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Forensic Chemistry 3 (2017) 7480

Contents lists available at ScienceDirect

Forensic Chemistry
journal homepage: www.elsevier.com/locate/forc

Case Reports

Analysis of illicit carfentanil: Emergence of the death dragon


John F. Casale , Jennifer R. Mallette, Elizabeth M. Guest
U.S. Drug Enforcement Administration, Special Testing and Research Laboratory, Dulles, VA 20166, USA

a r t i c l e i n f o a b s t r a c t

Article history: The United States is currently in the midst of an unprecedented illicit fentanyl crisis. In the last year alone
Received 8 December 2016 there have been more recent fentanyl-related overdose deaths than in the previous 60 years. The current
Available online 16 February 2017 crisis is multi-faceted and involves a global supply of fentanyl and related substances being smuggled
into the United States. Illicit carfentanil hydrochloride has recently entered the drug market causing mul-
Keywords: tiple overdoses and deaths across the U.S. To date, over 400 confirmed carfentanil cases have been iden-
Carfentanil tified. Carfentanil is approximately 100 times more potent that fentanyl with only 20 lg of material
Analysis
required to produce a lethal dose. Due to a lack of published spectra for carfentanil HCl, analytical profiles
Chromatography
Spectroscopy
are provided for three recent carfentanil submissions to our laboratory which include infrared spec-
Spectrometry troscopy, nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, isotope
Forensic chemistry ratio mass spectrometry, and quantitative determination via gas chromatographyflame ionization
detection. The three submissions were determined to contain 0.62%, 1.87%, and 0.31% carfentanil HCl,
respectively. Each exhibit also contained a fentanyl-related substance (fentanyl or 2-furanylfentanyl).
Acetylcarfentanil was characterized as an impurity in two exhibits. Isotopic analyses of two exhibits sug-
gest they are intimately related.
Published by Elsevier B.V.

1. Introduction ples. Two profiling methods were developed, one of which was
published [4]. In that work, 40 synthetic markers were identified;
Over the past 40 years there have been three significant 12 were Janssen route-specific, 7 were Siegfried route-specific,
fentanyl-related epidemics within the United States. The first epi- and 21 were non route-specific. The markers were significant in
demic began in the late 1970s and was dubbed China White due determining relationships between samples.
to the nature, purity, and color of the fentanyl and fentanyl-related The current outbreak of FFRCs began in late 2013 and accounted
compounds (FFRCs). The FFRCs were determined to be of Chinese for over 5000 overdose deaths within the U.S. during 2014, as
origin and caused hundreds of overdose deaths. Many of the FFRCs reported by the Center for Disease Control (CDC) [5,6]. A compila-
were new compounds not previously seen by the forensic commu- tion of FFRC overdose deaths in 2015 is not yet available, but Ohio
nity, and extensive research was conducted to characterize and has reported over 1100 for that state alone. The only 2016 data
identify these new substances [13]. These FFRCs were a major available at this time is for New Hampshire with 200 opiate/opioid
influence in the implementation of the Controlled Substance Ana- deaths in the first 8 months. However, the National Forensic Labo-
logue Enforcement Act of 1986, which controlled drug analogs of ratory Information System (NFLIS) has tracked a dramatic increase
listed Schedule I and II drugs. A second wave of over 1000 fatal in fentanyl submissions to local, state, and federal laboratories over
overdoses from illicit fentanyl occurred during the 20052007 the last 4 years (CY 2013 = 934, CY 2014 = 7864, CY 2015 = 13,059,
time frame. Illicit fentanyl was determined to be originating from and CY 2016 through September = 16,263). If the relationship of
a clandestine laboratory in Mexico. Our laboratory was tasked with NFLIS sample submissions to CDC overdose deaths is consistent
providing chemical intelligence on seized fentanyl exhibits. A between 2014 and 2016, there could well be over 10,000 overdose
fentanyl signature profiling research project was established to deaths during CY 2016. Recently, the extremely potent FFRC, car-
determine the clandestine synthetic routes and link seized sam- fentanil (Fig. 1) (10,000 more potent that morphine, 100 more
potent that fentanyl) has been detected in seizures and overdose
death toxicology samples. Carfentanil was implicated in 125
Corresponding author at: Special Testing and Research Laboratory, U.S. Drug deaths that occurred during the Moscow Theatre Siege of 2002
Enforcement Administration, 22624 Dulles Summit Court, Dulles, VA 20166-9509, [7]. A lethal dose of carfentanil is estimated at only 20 micrograms
USA. (20 one-millionths of a gram) and is barely visible to the naked eye.
E-mail address: John.f.casale@usdoj.gov (J.F. Casale).

http://dx.doi.org/10.1016/j.forc.2017.02.003
2468-1709/Published by Elsevier B.V.
J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480 75

Fig. 1. Structures of carfentanil (1), acetylcarfentanil (2), fentanyl (3), acetylfentanyl (4), and 2-furanylfentanyl (5).

During CY 2016, this laboratory has been notified of over 400 con- 2. Cases
firmed carfentanil cases within the U.S., with the overwhelming
majority (84%) occurring in Ohio (Table 1). Herein, we report ana- Exemplars of two cases comprised of three total exhibits were
lytical data for illicit carfentanil from three case exhibits which submitted to this laboratory for in-depth analysis. Specific details
include infrared spectroscopy (IR), nuclear magnetic resonance for each exhibit cannot be disclosed due to current and active
spectroscopy (NMR), gas chromatographymass spectrometry enforcement investigations; however, the following non-sensitive
(GC/MS), isotope ratio mass spectrometry (IRMS), and quantitative information is provided:
determination via gas chromatographyflame ionization detection Case #1: Two separate exhibits (1A and 1B) of white powder
(GCFID). seized in the central U.S. weighing approximately 2 and 20 g,

Table 1
Confirmed carfentanil cases examined by local, state, and federal forensic laboratories during 2016.a

Reported drug with carfentanil OH MI FL KY IL RI GA IN Total


Carfentanil only 48 1 8 7 0 0 3 0 67
Fentanyl 3 0 0 0 0 0 0 2 5
Furanylfentanyl 4 0 0 0 0 4 0 0 8
Heroin 20 0 2 5 0 0 0 0 27
Heroin/fentanyl 8 0 0 3 1 0 0 0 12
Heroin/furanylfentanyl 1 0 0 0 0 0 0 0 1
Heroin/fentanyl/furanyfentanyl 1 3 0 0 0 0 0 0 4
Other controlled substancesb 8 1 1 0 0 0 0 0 10
No detailed reporting 250 0 23 0 0 0 0 0 273
Total 343 5 34 15 1 4 3 2 407
a
Data compiled by DEA Special Testing and Research Laboratory as of October 26, 2016.
b
Includes mixtures containing cocaine, FUB-AMB, U47700, methamphetamine, and cannabinoids.
76 J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480

respectively. Carfentanil and lactose were identified by the submit- (St. Louis, MO). Carfentanil citrate (Wildnil) was a product of
ting laboratory; however, quantitative data was not acquired. Wildlife Pharmaceuticals (Fort Collins, CO). Carfentanil hydrochlo-
Case #2: One exhibit of brown powder seized in the north cen- ride was obtained through purification and authentication of mate-
tral U.S. weighing approximately one kilogram. Heroin, carfentanil, rial from one of the listed case exhibits.
fentanyl, and lactose were identified by the submitting laboratory; Carfentanil quantitation was performed using an Agilent (Palo
however, quantitative data was not acquired. Alto, CA) Model 7890A gas chromatograph with flame ionization
detection. The GC system was fitted with a 30 m  0.25 mm ID
3. Methods fused-silica capillary column coated with DB-1 (0.25 mm) (Agilent,
Santa Clara, CA) in constant flow mode at 43.5 cm/s of hydrogen
All solvents were distilled-in-glass products of Burdick and carrier gas. The injection port and flame ionization detectors were
Jackson Laboratories (Muskegon, MI). All other chemicals were of maintained at 280 C. The oven temperature was programmed as
reagent-grade quality and products of Sigma-Aldrich Chemical follows: Initial temperature, 250 C; initial hold, 12.0 min; program

Fig. 2. Partial GC/MS reconstructed total ion chromatograms for CH2Cl2 soluble extracts of (a) Case #1, Exhibit 1A, (b) Case #1, Exhibit 1B, and (c) Case #2, Exhibit 1. Peak
Identification: 1 = diphenhydramine, 2 = acetylcodeine, 3 = O6-monoacetylmorphine, 4 = heroin, 5 = fentanyl, 6 = acetylcarfentanil, 7 = carfentanil, 8 = 2-furanylfentanyl, and
9 = noscapine.
J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480 77

rate, 25.0 C/min; final temperature, 280 C; final hold, 3.8 min. each linearity solution was evaporated to dryness and reconsti-
Nitrogen was used as the auxiliary make-up gas for the detector. tuted in 100 mL of CHCl3 and placed into a 200 lL insert for injec-
Samples were injected (2 lL injection) in the split mode (21:1) tion. Linearity was evaluated and the method was shown to have
by an Agilent 7683 Series Auto Injector. Linearity was evaluated a linear response across the concentration range (5.25105.0 lg/
with carfentanil citrate. A stock solution was prepared by accu- mL) where m = 0.019, b = 0.016, and r = 0.99994. Illicit samples
rately weighing 1.05 mg of carfentanil citrate into a 15-mL cen- were accurately weighed (ca. 20 mg) into a 15-mL centrifuge tube,
trifuge tube and extracting from aqueous base with ether dissolved in 1 mL of water, rendered basic with 2 drops of concen-
(2  5 mL). The ether extracts were combined, evaporated to dry- trated NH4OH, extracted with 5 mL of ether containing 1.00 mL of
ness, and reconstituted to volume in a 10.0 mL volumetric flask CHCl3 (containing 20 lg/mL of tetracosane). The organic phase was
with CHCl3 containing 20 lg/mL of tetracosane. A set of serial dilu- passed through a cotton-plugged pipet, evaporated to dryness and
tions were made in order to provide 4 more solutions. One mL of reconstituted in 100 mL of CHCl3 for injection.

Fig. 3. Electron Ionization Mass Spectra of (a) carfentanil 1 and (b) acetylcarfentanil 2. Molecular ion or M-2 not observed for 2.
78 J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480

Gas chromatography/mass spectrometry (GC/MS) was per- 5.6 min. The injector was operated in the split mode (22:1) and at a
formed using an Agilent Model 5975C quadrupole mass-selective temperature of 280 C. The auxiliary transfer line to the MSD was
detector (MSD) interfaced with an Agilent 7890A gas operated at 280 C. An approximately 5 mg/mL equivalent of
chromatograph. The MSD was operated in the electron ionization CH2Cl2 extract of carfentanil was injected at a volume of 2 mL.
mode with an ionization potential of 70 eV, a scan range of Infrared spectra were obtained on a Thermo-Nicolet Nexus 670
34700 mass units, and at 1.34 scans/s. The GC system was fitted FT-IR equipped with a SensIR Dura-Scope single bounce attenuated
with a 30 m  0.25 mm ID fused-silica capillary column coated total reflectance (ATR) accessory. Instrument parameters were:
with DB-1 (0.25 mm) (Agilent, Santa Clara, CA) in constant flow Resolution = 4 cm 1; gain = 1; optical velocity = 0.4747; aper-
mode at 36.5 cm/s of helium. The GC oven was temperature pro- ture = 150; and scans/sample = 32.
grammed as follows: Initial temperature, 100 C; initial hold, NMR spectra were obtained using an Agilent 600MR-DD2
0.0 min; temperature program rate, 6 C/min to 300 C; final hold, 600 MHz NMR with a 5 mm OneNMR pulse field gradient probe

Fig. 4. Infrared Spectrum (FTIR-ATR) of (a) carfentanil citrate and (b) carfentanil hydrochloride.
J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480 79

Fig. 5. Proton spectra of (a) carfentanil citrate in 1.0 mL of CDCl3 containing 0.03% v/v tetramethylsilane (TMS, 0 ppm reference) and 2 drops of CD3OD and (b) carfentanil HCl
in CDCl3 containing 0.03% v/v tetramethylsilane. Insets for analyte peak groups are beneath each full spectrum. The letter i above a peak group indicates an impurity.

(Palo Alto, CA). The sample temperature was maintained at 25 C. A deuterated chloroform (CDCl3) containing 0.03% v/v tetramethylsi-
standard Agilent proton pulse sequence was used (90 degree pulse, lane (TMS, 0 ppm reference) (Cambridge Isotopes).
45 s delay, 6 s acquisition time, 8 scans). Carfentanil citrate was Carbon and nitrogen isotope ratio analyses were determined
dissolved in 1 mL deuterated chloroform (CDCl3) containing using an elemental analyzer (EA) (Costech Analytical Technologies
0.03% v/v tetramethylsilane (TMS, 0 ppm reference) with two Inc., Valencia, CA) interfaced with a Delta Plus XP isotope ratio
drops of deuterated methanol (CD3OD) to enhance solubility mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Ger-
(Cambridge Isotopes, Tewksbury, MA). 1,4-BTMSB-d4 (Wako Pure many). Typically, 0.81.1 mg of carfentanil was accurately weighed
Chemical Industries, Saitama, Japan) was used as the quantitative into a tin capsule (Costech Analytical Technologies Inc.) and
internal standard. Carfentanil hydrochloride was dissolved in introduced into the EA using a Costech Zero-Blank autosampler
80 J.F. Casale et al. / Forensic Chemistry 3 (2017) 7480

(Costech Analytical Technologies Inc.). The EA reactor tubes were for reference purposes. Carfentanil citrate is easily differentiated
comprised of two quartz glass tubes filled with chromium (III) from the HCl via the two citric acid doublet resonances at ca.
oxide/silvered cobaltous oxide and reduced copper, held at 2.7 ppm.
1040 C and 640 C for combustion and reduction, respectively. A Each exhibit was quantitatively determined for carfentanil via
trap filled with magnesium perchlorate was used to remove water GC-FID. Cases 1-1A, 1-1B and 2 were determined to contain
from generated combustion gases, and a post-reactor GC column 0.62%, 1.87%, and 0.31% carfentanil as the HCl, respectively. The
was maintained at 65 C for separation of evolved N2 and CO2. methods limit of detection was approximately 2 lg/mL. We note
Helium (99.999% purity, ARC3 Gasses, Richmond, VA) was used that the same quantitative values were obtained for each exhibit,
as the carrier gas, and the system head pressure was adjusted to whether at a 1000 or 100 lL final dilution. The concentrations of
achieve a measured flow of 90 mL/min. Data was acquired and pro- acetylcarfentanil in 1-1A and 1-1B were estimated to be approxi-
cessed using ISODAT 3.0 software (Thermo Fisher Scientific Inc., mately 0.001% and 0.004%, respectively, from the total ion current
Bremen, Germany). via GC/MS.
EA normalization and quality control materials consisted of Carfentanil was isolated and purified from both exhibits from
internally calibrated atropine (TCI, St. Louis, Missouri) and cocaine Case #1. Each was subjected to EA-IRMS analysis for nitrogen
base (DEA Laboratory, Dulles, Virginia). Sample sequences were and carbon isotope determination. Both exhibits gave virtually
bracketed by an internally calibrated atropine secondary standard, identical measurements: d13C = 1.1 (0.1) and d15N = 27.6
typically at intervals of one standard every seven samples. The (0.1) . Excessive heroin in Case #2 prevented the isolation of
atropine and cocaine secondary standards were calibrated to pri- carfentanil. Although not definitive, both exhibits in Case #1
mary isotopic standard materials IAEA-N-1, IAEA-N-2, USGS-25, appear to contain the same carfentanil or are intimately related,
LSVEC, and NBS-19 and corresponding scale normalized isotope although the GC/MS profiles contain different adulterants.
values are expressed as d13CVPDB-LSVEC for carbon and d15NAIR for It is probably not a sheer coincidence that Exhibit 1A is exactly
nitrogen. Overall system reproducibility of reference materials one-third the carfentanil concentration of 1B, and that Case #2 is
and replicate analyses of cocaine was consistently 0.1 and exactly one-half the concentration of 1A. Although we were unable
0.2 or better for all EA-IRMS d13C and d15N measurements, to obtain sufficient data on Case #2, we contend that Cases #1 and
respectively. #2 may be related.

4. Results and discussion 5. Addendum

All three exhibits were examined by GC/MS to determine their Our laboratory has recently initiated a Fentanyl Profiling Pro-
analytical trace profiles (Fig. 2). Each contained carfentanil (peak gram which utilizes multiple signature methodologies patterned
#7) and at least one other fentanyl-related compound. In addition after the DEAs Signature Programs. The objective is to enhance
to carfentanil, Case #1-1A was determined to contain traces of current capabilities towards tactical and strategic intelligence for
diphenhydramine (peak #1), fentanyl (peak #5), and acetylcarfen- the criminal justice system from a forensic chemistry perspective.
tanil (peak #6) which produced a base peak at m/z 289 (Fig. 3b), The current project will be the subject of a future communication.
with no apparent molecular ion or M-2 ion observed due to the
low concentration of the compound. Acetylcarfentanil gave the Acknowledgements
same relative retention time to carfentanil, similar to that of
acetylfentanyl to fentanyl. The spectrum of acetylcarfentanil vs. The authors are indebted to Patrick Hays and David Morello of
carfentanil (Fig. 3a vs. b) was analogous to that of acetylfentanyl this laboratory for their assistance in acquiring NMR and IRMS
vs. fentanyl, producing the same ions and relative abundances: data.
m/z 42, m/z 77, m/z 91, m/z 105, m/z 130, m/z 154, and m/z 187.
A mass difference of 14 Daltons was observed between acetylcar- References
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261 vs. m/z 275, m/z 289 vs. m/z 303, and m/z 321 vs. m/z 335, also [1] D. Cooper, M. Jacob, A. Allen, Identification of fentanyl derivatives, J. Forensic
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tures identified by Feasel et al. [8]. Case #1-1B was determined to [4] I.S. Lurie, A.L. Berrier, J.F. Casale, R. Iio, J.S. Bozenko, Profiling of illicit fentanyl
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[5] R. Frank. Rates of drug overdose deaths continue to rise, more action needed to
and 2-furanylfentanyl (peak #8). Case #2 was determined to con- reverse troubling trends. U.S. Department of Health and Human Services. 2015
tain diphenhydramine, heroin (Peak #4), fentanyl, and heroin- www.hhs.gov/blog/2015/12/10/rates-of-drug-overdose-deaths-continue-to-
related impurities. Direct FTIR-ATR spectra of the CH2Cl2 solubles rise.html> (Last accessed on 10-26-2016).
[6] R.M. Gladden, P. Martinez, P. Seth. Fentanyl law enforcement submissions and
from both exhibits in Case #1 gave absorbances in the range of
increases in synthetic opioid-involved overdose deaths 27 states, 20132014.
23003000 cm 1, indicative of an amine hydrochloride ion-pair. Center for Disease Control and Prevention Morbidity and Mortality Weekly
Their spectra were ultimately compared to a standard of carfen- Report. 65 (2016) 837885.
[7] J.R. Riches, R.W. Read, R.M. Black, N.J. Cooper, C.M. Timperley, Analysis of
tanil hydrochloride (HCl) which was prepared from ethereal HCl
clothing and urine from Moscow theatre siege casualties reveals carfentanil and
treatment of an ethereal basic extract of exhibit 1A (confirmed remifentanil use, J. Anal. Tox. 36 (2012) 647656.
via NMR). The salt form of Case #2 could not be spectroscopically [8] M.G. Feasel, A.W. Wohlfarth, J.M. Nilles, S. Pang, R.L. Hristovich, M.A. Huestis,
determined; however, the carfentanil was CH2Cl2 soluble, indicat- Metabolism of carfentanil, an ultra-potent opioid, in human liver microsomes
and human hepatocytes by high-resolution mass spectrometry, AAPS J. (2016),
ing it was not the citrate. FTIR-ATR spectra for carfentanil citrate http://dx.doi.org/10.1208/s12248-016-9963-5, Published online 05 August
and carfentanil HCl are illustrated in Fig. 4. The proton spectra 2016.
for carfentanil citrate and carfentanil HCl are illustrated in Fig. 5