This document summarizes a study that used Direct Sample Analysis Time-of-Flight Mass Spectrometry (DSA-TOF MS) to rapidly identify synthetic opiates including fentanyl, acetylfentanyl, and others. DSA is a direct ambient ionization technique that requires no chromatography and minimal sample preparation. It was able to identify 18 opiates in under 20 seconds with high mass accuracy. In-source collision-induced dissociation provided additional structural information. The method showed potential for screening drug samples and emerging synthetic drugs.

1. Identification of Fentanyl and Other Synthetic Opiates using Ambient Ionization
High Resolution Time-of-Flight Mass Spectrometry
Amanda Moore1
, Jamie Foss2,3
, Sabra Botch-Jones1
, Frank Kero3
1.Boston University School of Medicine, Boston, MA 2. Maine Health and Environmental Testing Laboratory, Augusta, ME 3. PerkinElmer, Shelton, CT
Fig 3. Glass capillary and mesh target screen sample trays for DSA (left). Schematic of DSA ionization
source (right).
Fig 2. (A) Close-up of mesh target screen sample tray and inlet to AxION TOF MS. (B) DSA
ionization source (C) DSA-TOF MS instrument.
A
B
CIntroduction
As of March 2016, the Drug Enforcement Administration placed
two fentanyl analogs (beta-hydroxythiofentanyl and butyryl
fentanyl) under Schedule I due to their imminent threat to public
health. These drugs elicit analgesic effects similar to heroin making
them desirable drugs to abuse. Novel fentanyl analogs and designer
opiates are expected to be more prominent in forensic casework in
the near future. These drugs can be seen in forensic casework either
alone or can be mixed with other drugs of abuse such as heroin. It is
therefore necessary to have an efficient methodology to identify
these compounds. Currently, Gas Chromatography-Mass
Spectrometry (GC-MS) is used to identify drugs of abuse and is
considered the “gold standard” in forensic casework. However,
analysis times can often range from 15-60 minutes in length.
Another drawback is need for spectral library matching, requiring
analytical reference materials for identification, meaning these new
designer drugs cannot be identified until a reference material is
available. In this study, Direct Sample Analysis Time-of-Flight
Mass Spectrometry (DSA-TOF) was utilized to provide rapid
identification of fentanyl and related synthetic opiates. DSA is a
direct ambient ionization source, requiring no chromatography and
minimal sample preparation. High resolution time of flight mass
spectrometry generates empirical formula information allowing for
substance identification without a reference material. Applying in-
source collision-induced dissociation (CID) produces additional
structural information for confirmation. The analytes explored in
this study include: heroin, 6-MAM, morphine, fentanyl,
norfentanyl, acetylfentanyl, butyrylfentanyl, beta-
hydroxythiofentanyl, furanylfentanyl, valerylfentanyl, AH-7921, U-
47700, buprenorphine, norbuprenorphine, desomorphine, MT-45,
W-15, and W-18.
Conclusion
•For the screening of synthetic opiates, DSA-TOF was successful at
reducing analysis time from minutes to seconds for qualitative
analyte identification.
• Analysis time was only 20 seconds.
•18 opiates and related compounds were evaluated, and in all
measurements, mass accuracy was below 5 ppm.
•In-source collision-induced dissociation (CID) can be used to
generate compound specific fragmentation for further
confirmation.
•Limit of detection (LOD) was determined to be 0.1 ppm. LOD was
determined by analyzing standards at 0.1 ppm, 0.05 ppm, and 0.01
ppm concentrations a minimum of 10 times. At 0.1 ppm, there was
only one mass accuracy failure, but no fragment identification
failure. At concentrations of 0.05 and 0.01 there was fragment
identification failure (signal below 500 counts) over 50% of the
time.
•Application of this method to a forensic case sample was successful,
demonstrating its utility in the forensic laboratory for these types of
compounds
•TOF allows for both targeted and non-targeted analysis
•As designer drugs emerge in communities, TOF methods allow the
flexibility to scale methods to include emerging public health
threats
•TOF allows the user to re-analyze sample data without the need for
reinjection
•Future Directions:
• Exploration of DSA-TOF MS as a rapid method to screen
urine samples for opiates
• DSA-TOF MS is currently being explored for use in the
identification of other street drugs.
Compound Formula Error (ppm)
Heroin C21H23NO5 0.387
6-MAM C19H21NO4 -4.44
Morphine C17H19NO3 -1.64
Buprenorphine C29H41NO4 -3.11
Norbuprenorphine C25H35NO4 -1.26
Fentanyl C22H28N2O -3.38
Norfentanyl C14H20N2O 4.71
Acetylfentanyl C21H26N2O -0.113
β-hydroxythiofentanyl C20H26N2O2S 1.73
Butyrylfentanyl C23H30N2O -1.3
Furanylfentanyl C24H26N2O2 0.529
Valerylfentanyl C24H32N2O -2.21
AH-7921 C16H22Cl2N2O -3.3
U-47700 C16H22Cl2N2O 0.819
Desomorphine C17H21NO2 -2.47
MT-45 C24H32N2 2.58
W-15 C19H21ClN2O2S -1.26
W-18 C19H20ClN3O4S -4.55
94.0631
105.0699
188.1445
189.1471
281.2007
337.2286
338.2318
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
100 200 300 400 500
Mass
GEN (00.09458:00.10791)
Amplitude
m/z
Curve 1
94.0405
105.0684
188.1437
189.1465
322.0484
323.2118
324.2150
0
5000
10000
15000
20000
25000
30000
35000
100 200 300 400 500
Mass
GEN (00.11150:00.12150)
Amplitude
m/z
Curve 1
94.0404
121.0497
188.1446
375.2065
376.2096
0
1000
2000
3000
4000
5000
6000
100 200 300 400 500
Mass
GEN (00.19458:00.20791)
Amplitude
m/z
Curve 1
105.0712
188.1455
189.1494
351.2435
352.2475
353.2532
0
5000
10000
15000
20000
25000
100 200 300 400 500
Mass
GEN (00.08116:00.09791)
Amplitude
m/z
Curve 1
94.0408
105.0438
121.0509
192.0862
210.0945
285.1418
341.1682
359.1782
360.1818
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
100 200 300 400 500
Mass
GEN (00.19550:00.21216)
Amplitude
m/z
Curve 1
94.0416
121.0509
188.1445
189.1498
365.2596
366.2630
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
100 200 300 400 500
Mass
GEN (00.14016:00.16016)
Amplitude
m/z
Curve 1
95.0856
172.9575
174.9546
189.9843 191.9820
284.0625
285.0667
286.0597
287.0611
329.1193
331.1169
0
5000
10000
15000
20000
25000
30000
35000
100 200 300 400 500
Mass
RET (00.13750)
Amplitude
m/z
Curve 1
94.0412
121.0509
167.1550
169.1699
181.1015
322.0456
350.2672
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
100 200 300 400 500
Mass
GEN (00.14541:00.15541)
Amplitude
m/z
Curve 1
94.0375
121.0509
174.9557
284.0606
285.0620
286.0578
287.0614
0
2000
4000
6000
8000
10000
12000
14000
16000
100 200 300 400 500
Mass
GEN (00.13783:00.14783)
Amplitude
m/z
Curve 1
273.0461
392.1215
394.1193
422.0955
423.0988
424.0933
0
1e4
2e4
3e4
4e4
5e4
6e4
7e4
8e4
9e4
10e4
100 200 300 400 500
Mass
RET (00.09308)
Amplitude
m/z
Curve 1
105.0710
121.0509 322.0459
377.1090
378.1108
379.1052
380.1089
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
100 200 300 400 500
Mass
RET (00.10908)
Amplitude
m/z
Curve 1
377.1090
378.1108
379.1052
380.1089
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
376 377 378 379 380 381 382
Mass
RET (00.10908)
Amplitude
m/z
Curve 1
Fig 4. Diagram of a TOF Mass Spectrometer showing the separation of ions of different mass (top).
Diagram of a TOF Mass Spectrometer showing how a Reflector improves mass resolution (bottom).
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
4 0 0 0 0 0
4 5 0 0 0 0
m / z - - >
A b u n d a n c e
S c a n 2 1 5 2 (1 6 .1 1 1 m in ) : 0 0 6 .D d a ta .m s
2 8 3 . 1
2 4 0 . 1
9 5 . 1
1 5 8 . 14 2 . 2
1 8 7 . 1
3 7 2 . 11 2 8 . 1 3 3 1 . 1 4 0 5 . 2 4 4 5 . 9
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
5 0 0 0 0
6 0 0 0 0
7 0 0 0 0
8 0 0 0 0
9 0 0 0 0
1 0 0 0 0 0
1 1 0 0 0 0
1 2 0 0 0 0
1 3 0 0 0 0
1 4 0 0 0 0
1 5 0 0 0 0
1 6 0 0 0 0
1 7 0 0 0 0
1 8 0 0 0 0
1 9 0 0 0 0
m / z - - >
A b u n d a n c e
S c a n 2 1 5 3 ( 1 6 .1 1 7 m i n ) : 0 1 0 C A Y M A N 0 4 7 5 9 9 3 - 3 4 .D d a ta .m s
2 8 3 . 1
2 4 0 . 1
9 5 . 1
1 5 8 . 14 2 . 1
1 3 0 . 1 1 8 7 . 1
3 7 2 . 23 4 1 . 0 4 0 5 . 2
85.0280
105.0683
188.1441
189.1471
322.0487
325.1897
375.2069
376.2092
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
100 200 300 400 500
Mass
GEN (00.07450:00.08791)
Amplitude
m/z
Curve 1
375.2069
376.2092
377.2113
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
375 380 385 390
Mass
GEN (00.07450:00.08791)
Amplitude
m/z
Curve 1
Fig 1. NFLIS regional trends in fentanyl reported per 100,000 persons aged 15 or older, January
2009 – June 2014
Fig 5. DSA-TOF spectra at 10ppm concentration: a) fentanyl b) acetylfentanyl c)furanylfentanyl d) butyrylfentanyl e) β-
hydroxythiofentanyl f) valerylfentanyl g) AH-7921 h) MT-45 i) U-47700 j) W-18
Table 1. Molecular formula and mass accuracy for 18 compounds evaluated in this study.
IS
Mannitol
a
b
c
d
e
f
g
h
i
j
Acknowledgements
•Maria Pease, Maine HETL Forensic Chemistry Section
•Maine Drug Enforcement Agency
•David Francis, PerkinElmer, for troubleshooting assistance
•Charlie Schmidt and Bill Hahn for continued support
References
1.NATIONAL FORENSIC LABORATORY INFORMATION SYSTEM. Special Report:
Opiates and Related Drugs Reported in NFLIS, 2009–2014. Office of Diversion Control,
DOJ, DEA. 2015
2.Ohta, H.; Suzuki, S.; Ogasawara, K. Journal of Analytical Toxicology 1999, 23, 280–285.
3.Vardanyan, R.; Hruby, V.; Future Med Chem. 2014 March, 6(4), 385-412.
4.Winter, G.; Wilhide, J.; LaCourse, W.; J. Am. Soc. Mass Spectrom. 2015
5.PerkinElmer resources for AxION DSA-TOF
Instrumentation
PerkinElmer AxION 2 TOF Time of Flight Mass Spectrometer
PerkinElmer AxION Direct Sample Analysis module
Sample Preparation
Standard Solutions: 5 uL of dilute analytical standard was applied
directly to the mesh target screen for DSA-TOF analysis. Analytical
standards were purchase from Cerilliant Corporation (Round
Rock, TX) and Cayman Chemical (Ann Arbor, MI) at a
concentration of 1.0 mg/mL in methanol and diluted with LC-MS
Grade water (Sigma Aldrich).
Solid Samples: A small amount of sample was taken on the tip of a
spatula, added to 3 mL of Methanol and vortexed. 5 uL was applied
directly to the mesh target screen for DSA-TOF analysis.
AxION DSA/TOF MS Instrument Parameters
The AxION DSA conditions were as follows: a corona current of
5 µA, heater temperature of 325°C, auxiliary gas (N2) pressure of
80 psi, drying gas (N2) flow of 3 L/min, and drying gas (N2)
temperature of 25°C. The AxION 2 TOF MS was run in positive
mode with a flight tube voltage of -10,000 V. The capillary exit
voltage was set to 175V for MS analysis. Mass spectra were
acquired with a mass range of 85-2000 m/z and an acquisition rate
of 5 spectra/sec. To maintain mass accuracy, two lock mass ions
were used (m/z 121.0509 and m/z 622.0290). All samples were
analyzed for only 20 seconds.
Fig 6. DSA-TOF spectra of W-15 analytical standard (10 ppm). Fig 7. Close up of DSA-TOF spectra of W-15 [M+H]+
and isotopes.
Fig 8. AxION EC ID software determination of molecular formula for W-15 based on mass
accuracy, isotope ratios, and isotope spacing. Demonstration using analytical reference
material.
Fig 14. AxION EC ID software determination of molecular formula for unknown based on
mass accuracy, isotope ratios, and isotope spacing. Molecular formula for furanylfentanyl was
determined with a mass accuracy of -0.598 ppm. Following TOF analysis, an analytical
standard was run on GC-MS for confirmation.
Fig 9. TIC from GC-MS analysis of unknown substance.
Fig 10. EI-MS spectra of unknown (top). EI-MS spectra of Furanylfentanyl analytical standard (bottom).
Fig 12. DSA-TOF spectra of unknown sample. Fig 13. Close up of DSA-TOF spectra of unknown sample [M+H]+
and isotopes.
Fig 11. Unknown powder seized by law enforcement in Maine.
349.2629
[M+H]+
329.1179