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Identification Of Fentanyl-Type Opioids Using GC-MS Fragmentation Data
- 1. Identification Of Fentanyl-TypeOpioids Using GC-MS Fragmentation Data Holly Grenke Pierzynski1 , M.S., Laura Neubauer1 , Chung Choi1 , Roxanne Franckowski1 , M.S., Nathan Layle1 , M.S., Steven D. Augustin2 , M.S., and Donna M. Iula1 , Ph.D. 1 Cayman Chemical; 2 Velesco Pharmaceutical Services Exceptions While most fentanyl-type unknowns may be discerned using these predictive patterns, there are always a few exceptions to the rules. For instance, when there is a substitution in the 4-position within the R3 region on the piperidine ring, such as carfentanil. Pattern 1 still applies because the BP is shown at 303, yet the functional group in the 4-position is either eliminated during fragmentation or is retained, causing rearrangement leading to other fragments. Further investigation is needed for compounds similar to carfentanil that have substitutions in the 4-position on the piperidine ring (Figure 5). In summary, by following the patterns outlined above (and taking into consideration notable exceptions) we hope that we have provided useful tips for interpreting the GC-MS of novel 4-anilidopiperidines. Figure 1 – General synthesis and comparison of fentanyl and the four different areas on the fentanyl scaffold that have been modified in new fentanyl varieties. Figure 2 – A comparison of the GC-MS of fentanyl with its corresponding cation fragments (left) and the GC-MS of benzyl fentanyl (right). Figure 4 – Cleavage at the amide C-N bond will generate the BP if a highly stabilized group has been substituted in the acyl region (R1 ). The primary cleavage of the highly resonance-stabilized cation gives the BP fragments shown at 95 and 149 for furanyl fentanyl (top left), benzodioxole fentanyl (top right), phenyl fentanyl (bottom left), and tetramethyl cyclopropyl fentanyl (bottom right). Figure 3 – The GC-MS for isobutyryl fentanyl is showing a 1:1 ratio of cations 146 to 189, while the GC-MS for cyclopentyl fentanyl is showing a ratio of less than one, where the cation 189 is in larger abundance than the 146. Figure 5 – GC-MS of carfentanil. References 1. Huang, B., Deutsche, K.H., Lalinde, N.L., et al. European Patent EP 0160422 B1 (1985). 2. Vardanyan, R.S. and Hruby, V.J. Future Med. Chem. 6(4), 385-412 (2014). 3. Vuckovic, S., Prostran, M., Ivanovic, M., et al. Curr. Med. Chem. 16(19), 2468-2474 (2009). 4. Wang, L. and Bernert, J.T. J. Anal. Toxicol. 30(5), 335-341 (2006). 5. Valaer, A.K., Huber, T., Andurkar, S.V., et al. J. Chromatogr. Sci. 35(10), 461-466 (1997). 6. Higashikawa, Y. and Suzuki, S. Forensic Toxicol. 26(1), 1-5 (2008). 7. Katselou, M., Papoutsis, I., Nikolaou, P., et al. Forensic Toxicol. 34(2), 201-212 (2016). 8. Ohta, H., Suzuki, S., and Ogasawara, K.J. Anal. Toxicol. 23(4), 280-285 (1999). 9. Breindahl, T., Kimergard, A., Andreasen, M.F., et al. Drug Test. Anal. 9(3), 415-422 (2017). 10. Goromaru, T., Katashima, M., Matsuura, H., et al. Chem. Pharm. Bull. 33(9), 3922-3928 (1985). Abstract Introduction Results Conclusion Predictive fragments and fragmentation patterns were determined after analyzing the mass spectral data of 60+ fentanyl-like substances via GC-MS. Differences in retention times between structurally related items and positional isomers were also noted. This data can assist in the identification of novel opioids of the fentanyl structural class when standards or library matches are unavailable. · Fentanyl is a powerful synthetic opioid developed by Janssen Pharmaceutica in 1959 that has roughly one hundred times the potency of morphine. · Structurally, fentanyl can be described as a 4-anilidopiperidine. Extensive structure-activity relationship (SAR) studies are well documented in scientific and patent literature.1-3 Some additional 4-anilidopiperidines are safely used in human and/or veterinary medicine (such as alfentanil, sufentanil, and remifentanil4,5 ) but most, however, have never advanced to clinical trials. · Until recently, only a handful of 4-anilidopiperidines were found on the illicit market for recreational drug use within the U.S. and overseas.6,7 · The rapid proliferation on the gray market of new molecular entities containing the fentanyl scaffold—designed to skirt existing regulations—has certainly made the identification of new psychoactive substances a challenge and a necessity. · The majority of the new fentanyl varieties have been modified at the areas defined in the figure as R1 , R2 , R3 , and R4 (Figure 1). · Thus, if the 146 and 189 peaks are present, your unknown will most likely be a fentanyl-like compound without substitutions on the piperidine or aniline groups. · Often with fentanyl compounds, the molecular weight (MW) ion is not generally detected in the spectra. · In such cases, the BP corresponds to the primary cleavage of the compound, which occurs between the α/β site on the phenethyl moiety or similar chain. · The presence of 91, the tropylium ion, in the spectra of fentanyl-like compounds strongly suggests the presence of a phenethyl group. · A noteworthy exception to this first pattern occurs when the phenethyl moiety is replaced with a N-benzyl or N-methyl moiety, as with benzyl fentanyl.8 · In the case of benzyl fentanyl, the MW ion (322) is noticeable along with the BP of 91 resulting from the cleavage between the piperidine ring and the benzyl group (Figure 2). · The BP cation undergoes additional cleavage in two areas. · The first is along the piperidine ring, Pattern 2A, eliminating the nitrogen and two carbons to form a cyclobutyl cation (mass 202 for fentanyl).8,10 · The second cleavage occurs along the C-N amide bond to produce a peak with an abundance of 30-60% of the BP (mass 189 for fentanyl), Pattern 2B.8 · The third characteristic fragment results from cleavage along the amide bond and additional cleavage within the piperidine ring. · The ratio between 146 and 189 is almost always greater than one unless there is a branched alkyl group or a closed alkyl ring system as seen with isobutyryl fentanyl and cyclopentyl fentanyl (Figure 3). · This is the case for furanyl fentanyl (95) and benzodioxole fentanyl (149). · Incidentally, cleavage also occurs at the α/β site for furanyl fentanyl, benzodioxole fentanyl, phenyl fentanyl, and tetramethyl cyclopropyl fentanyl, generating less abundant fragments 283, 337, 293, and 313, respectively (Figure 4). · Instrumentation: · Agilent 6890 Gas Chromatograph with a 5973 Mass Selective Detector (GC-MSD) · Restek Rtx-5MS (30 m x 0.32 mm x 0.5 μm) column, helium as carrier gas, flow at 2 ml per minute, and a split injection at a 15:1 ratio. · The temperature ramp: 50°C with a 1 minute hold, from 50°C to 300°C at 30°C per minute, holding at 300°C for 10 minutes. The inlet and transfer line temperatures are at 300°C. The MSD is tuned using Agilent’s standard tune (S-Tune). · As new forensic materials go through the quality control process, the GC-MS data are added to Cayman’s GC-MS Spectral library. · Four major predictive patterns have emerged that provide clues to help in identifying unknown cases. Item No. Product Name MW (free base) BP 22752 para-fluoro Tetrahydrofuran fentanyl HCl 396.5 305 19410 Carfentanil 394.5 303 22390 Cyclohexyl fentanyl HCl 390.5 299 20018 para-Chloroisobutyryl fentanyl HCl 384.9 293 18089 para-methoxy-Butyryl fentanyl HCl 380.5 289 20859 Tetrahydrofuran fentanyl HCl 378.5 287 22664 Tetrahydrofuran fentanyl 3-tetrahydro- furancarboxamide HCl 378.5 287 20439 Cyclopentyl fentanyl HCl 376.5 285 20034 para-Chlorofentanyl HCl 370.9 279 21740 meta-fluoro Methoxyacetyl fentanyl HCl 370.4 279 21741 para-fluoro Methoxyacetyl fentanyl HCl 370.4 279 18583 Ocfentanil HCl 370.4 279 19313 FIBF HCl 368.5 277 19566 meta-Fluorobutyryl fentanyl HCl 368.5 277 19567 ortho-Fluorobutyryl fentanyl HCl 368.5 277 17049 para-Fluorobutyryl fentanyl HCl 368.5 277 20206 meta-Fluoroisobutyryl fentanyl HCl 368.5 277 20207 ortho-Fluoroisobutyryl fentanyl HCl 368.5 277 22749 para-fluoro Cyclopropyl fentanyl HCl 366.4 275 20035 para-Methoxyfentanyl HCl 366.5 275 20818 α-methyl Butyryl fentanyl HCl 364.5 273 20817 (±)-cis-3-methyl Butyryl fentanyl HCl 364.5 273 18934 Valeryl fentanyl HCl 364.5 273 22389 Cyclobutyl fentanyl HCl 362.5 271 21952 3-Fluorofentanyl HCl 354.4 263 19424 meta-Fluorofentanyl HCl 354.4 263 19425 ortho-Fluorofentanyl HCl 354.4 263 15496 para-Fluorofentanyl HCl 354.4 263 21932 ortho-fluoro Acrylfentanyl HCl 352.4 261 21116 para-fluoro Acrylfentanyl 352.5 261 20782 Methoxyacetyl fentanyl HCl 352.4 261 20821 α-methyl Thiofentanyl HCl 356.5 259 14728 Butyryl fentanyl HCl 350.5 259 18280 α-methyl Fentanyl HCl 350.5 259 9002747 (±)-cis-3-methyl Fentanyl HCl 350.5 259 9002482 (±)-trans-3-methyl Fentanyl HCl 350.5 259 18584 Isobutyryl fentanyl HCl 350.5 259 22633 meta-Methylfentanyl HCl 350.5 259 22634 ortho-Methylfentanyl HCl 350.5 259 20038 para-Methylfentanyl HCl 350.5 259 21739 Cyclopropyl fentanyl HCl 348.4 257 17421 β-Hydroxythiofentanyl HCl 358.5 245 20789 β-hydroxy Fentanyl HCl 352.4 245 9002860 β-methyl Fentanyl HCl 350.5 245 21914 4'-methyl Fentanyl HCl 350.5 245 20786 Thiofentanyl HCl 342.5 245 20816 α-methyl Acetyl fentanyl HCl 336.4 245 14719 Fentanyl HCl 336.4 245 20819 Furanylethyl fentanyl HCl 326.4 245 19312 Acrylfentanyl HCl 334.4 243 9002271 4'-methyl Acetyl fentanyl HCl 336.4 231 ISO00128 Acetyl fentanyl HCl 322.4 231 20858 Benzodioxole fentanyl 428.5 149 22744 2,2,3,3-tetramethyl-Cyclopropyl fentanyl HCl 404.6 125 22551 Phenyl fentanyl HCl 384.5 105 20785 Thienyl fentanyl HCl 328.4 97 22779 ortho-methyl Furanyl fentanyl HCl 388.5 95 18705 Furanyl fentanyl HCl 374.4 95 21213 Furanyl fentanyl 3-furancarboxamide isomer HCl 374.4 95 20350 Benzyl Carfentanil HCl 380.4 91 19883 Benzyl fentanyl HCl 322.4 91 22660 Benzyl Acrylfentanyl HCl 320.4 91 HN N Cl O NH O O H2 N NBr N NO p m o 2 3 4 2' 3' 4' N NO R1 R2 R3 R4 Fentanyl Scaffold RegionsFentanyl General Synthesis (4-Piperidinone) (Phenethyl bromide) Cs2 CO3 acetonitrile Cs2 CO3 acetonitrile (1-(2-Phenylethyl)-4-piperidinone) Na(OAc)3 BH HOAc toluene (Aniline) (4-Anilino-N-phenethylpiperidine) "4-ANPP" (Propionyl chloride) α β Pattern 1: Fentanyl-like compounds cleave between the α and β carbons of the ethyl heterocyclic linker, which results in the base peak (BP) value. Pattern 2: Additional cleavage of the BP cation occurs along the piperidine ring and at the amide C-N bond. Pattern 3: Subsequent cleavage at either the piperidine ring or the amide C-N bond of the secondary fragments results in a third characteristic fragment. Table 1: Comprehensive list of fentanyl-like compounds with corresponding molecular weight (MW) and Base Peaks (BP). Pattern 4: Cleavage at the amide C-N bond will generate the BP if a highly stabilized or highly substituted group is in the acyl region (R1 ). N O N α site β site N O N Base Peak Cation 245Fentanyl, MW 336 Pattern 1 + HN HN N N O N N O N O N MW 336 O 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 m/z--> Abundance 245.2 146.1 189.1 91.1 105.157.0 77.0 202.142.1 132.1 118.1 160.1 334.4216.1173.1 281.1229.2 267.1 307.3 347.1320.4294.4 N O N MW 322 HN N N NH HN N 82 O 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1000000 1100000 1200000 m/z--> Abundance 91.0 173.1 146.1 57.0 132.0 118.1 77.0 265.1 104.042.1 322.2249.1190.1 231.1 159.1 293.1217.1203.1 281.0 306.1 334.3 N O N Base Peak Cation 245 Pattern 2B Pattern 2A N O 202 HN N 189 A B + + + N O 202 HN N 189 HN 146 Pattern 3A Pattern 3B + + + N O N N O N 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 2200000 2400000 m/z--> Abundance 259 189 146 43 10591 77 132 118 65 216 16055 207 281173 348230 243 307 335198 269 327 357295 316 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 m/z--> Abundance 285 69 189 146 105 41 91 132 79 55 118 160 207 253242173 355 374327231 267 343221 315295 385276 305 MW 428 Base Peak 149 N O N O O Pa‚ern 4 O O O + N O N O N O O O N O N O MW 374 N O N O O MW 428 N O N O O O O O O 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 0 50000 100000 150000 200000 250000 300000 350000 m/z--> Abundance 95.0 283.1 240.0 158.077.042.0 130.0 187.0 206.956.0 144.0115.0 171.9 331.0253.8 372.1268.9 346.0222.8 315.1297.9 386 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 220000 240000 260000 280000 m/z--> Abundance 149 337 12165 91 105 7742 132 187160 241207 22453 294279253177 265 327 355 426315 405377 389 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 0 50000 100000 150000 200000 250000 300000 350000 400000 m/z--> Abundance 105 293 77 91 250 42 158 13051 65 180118 197146 207 281 382327267 341170 355222 311239 367 393 N O + + N O N + N O N MW 384 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 0 50000 100000 150000 200000 250000 300000 350000 400000 m/z--> Abundance 125 313 189 105 14655 91 77 41 67 158 207115 135 279253172 217 402231 341267 327 355 389297241 415377366 N O N + + + + + + N O N MW 404 + O +O 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 m/z--> Abundance 303 105 18757 91 15442 77 243 130 275 118 144 33567 202 230219170 392363253 289 325315265 347 403377 N O N O + ON O N O O MW 394