Identification of Fentanyl and Other Synthetic Opiates using Ambient Ionization High Resolution Time-of-Flight Mass Spectrometry Amanda Moore 1 , Jamie Foss 2,3 , Sabra Botch-Jones 1 , Frank Kero 3 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 C Introduction 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 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 G EN (0 0 .0 9 45 8 :0 0 .1 0 7 9 1 ) A m plitude m /z 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 A m plitude m /z 94.0404 121.0497 188.1446 375.2065 376.2096 0 1000 2000 3000 4000 5000 6000 100 200 300 400 500 A m plitude m /z 105.0712 188.1455 189.1494 351.2435 352.2475 353.2532 0 5000 10000 15000 20000 25000 100 200 300 400 500 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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 A m plitude m /z 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). 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 m /z--> Scan 2152 (16.111 m in): 006.D \data.m s 283.1 2 4 0.1 9 5.1 158.1 4 2.2 187.1 372.1 12 8.1 331.1 405.2 445.9 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 160000 170000 180000 190000 m /z--> Scan 2153 (16.117 m in): 010 C A YM AN 0475993-34.D \data.m s 283.1 240.1 95.1 158.1 42.1 130.1 187.1 372.2 341.0 405.2 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 A m plitude m /z 375.2069 376.2092 377.2113 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 55000 375 380 385 390 A m plitude m /z 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. I S Mannito l 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 (N 2 ) pressure of 80 psi, drying gas (N 2 ) flow of 3 L/min, and drying gas (N 2 ) 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. 349.262 9 [M+H] + 329.11 79
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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
MassGEN (00.09458:00.10791)
Ampl
itude
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
MassGEN (00.11150:00.12150)
Ampl
itude
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
MassGEN (00.19458:00.20791)
Ampl
itude
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
MassGEN (00.08116:00.09791)
Ampl
itude
m/z
Curve 1
94.0408 105.0438
121.0509 192.0862
210.0945
285.1418
341.1682
359.1782
360.1818
0100020003000400050006000700080009000
1000011000
100 200 300 400 500
MassGEN (00.19550:00.21216)
Ampl
itude
m/z
Curve 1
94.0416 121.0509
188.1445
189.1498
365.2596
366.2630
02000400060008000
10000120001400016000180002000022000
100 200 300 400 500
MassGEN (00.14016:00.16016)
Ampl
itude
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
MassRET (00.13750)
Ampl
itude
m/z
Curve 1
94.0412 121.0509
167.1550
169.1699
181.1015
322.0456
350.2672
02000400060008000
100001200014000160001800020000
100 200 300 400 500
MassGEN (00.14541:00.15541)
Ampl
itude
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
MassGEN (00.13783:00.14783)
Ampl
itude
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
MassRET (00.09308)
Ampl
itude
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
MassRET (00.10908)
Ampl
itude
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
MassRET (00.10908)
Ampl
itude
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).
40 60 80 1001201401601802002202402602803003203403603804004204400
50000
100000
150000
200000
250000
300000
350000
400000
450000
m/z-->
Abundance
Scan 2152 (16.111 min): 006.D\data.ms283.1
240.195.1
158.142.2
187.1372.1128.1 331.1 405.2 445.9
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 4200
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
m/z-->
Abundance
Scan 2153 (16.117 min): 010 CAYMAN 0475993-34.D\data.ms283.1
240.195.1
158.142.1
130.1 187.1
372.2341.0 405.2
85.0280 105.0683
188.1441
189.1471 322.0487 325.1897
375.2069
376.2092
05000
10000150002000025000300003500040000450005000055000
100 200 300 400 500
MassGEN (00.07450:00.08791)
Ampl
itude
m/z
Curve 1375.2069
376.2092
377.2113 0
500010000150002000025000300003500040000450005000055000
375 380 385 390
MassGEN (00.07450:00.08791)
Ampl
itude
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. 20152.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. 20155.PerkinElmer resources for AxION DSA-TOF
InstrumentationPerkinElmer AxION 2 TOF Time of Flight Mass SpectrometerPerkinElmer AxION Direct Sample Analysis module
Sample PreparationStandard 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 ParametersThe 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
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