/v acetonitrile – water–trifluoroacetic acid
(TFA).

LC –MS-MS method
A Surveyor LC system (Thermo-Finnigan, Hemel Hempstead,
UK) comprising a quaternary pump (but using two channels)
and an autosampler was used. A Betasil Silica-100 (50   3 mm)
column (Thermo Hypersil Keystone) was used with a flow rate
of 200 mL/min. The isocratic mobile phase comprised 7%
0.05% TFA in water (v/v) and 93% 0.05% TFA in acetonitrile
(v/v). The injection volume was 10 mL. A TSQ Quantum triple
quadrupole mass spectrometer (Thermo-Finnigan) was operated in positive ion electrospray mode with the electrospray
needle maintained at 3 kV. The instrument was tuned by infusing
a 100 ng/mL solution of fentanyl in 1:1 v/v water–acetonitrile at
a flow rate of 10 mL/min. Sheath gas and auxiliary flow rates
were set to 30 and 10, respectively, and capillary temperature
to 3508C. Both quadrupoles were maintained at unit resolution
(0.7 at half-peak height). Fentanyl and analogues produced
abundant [M þ H]þ ions. The tube lens and capillary offset settings were optimised for the transitions m/z 337 ! 188 and
m/z 337 ! 105 (optimum collision energy for these transitions was 28 and 44 V, respectively). For cis-3-methylfentanyl
and carfentanil, selected reaction monitoring (SRM) transitions
were selected by acquiring the product ion spectra of the
parent ions m/z 351 and 395, respectively. Carfentanil gave a
wider range of fragment ions than fentanyl and cis-3methylfentanyl; thus, the overall sensitivity of the SRM method
was lower.

LC –MS-MS analysis of clothing extracts
The LC– MS-MS method was very sensitive for fentanyl and
cis-3-methylfentanyl. Its linearity determined via a six-point
calibration curve, evaluated over the concentration range 0.025
to 0.5 ng/mL, gave a line of regression with a correlation coefficient (r 2) of 0.999 (Supplementary Figure S1). The lowest concentration detected for each analyte was 0.01 ng/mL, close to
the detection limit of the instrument. Samples used for the
GC –MS study were dried and redissolved in 95:5 acetonitrile –
water (containing 0.05% TFA). They were screened for fentanyl
and cis-3-methylfentanyl using a method that monitored for
two transitions (negative for all samples) and for a carfentanil

standard in full scan mode. Appropriate transitions were added
to the SRM method for carfentanil and a strong signal was
obtained for the water extract of the shirt at the correct retention time for carfentanil. A portion (5 mL) of the original water
extract of the shirt was processed using SPE and analyzed
for carfentanil using a method containing four transitions for
carfentanil, and a positive signal was obtained for all four transitions. To confirm this, a fresh sample of the shirt was processed
by SPE with a full glassware blank. Again, a positive result was
obtained. The jumper extract and leather jacket swab samples
were analyzed under identical conditions. The shirt was also
extracted with dichloromethane and methanol, but carfentanil
was not detected in either extract.
Shirt sample extracts were examined for remifentanil based
on the transitions m/z 377 to 228, 261, 285 and 317. The transition m/z 377 ! 285 was adversely affected by chemical
noise, so the transition m/z 377 ! 116 (40 eV) was selected
instead. Remifentanil was less sensitive than fentanyl, but a
0.1 ng/mL standard offered good signal-to-noise. Analysis of
this sample resulted in a strong signal for four and three SRM
transitions. A remifentanil standard was analyzed by SRM in
positive ion chemical ionization mode on the benchtop GC –
MS system (Finnigan MAT GCQ ion trap mass spectrometer).
The limit of detection was  10 ng/mL, approximately 100
times higher than from the LC –MS-MS method.

SPE efficiency
Recovery of the fentanyls was studied using the SPE protocol
used for the clothing extracts. A water sample was spiked with
all analytes at a concentration of 10 ng/mL. This sample was
processed as normal. A second clean water sample was processed and spiked at the same concentration after elution from
an SPE cartridge. The ratio of the peak areas for the cartridge
spiked before the SPE procedure with that spiked afterwards
was calculated as a percentage (recovery) and offered the following results: fentanyl (71%), cis-3-methylfentanyl (62%), carfentanil (67%), remifentanil (102%) and lofentanil (67%). These
data confirmed that the SPE method provided good recoveries
of all the analytes.

Analysis of blood samples
Aliquots of plasma (500 mL) from the EDTA-treated blood were
pipetted into 2 mL Aquasil-treated vials, aqueous NaOH (4 M,
50 mL) and then 1-chlorobutane (1 mL) were added to each
vial, and the samples were vortexed and left for 10 min, and
vortexed again and left for a further 5 min. The organic layer
was removed and placed in another 2 mL Aquasil-treated vial
and the solvent was removed under a gentle stream of nitrogen
at 408C. The residue was dissolved in toluene (20 mL). Plasma
for control and spiking experiments was obtained with
consent from one of the authors.
Plasma samples were spiked with low concentrations of fentanyl salts, cleaned up as described earlier, and analyzed by
GC– MS-MS. Again, good linearity was obtained for the fentanyl
standards (r 2   0.99). The samples spiked with the lowest concentration standard (0.2 ng/mL) showed a detectable peak for
all the analytes, except carfentanil (which was identified in the
sample spiked at the 0.4 ng/mL level).

Analysis of Clothing and Urine from Moscow Theatre Siege Casualties Reveals Carfentanil and Remifentanil Use 649

 Plasma samples spiked with fentanyl and cis-3-methylfentanyl
were processed by the SPE method and analyzed by LC –MS-MS
(r 2   0.99). The lower limit of detection achieved for both analytes was 0.05 ng/mL, which is in agreement with that of the
study by Shou (8) on which this method was based. Because
of the limited sample available, the samples used for the
GC –MS-MS analysis were evaporated to dryness, dissolved in
acetonitrile –water, and analyzed by LC –MS-MS using a method
incorporating transitions for fentanyl, cis-3-methylfentanyl and
carfentanil.

LC –MS-MS analysis of urine
All extractions were performed manually. Control urine was a
commercial freeze-dried product (Randox Laboratories Ltd,
Crumlin, County Antrim, UK). Blank and spiked control
samples were analyzed together with aliquots of urine from
Casualty 3. Briefly, the urine (1 mL) was diluted with 0.2 M
ammonium acetate buffer ( pH 4, 1 mL) and loaded onto an
Oasis HLB (60 mg/3 mL) cartridge (Waters Ltd, Elstree, UK)
pre-conditioned with methanol and water. The cartridge was
washed with water and 20:80 methanol –water. Excess solvent
was expelled and the cartridge was eluted with methanol. The
eluate was evaporated to dryness in a centrifugal evaporator
(SPD 121P Speedvac, ThermoSavant, Holbrook, NY) at 508C.
Residues were dissolved in 0.05% TFA in water and transferred
to glass autosampler vials with 250 mL glass inserts.
LC–MS-MS was performed using a Surveyor MS pump and
autosampler (Thermo Scientific) with a Halo C8, 50   2.1 mm,
2.7 mm particle size column (Hichrom, Reading, UK), interfaced to a Deca XP Plus ion trap mass spectrometer (Thermo
Scientific). The mass spectrometer was equipped with an electrospray ionization source. Mobile phase consisted of 0.05%
TFA in water and acetonitrile at a flow rate of 0.25 mL/min.
Elution used a gradient of 0.05% TFA in 5% acetonitrile –95%
water for 2 min, which was increased to 75% acetonitrile –25%
water at 24 min and held for 2 min. The column was fitted with
a C8 SecurityGuard guard cartridge (Phenomenex, Macclesfield,
Cheshire, UK) and maintained at 258C. The injection volume
was 10 mL.
The mass spectrometer was operated in positive ion SRM
mode. Source conditions were: spray voltage, 5.5 kV; sheath gas
(nitrogen) flow, 50; sweep gas (nitrogen) flow, 20; heated capillary temperature, 2508C. The monitored SRM transitions were
m/z 291.0 ! 259.0 for norcarfentanil and m/z 363.0 ! 330.9
for remifentanil acid. The limit of detection was approximately
0.02 ng/mL for both norcarfentanil and remifentanil acid.

Results
Fentanyl, first synthesized over 45 years ago (9), is a short-acting
analgesic used clinically in humans to treat acute and chronic pain
(10). One of a family of related compounds (11, 12) it binds like
morphine to m-opioid receptors (13). It is 80–100 times more
potent than morphine and some of its relatives are more powerful
still. The fentanyls that ranked highest among contenders used to
end the siege were fentanyl, cis-3-methylfentanyl and carfentanil
(Figure 1A), which various media spokespeople speculated had
been used. Cis-3-methylfentanyl (14) had appeared on the
650 Riches et al.

Figure 1. Structures of fentanyl compounds: fentanyl, sufentanil and remifentanil
are analgesic-anaesthetics used in humans, and carfentanil is used in veterinary
medicine to tranquillise large mammals (A); fentanyl and carfentanil are metabolized
by the liver to nor compounds, and remifentanil reacts with blood enzymes to
produce remifentanil acid; the metabolites are not especially biologically active and
are excreted unchanged in urine (B).

Russian black market (15) under the name of “Krokodil” (crocodile) and the Russian press had already commented that a
trimethylfentanyl (allegedly investigated under the codename
“Kolokol-1” by the KGB) had been used. Other candidate
fentanyls included sufentanil (16), lofentanil and remifentanil
(17), a metabolically-labile variant that had been introduced into
clinical practice by GlaxoSmithKline in the 1990s.
The GC –MS and GC –MS-MS results after analysis for fentanyl, cis-3-methylfentanyl, carfentanil, sufentanil, lofentanil and
remifentanil were negative for the clothing and blood samples.
Further analyses were undertaken after development of a very
sensitive analytical method using LC–MS-MS; analyte concentrations approximately 50 times lower could be detected in
comparison to the less sensitive GC –MS-MS protocol. Extracts
were specifically analyzed for fentanyl, cis-3-methylfentanyl,
carfentanil and lofentanil. A subsequent analysis was undertaken for remifentanil (clothing only). None of the analytes
were detected in extracts of the jacket of Casualty 1. A suggestion of carfentanil was observed in the jumper extracts.
However, the chromatographic peaks were too obscured by
interfering components to allow a positive identification to
be made (Supplementary Figure S2). Low concentrations
(, 0.5 ng/mL) of carfentanil and remifentanil were detected in
the concentrated aqueous extracts of the shirt of Casualty
2. No fentanyls were found in the blood samples.
In a confirmatory analysis, monitoring four structurally specific fragmentations of the protonated molecule, carfentanil
and remifentanil were detected in fresh shirt extracts. Good
signal-to-noise ratios were obtained for each at the correct retention time. Relative peak areas agreed closely with those of a
carfentanil standard (Table I). Those for remifentanil showed a

 with the other results of these analyses and provides additional
evidence for the use of carfentanil. Analyses including SRM
transitions for intact remifentanil and carfentanil provided no
evidence for their presence.

Table I
LC –MS-MS MRM Detection of Carfentanil and Remifentanil in Shirt Extract*
Production

Relative peak areas (%)†

Carfentanil
335.1
279.2
246.2
134.1
Remifentanil
228.1
116.0
261.0

Sample
—
100
40
58
37
—
100
41
82

Standard
—
100
36
50
35
—
100
57
77

*Note: Relative peak areas of product ions m/z 395.3 for carfentanil and m/z 377.1 for
remifentanil in an extract of shirt sample are shown versus those measured for the pure
analytical standards dissolved in ultra-high-purity water (0.5 ng/mL).
†
Reference ion ratios were from standards selected to provide an equivalent signal to that from
the sample as far as possible.

greater variability due to a lower concentration and greater
background interference. Closer agreement with a standard
was obtained by monitoring three fragmentations (Table I). No
carfentanil or remifentanil was detected in the glassware
blanks. The total amounts of carfentanil and remifentanil
extracted were estimated as ,5 ng, with carfentanil present at
the higher concentration. In fact, comparison of the signal
from the shirt sample with standard solutions indicated that
the concentration of carfentanil in the concentrated shirt
extract was approximately 0.3 ng/mL. This corresponded to a
concentration of 10 pg/mL in the original extract. Examples of
chromatograms derived from the shirt extracts, and from positive and negative controls, appear in Figures 2 and 3. Following
these results, more concentrated extracts of the shirt after SPE
clean-up were specifically analyzed by GC –MS-MS for carfentanil and remifentanil (data not shown). These analyses were
negative, but the detection limits were not as low as those
obtained by LC–MS-MS and the chromatograms revealed many
more extraneous components, which adversely affected detection limits and degraded column performance.
The urine from Casualty 3 was analyzed for the major
metabolites of carfentanil and remifentanil. Although the main
metabolite of carfentanil had not been identified unequivocally
in humans before, based on its resemblance to fentanyl, which
is metabolized to norfentanyl (18, 19), it was possible to
predict a likely candidate: norcarfentanil (20) (Figure 1B).
Remifentanil, characterized by very short action and potency
similar to fentanyl, is metabolized quickly in blood to remifentanil acid (17, 21). The metabolites of fentanyls generally lack
appreciable physiological action (17).
A known procedure for automated SPE of fentanyls and
metabolites from urine was used (22), modified slightly to
allow for a larger sample volume. An initial analysis for norcarfentanil and remifentanil acid showed a peak corresponding to
norcarfentanil in chromatograms from the urine sample. The
peak was comparable ( 75% height) to that from a sample of
control urine spiked with 0.1 ng/mL norcarfentanil. No remifentanil acid was detected (Figure 4A). Repeat analysis for norcarfentanil alone confirmed the presence of this metabolite
(Figure 4B). Although it may be argued that the identification
of norcarfentanil in the urine sample is not definitive because
only a single transition was monitored, it is entirely consistent

Discussion
The case histories of Casualties 1 to 3 agree with those of
other Moscow Theatre Siege survivors (5). The rapid unconsciousness upon inhalation of the aerosol and loss of pain sensation upon awakening experienced by Casualties 1 and 2 are
consistent with exposure to fentanyl/s. The effects of such
compounds are well known: in moderate doses, pain suppression, and at higher doses, a sleep-like state that can progress to
coma. Fentanyls depress the urge to breathe in a dosedependent manner, and when unconscious, breathing may
slow to the point that sufficient oxygen cannot be maintained
in the blood to sustain normal function. Even if breathing continues at a lowered rate, fentanyls can cause the neck and
tongue to become limp, which can lead to airway obstruction.
Additionally, when administered rapidly, fentanyls can cause
muscular rigidity, which can result in cessation of breathing. It
is uncertain whether the three casualties were treated with naloxone post-aerosol exposure, as evidence suggests for some
survivors (4, 5). Naloxone was not among the drugs given to
Casualty 3 while he was hospitalized. However, he did receive
ipratropium, which is used for treating chronic obstructive
lung disease, and this may have helped him to breathe normally. While in the hospital, Casualties 1 and 3 appear to have
received diuretic drugs, presumably to help excrete any
fentanyls.
The LC–MS-MS results for the shirt and urine sample resolve
the mystery of whether the Russian Special Forces used a
single chemical or a combination of chemicals. Positive identification of carfentanil and remifentanil on the shirt of Casualty 2
suggests that the aerosol contained these compounds.
Norcarfentanil, identified in the urine sample from Casualty 3
after he donated it five days after he had been exposed, implies
that carfentanil was used. Remifentanil is not appreciably metabolized by the lung (23), but has a methyl ester linkage that is
hydrolyzed by blood and tissue enzymes to give remifentanil
acid. Piperidine N-dealkylation of remifentanil to produce norcarfentanil is not a significant metabolic pathway in humans
(23). The detection of norcarfentanil in urine donated so many
days after inhalation of the aerosol is presumably due to metabolic N-dealkylation of carfentanil (20) and the high fatsolubility of carfentanil; elimination of lipophilic fentanyls is
limited by their redistribution in the body (17). An absence of
remifentanil acid in the urine was unsurprising: remifentanil is
rapidly metabolized with a mean half-life in humans of  2 h
(23), and this casualty had undergone forced diuresis in hospital. The blood samples were collected 19 h (Casualty 1) and
12 h (Casualty 2) after exposure to the aerosol, and detection
of any agent in the samples was considered unlikely.
Carfentanil is more potent than fentanyl (24). Its sole
approved use is by veterinarians to incapacitate wildlife for
examination and procedures; it is not approved for use in
humans. The first documented human exposure to carfentanil
in the open literature became available only recently (24).

Analysis of Clothing and Urine from Moscow Theatre Siege Casualties Reveals Carfentanil and Remifentanil Use 651

 Figure 2. LC–MS-MS MRM detection of carfentanil on the shirt of Casualty 2: detection of carfentanil, monitoring fragmentation m/z 395.3 ! 335.1 (top), glassware control
(middle, baseline magnified  10 times) and a carfentanil standard (bottom) (A); detection of carfentanil in the shirt extract monitoring four fragmentations of the protonated
molecule m/z 395.3 (B).

652 Riches et al.

 Figure 3. LC– MS-MS MRM detection of remifentanil on the shirt of Casualty 2: detection of remifentanil, monitoring the fragmentation m/z 377.1 ! 317.1 (top), the
glassware control (middle) and a remifentanil standard (bottom) (A); detection of remifentanil in a shirt extract monitoring four fragmentations of the protonated molecule m/z
377.1 (B).

Analysis of Clothing and Urine from Moscow Theatre Siege Casualties Reveals Carfentanil and Remifentanil Use 653

 Figure 4. LC– MS-MS SRM chromatograms for the urine sample from Casualty 3: transition m/z 363.0 ! 330.9 from an extract of blank urine (top), urine from Casualty 3
(middle) and spiked urine (bottom) showing the absence of remifentanil acid (A); transition m/z 291.0 ! 259.0 from an extract of blank urine (top), urine from Casualty 3
(middle) and spiked urine (bottom) showing the presence of norcarfentanil (B).

A 47-year-old male splashed his eyes and mouth accidently
with the contents of a tranquillizer dart containing 1.5 mg carfentanil citrate. He washed his face immediately with water,
but became drowsy two minutes later. His colleagues,
654 Riches et al.

recognizing that he had been poisoned, administered 100 mg
of the reversal agent naltrexone (whose antagonistic potency is
approximately twice that of naloxone). This presumably saved
his life: the man complained an hour later, while in the

 hospital, of mild and transient chest discomfort, but this disappeared and he was discharged in a stable condition a day later.
This account suggests that carfentanil exposures may cause
severe intoxication or fatality in the absence of prompt and
appropriate medical treatment. The same conclusion can be
drawn for remifentanil. Both carfentanil and remifentanil have
narrow safety margins, meaning that potentially fatal side
effects, including respiratory depression, can occur at doses
only slightly higher than those that impart medical benefits.
Carfentanil may have been mixed with the less potent,
shorter-acting remifentanil in an attempt to lessen the number
of fatalities that may have otherwise resulted from exposure to
carfentanil alone. If the remifentanil was used to dilute the carfentanil, then presumably it was disseminated in higher concentration (this was not reflected by the analysis results,
possibly due to its lower stability). An advantage of remifentanil
for surgical use is that the depth of narcosis can be titrated so
that the dose rate approximates the metabolism rate. Its use by
the Russian Special Forces may partially explain why casualties
survived an estimated aerosol exposure of 30 –45 min. Human
data for other fentanyls suggest that once patients lose consciousness, life-threatening respiratory depression can occur.
Scientific papers published by Russian military officers indicate an interest in fentanyls extending back 12 years: opioid receptor studies (7, 25), fentanyl analysis (26) and synthesis of
fentanyl precursors (27, 28). That Russian military research on
fentanyls occurred before 1994 is evident from a passage in a
book by General Antonov (29), a former director of the
Military Chemical Institute in Shikhany, which states that “the
action of analgesics is a knock-out blow—personnel subject to
an attack of forces only a few minutes after the beginning of a
chemical attack will lose their capacity to stand, not to
mention move about. In severe cases people will enter an ‘unconscious state’ and ‘carfentanil is one of the most active substances of the entire group of the studied derivatives of
fentanyl. It manifests its activity for different pathways of entry
into the organism, including inhalation of vapours or aerosol.’”
To the authors’ knowledge, this is the only information on carfentanil aerosolization in the public domain. One further use of
an incapacitant has been reported since the theatre siege (30).
On October 13, 2005, armed Chechen separatists attacked the
Russian town of Nalchik. In response, Russian Special Forces
employed a “narcotic agent” against the separatists who were
holding two women hostage in a shop. No information about
the narcotic agent has emerged, although the affected hostages
were rescued and revived successfully by an unidentified
antidote.

Conclusions
The detection of carfentanil and remifentanil by LC –MS-MS on
the shirt of Casualty 2 worn throughout the siege, monitoring
four and three specific fragmentations, respectively, provides
high confidence in the identification. Differences between the
relative peak areas for the fentanyls identified in extracts of the
sample and those of analytical standards were between 2 –8%
for carfentanil and 5 –16% for remifentanil (Table I). In other
areas of trace analysis, monitoring of two fragmentations at the
correct retention time, with relative peak areas within 20% of

those of a standard, is usually considered sufficiently specific
for unequivocal identification. Ideally, identification of this
mixture by two independent techniques would have been
desirable, but complementary GC –MS-MS results were not
achieved due to the lower sensitivity of the GC instrument
coupled with high levels of chemical noise. The identification
of the metabolite norcarfentanil by LC–MS-MS analysis of urine
donated by a separate casualty, considered alongside the clothing results and the expected metabolic differences between
carfentanil and remifentanil, suggest that this metabolite most
likely originated from carfentanil. Similar searches in the urine
sample for intact carfentanil and remifentanil, and its metabolite remifentanil acid, were unsuccessful, as were analogous
searches among the blood samples.
The finding that the aerosol comprised carfentanil and remifentanil is consistent with the outcome arising from its use, for
literature data suggest little margin of safety between their
therapeutic and lethal doses in humans, and a high lethality in
the absence of prompt and appropriate medical intervention. It
is highly improbable that a chemical agent exists for which a
dose can be calibrated in a tactical environment to incapacitate
opponents reliably and without substantial mortality. Mass
spectrometric techniques such as those described are sufficiently powerful to detect the presence of fentanyls in a range
of matrices at trace levels and allow their use to be verified and
attributed to individuals or states.

Acknowledgments
We are grateful to the UK Ministry of Defence (MoD) for
funding this study. # Crown Copyright 2012. Published with
permission of the Defence Science and Technology Laboratory
on behalf of the Controller of Her Majesty’s Stationary Office.

References
1. (2002) “Russian Health Minister says substance used in raid is not
prohibited by CWC.” Russian News Agency (ITAR-TASS), Moscow,
news bulletin in English, 30 October, 1540 GMT.
2. Schiermeier, Q. (2002) Hostage deaths put gas weapons in spotlight. Nature, 420, 7.
3. Rieder, J., Keller, C., Hoffmann, G., Lirk, P. (2003) Moscow theatre
siege and anaesthetic drugs. The Lancet, 361, 1131.
4. Wax, P.M., Becker, C.E., Curry, S.C. (2003) Unexpected “gas” casualties in Moscow: A medical toxicology perspective. Annals of
Emergency Medicine, 41, 700– 705.
5. Finogenov and Others v. Russia (2011) Judgment of the European
Court of Human Rights (First Section), Applications Nos. 18299/03
and 27311/03, Strasbourg. http://cmiskp.echr.coe.int/tkp197/
viewhbkm.asp?sessionld=83685113&skin=hudoc-en&action=html
&table=F69A27FD8FB86142BF01C1166DEA398649&key=95152&
highlight= (accessed 16 January 2012).
6. New Scotland Yard (NSY) (2002) British hostage debrief reports:
Casualties 1 and 2 interviewed by a detective inspector from the
NSY Hostage & Crisis Negotiation Unit and a clinical psychologist
from the British National Crime Faculty, London, UK.
7. Kuzmina, N.E., Kuzmin, V.S. (2011) Development of concepts on
the interaction of drugs with opioid receptors. Russian Chemistry
Reviews, 80, 145– 169.
8. Shou, W.Z. (2001) A highly automated 96-well solid phase extraction and LC tandem MS method for the determination of fentanyl
in human plasma. Rapid Communications in Mass Spectrometry,
15, 466–476.

Analysis of Clothing and Urine from Moscow Theatre Siege Casualties Reveals Carfentanil and Remifentanil Use 655

 9. Janssen, P.A.J. (1964) Method for producing analgesia. US Patent
3,141,823.
10. Drdla-Schutting, R., Benrath, J., Wunderbaldinger, G., Sandkühler, J.
(2012) Erasure of a spinal memory trace of pain by a brief, highdose opioid administration. Science, 335, 235– 238.
11. Van Daele, P.G.H., De Bruyn, M.F.L., Boey, J.M., Sanczuk, S., Agten,
J.T.M., Janssen, P.A.J. (1976) Synthetic analgesics: N-(1-[2-arylethyl]4-substituted 4-piperidinyl) N-arylalkanamides. Arzneimittel Forschung,
26, 1521–1531.
12. Van Bever, W.F.M., Niemegeers, C.J.E., Schellekens, K.H.L., Janssen,
P.A. J. (1976) N-4-Substituted 1-(2-arylethyl)-4-piperidinyl-N-phenylpropanamides, a novel series of extremely potent analgesics
with unusually high safety margin. Arzneimittel Forschung, 26,
1548–1551.
13. Dosen-Micovic, L., Ivanovic, M., Micovic, V. (2006) Steric interactions and the activity of fentanyl analogs at the m-opioid receptor.
Bioorganic and Medicinal Chemistry, 14, 2887–2895.
14. Gergov, M., Nokua, P., Vuori, E., Ojanperä, I. (2009). Simultaneous
screening and quantification of 25 opioid drugs in post-mortem
blood and urine by liquid chromatography-tandem mass spectrometry. Forensic Science International, 186, 36 –43.
15. Godunov, A.V., Kireeva, A.V., Volchenko, S.V. (2007). Determination
of 3-methylfentanyl by GC/MS in biological and non-biological
objects. Forensic Science International, 169S, S29–S35.
16. Liang, L., Wan, S., Xiao, J., Zhang, J., Gu, M. (2011) Rapid UPLC-MS/
MS method for the determination of sufentanil in human plasma
and its application in target-controlled infusion system. Journal of
Pharmaceutical and Biomedical Analysis, 54, 838– 844.
17. Feldman, P.L., James, M.K., Brackeen, M.F., Bilotta, J.M., Schuster,
S.V., Lahey, A.P. et al. (1991) Design, synthesis, and pharmacological
evaluation of ultrashort- to long-acting opioid analgetics. Journal
of Medicinal Chemistry, 34, 2202– 2208.
18. Labroo, R.B., Paine, M.F., Thummel, K.E., Kharasch, E.D. (1997).
Fentanyl metabolism by human hepatic and intestinal cytochrome
P450 3A4: Implications for interindividual variability in disposition,
efficacy, and drug interactions. Drug Metabolism and Disposal,
25, 1072–1080.
19. Coopman, V., Cordonnier, J., Pien, K., Van Varenbergh, D. (2007)
LC-MS/MS analysis of fentanyl and norfentanyl in a fatality due to
application of multiple Durogesicw transdermal therapeutic
systems. Forensic Science International, 169, 223–227.

656 Riches et al.

20. Endres, C.J., Bencherif, B., Hilton, J., Madar, I., Frost, J.J. (2003)
Quantification of brain m-opioid receptors with [11C]carfentanil:
Reference-tissue methods. Nuclear Medicine and Biology, 30,
177–186.
21. Said, R., Pohanka, A., Andersson, M., Beck, O., Abdel-Rehim, M.
(2011) Determination of remifentanil in human plasma by liquid
chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation. Journal of
Chromatography B, 879, 815– 818.
22. Wang, L., Bernert, J.T. (2006) Analysis of 13 fentanils, including
sufentanil and carfentanil, in human urine by liquid chromatography-atmospheric-pressure ionization-tandem mass spectrometry.
Journal of Analytical Toxicology, 30, 335–341.
23. Navapurkar, V.U., Archer, S., Gupta, S.K., Muir, K.T., Frazer, N., Park,
G.R. (1998) Metabolism of remifentanil during liver transplantation.
British Journal of Anaesthesia, 81, 881– 886.
24. George, A.V., Lu, J.J., Pisano, M.V., Metz, J., Erickson, T.B. (2010)
Carfentanil—An ultra potent opioid. American Journal of
Emergency Medicine, 28, 530– 532.
25. Dukhovich, F.S., Darkhovskii, M.B., Gorbatova, E.N., Polyakov, V.S.
(2005) The agonist paradox: Agonists and antagonists of acetylcholine receptors and opioid receptors. Chemistry & Biodiversity, 2,
354– 366.
26. Zlobin, V.A., Bukreeva, L.P., Kuznetsov, P.E., Panfilov, A.V., Nazarov,
G.V., Kirsanov, A.T. (2000) Analysis of opiates by HPLC with indirect spectrophotometric detection. Pharmaceutical Chemistry
Journal, 34, 279–280.
27. Panfilov, A.V., Markovich, Y.D., Ivashev, I.P., Zhirov, A.A., Eleev, A.F.,
Kurochkin, V.K. et al. (2000) Sodium borohydride in reductive
amination reactions. Pharmaceutical Chemistry Journal, 34,
76– 78.
28. Panfilov, A.V., Markovich, Y.D., Zhirov, A.A., Ivashev, I.P., Kirsanov,
A.T., Kondrat’ev, V.B. (2000) Reactions of sodium borohydride in
acetic acid: Reductive amination of carbonyl compounds.
Pharmaceutical Chemistry Journal, 34, 371– 373.
29. Antonov, N.S. (1994) Chemical weapons at the turn of the century.
English Translation LN72-96, Progress Publisher, Moscow, Russia.
30. Crowley, M. (2009) Dangerous ambiguities: Regulation of riot
control agents and incapacitants under the Chemical Weapons
Convention. Bradford Non-Lethal Weapons Research Project,
University of Bradford, UK, p. 76.