Drug Testing and Analysis
Research article Received: 11 December 2013
Revised: 27 March 2014
Accepted: 12 April 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1667
Qualitative analysis of seized synthetic cannabinoids and synthetic cathinones by gas chromatography triple quadrupole tandem mass spectrometry Seongshin Gwak, Luis E. Arroyo-Mora and José R. Almirall* Designer drugs are analogues or derivatives of illicit drugs with a modification of their chemical structure in order to circumvent current legislation for controlled substances. Designer drugs of abuse have increased dramatically in popularity all over the world for the past couple of years. Currently, the qualitative seized-drug analysis is mainly performed by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) in which most of these emerging designer drug derivatives are extensively fragmented not presenting a molecular ion in their mass spectra. The absence of molecular ion and/or similar fragmentation pattern among these derivatives may cause the equivocal identification of unknown seized-substances. In this study, the qualitative identification of 34 designer drugs, mainly synthetic cannabinoids and synthetic cathinones, were performed by gas chromatography-triple quadrupole-tandem mass spectrometry with two different ionization techniques, including electron ionization (EI) and chemical ionization (CI) only focusing on qualitative seized-drug analysis, not from the toxicological point of view. The implementation of CI source facilitates the determination of molecular mass and the identification of seized designer drugs. Developed multiple reaction monitoring (MRM) mode may increase sensitivity and selectivity in the analysis of seized designer drugs. In addition, CI mass spectra and MRM mass spectra of these designer drug derivatives can be used as a potential supplemental database along with EI mass spectral database. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: designer drugs; synthetic cathinones; synthetic cannabinoids; GC-MS/MS
Introduction
Drug Test. Analysis (2014)
* Correspondence to: José R. Almirall, Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, Miami, FL 33199, USA. E-mail: almirall@fiu.edu Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, Miami, Florida, 33199, USA
Copyright © 2014 John Wiley & Sons, Ltd.
1
Designer drugs are structurally and chemically modified substances manufactured illicitly to circumvent the current legislation for controlled substances.[1] Some early examples of drugs that could today be considered as designer drugs are 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxy-amphetamine (MDA), first synthesized in the 1910s; they have been widely abused since the beginning of 1970s. Recently, the abuse of designer drugs has proliferated with the ease of purchase over the Internet and in head shops at relatively low cost, especially for the synthetic cannabinoid and synthetic cathinone classes of designer drugs.[2–4] Synthetic cannabinoids are the main components used to produce herbal marijuana alternatives (HMAs), also called synthetic marijuana.[5] One or more synthetic cannabinoids are dissolved in solution and sprayed onto herbs or plants for a natural appearance resembling an herbal product.[6] ‘K2’ and ‘Spice’ are the most common street names for HMAs, which are known to be more potent than delta-9-tetrahydrocannabinol (Δ9-THC), an active component in marijuana. Although there is no structural similarity with Δ9-THC, these synthetic cannabinoids act as cannabinoid receptor agonists.[7] Synthetic cathinones are derivatives of cathinone, an active component found in the Khat leaves.[8] The individual synthetic cathinones or the mixture of different synthetic cathinones are called ‘bath salts’ and generally sold in the form of a white powder, but may be found in other forms as well.[9] As a betaketone amphetamine analogue, the effects of synthetic cathinones
are similar to illicit stimulants such as amphetamine, cocaine, and MDMA (ecstasy).[9] In the United States, the number of emergency response calls related to synthetic cannabinoid exposures has dramatically increased from 2906 (2010) to 5205 (2012) and from 304 (2010) to 2656 (2012) related to synthetic cathinone exposures, according to the American Association of Poison Control Centers (AAPCC).[10,11] Despite the fact that some of these classes of designer drugs are currently regulated by the Federal Controlled Substances Act, new compounds with similar structures and effects continue to appear on the streets, replacing currently controlled substances as of May 2013.[12] This recent prevalence of seized designer drugs, especially of synthetic cannabinoids and cathinones, has led to research interest at various forensic laboratories across the world focusing on the identification and characterization of these drugs, including the clinical case series of synthetic cathinones.[13–21] As the gold standard technique that is most widely available in the forensic laboratory, gas chromatographymass spectrometry (GC-MS) has been widely implemented for the analysis of designer drugs. The identification of synthetic
Drug Testing and Analysis
S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
cathinones was reported by several research groups using GC-MS with the electron ionization (EI) source and with gas chromatography-ion trap-mass spectrometry (GC-IT-MS) with the EI and chemical ionization (CI) sources.[13–16] Extensive fragmentation with the EI source resulted in the absence of molecular ions (M•+), whereas the protonated molecular ions ([M + H]+) were determined with the use of CI source for those synthetic cathinone derivatives, such as 4-fluoromethcathinone, 4-methyl-Nethylcathinone, mephedrone, MDPV, butylone, and naphyrone. A number of synthetic cannabinoids have also been studied in various research groups using GC-MS with an EI source.[17–21] Gas chromatography-tandem mass spectrometry (GC-MS/MS) has been applied to the identification and quantitation of traditional drugs of abuse, especially for the presence of GHB, opioids, cocaine, and amphetamine derivatives in human hair where a method of high sensitivity is required.[22–24] More recently, GC-MS/MS has been used for the mass spectrometric differentiation of designer drug regioisomers.[25–28] In this study, the qualitative identification of 34 designer drugs was performed by gas chromatography-triple quadrupole-tandem mass spectrometry (GC-MS/MS) with two different ionization
techniques, including EI and CI, only focusing on qualitative seized-drug analysis. The advantage of using the soft CI source is the formation of molecular ions in order to assist with the easier identification for emerging designer drug derivatives. We report a sensitive and selective method using GC-MS/MS that can be used for the positive identification of these increasingly important classes of designer drugs at the level of qualitative seized-drug analysis and also report the corresponding mass spectra expected for these designer drugs.
Material and methods Chemicals Methanol (Optima®, LC/MS grade) and proadifen were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA), respectively. Proadifen was prepared in methanol at 10 μg/mL and used to lock the retention time in GC system due to the structural similarity to the target analytes. Reference materials of 34 designer drugs were provided by Cayman Chemical (Ann Arbor, MI, USA), including AKB48 (APINACA), AM694, AM1220,
Table 1. Summary of major peaks in EI and CI full scan MS for the analytes of interest with the molecular ions, shown in bold. Molecular ions are absent for the highlighted analytes in EI full scan MS No.
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Analyte 3-FMC 4-MMC 4-MEC 3,4-DMMC Methedrone Methylone MDPV CP-47,497 XLR-11 CP-47,497 C8 JWH-251 JWH-203 JWH-250 JWH-302 RCS-4 JWH-249 JWH-201 AM694 JWH-073 APINACA JWH-022 JWH-018 JWH-019 AM2201 CPE JWH-122 RCS-8 JWH-398 AM2233 CB-13 APICA JWH-081 AM1220 JWH-200
Retention Time (min)
Molecular Weight (amu)
Major Peaks in EI Full Scan MS in order of Relative Abundance (%)
Major Peaks in CI Full Scan MS in order of Relative Abundance (%)
9.715 11.584 12.307 13.143 13.634 14.639 19.101 22.897 22.970 23.648 24.777 25.376 25.461 25.732 25.862 26.070 26.093 26.562 26.590 26.923 27.144 27.155 27.759 27.855 28.002 28.030 28.256 28.369 28.408 28.425 28.442 29.115 30.035 30.431
181.09 177.12 191.13 191.13 193.11 207.09 275.15 318.26 329.22 332.27 319.19 339.14 335.19 335.19 321.17 383.10 335.19 435.05 327.16 365.25 339.16 341.18 355.19 359.17 376.22 355.19 375.22 375.14 458.09 368.18 364.25 371.19 382.20 384.18
58 (100), 95 (12.28), 123 (6.10) 58 (100), 91 (8.68), 119 (5.79) 72 (100), 91 (8.27), 119 (5.48) 58 (100), 133 (5.47) 58 (100), 135 (8.26), 77 (6.24) 58 (100), 149 (6.17) 126 (100), 149 (5.52) 215 (100), 233 (72.85), 318 (5.90) 232 (100), 144 (24.86), 329 (7.17) 215 (100), 233 (75.07), 332 (6.25) 214 (100), 144 (23.47), 116 (6.47) 214 (100), 144 (24.60), 116(6.93) 214 (100), 144 (24.31), 116 (6.01) 214 (100), 144 (23.92), 116 (5.88) 135 (100), 321 (87.28), 264 (78.89) 214 (100), 144 (21.44), 116 (5.36) 214 (100), 144 (22.53) 232 (100), 435 (51.84), 220 (49.53) 200 (100), 327 (92.61), 284 (66.56) 215 (100), 294 (29.79), 365 (16.27) 155 (100), 127 (72.01), 339 (58.51) 214 (100), 284 (82.99), 341 (75.16) 355 (100), 284 (96.12), 228(94.36) 359 (100), 232 (98.72), 284 (93.74) 98 (100), 70 (5.87) 355 (100), 214 (83.82), 298 (74.97) 254 (100), 144 (21.02), 55 (9.19) 214 (100), 375 (67.11), 318 (63.38) 98 (100), 70 (6.11) 171 (100), 368 (47.68), 297 (44.77) 214 (100), 307 (28.30), 364 (21.64) 371 (100), 214 (71.12), 314 (64.96) 98 (100), 70 (6.32) 100 (100), 127 (5.89), 56 (5.53)
182 (100), 164 (36.02), 58 (7.69) 160 (100), 178 (72.11), 58 (17.83) 192 (100), 174 (69.25), 72 (13.96) 174 (100), 192 (82.34), 163 (23.95) 176 (100), 194 (60.63), 165 (29.20) 190 (100), 208 (99.96), 160 (41.26) 276 (100), 126 (54.93), 207 (42.39) 301 (100), 233 (39.33), 318 (5.76) 330 (100), 310 (67.56), 125 (18.59) 315 (100), 247 (25.72), 332 (5.45) 320 (100), 214 (22.84), 304 (22.07) 340 (100), 214 (24.05), 127 (13.09) 336 (100), 214 (30.53), 320 (18.77) 336 (100), 214 (19.18), 320 (5.01) 322 (100), 188 (34.07), 135 (21.50) 384 (100), 288 (66.76), 214 (46.49) 336 (100), 188 (36.23), 214 (27.01) 436 (100), 310 (23.68), 231 (12.30) 328 (100), 200 (9.92) 135 (100), 366 (23.65), 214 (14.09) 340 (100), 212 (10.16) 342 (100), 214 (14.42), 155 (9.62) 356 (100), 202 (20.88), 157 (8.84) 360 (100), 340 (11.75), 232 (10.71) 377 (100), 98 (65.24), 359 (7.33) 356 (100), 214 (14.17), 169 (8.45) 376 (100), 360 (33.56), 254 (29.15) 376 (100), 214 (16.50), 189 (8.41) 459 (100), 98 (64.98), 331 (8.20) 369 (100), 241 (10.73), 155 (8.29) 365 (100), 135 (43.38), 213 (22.34) 372 (100), 188 (34.54), 355 (17.62) 383 (100), 98 (96.57), 286 (14.48) 385 (100), 100 (14.98), 281 (10.31)
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Analysis of Cannabinoids and Cathinones AM2201, AM2233, Cannabipiperidiethanone (CPE), CB-13, CP-47,497, CP-47,497 C8 homolog, 3,4-dimethylmethcathinone (3,4-DMMC), 3-fluoromethcathinone (3-FMC), 4-methoxymethcathinone (methedrone), 3,4-methylenedioxy-N-methcathinone (methylone), 3,4-methylenedi oxypyrovalerone (MDPV), 4-methylethcathinone (4-MEC), 4-methyl methcathinone (4-MMC), JWH-018, JWH-018 adamantyl carboxamide (APICA), JWH-019, JWH-022, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-249, JWH-250, JWH-251, JWH-302, JWH-398, RCS-4, RCS-8, and XLR-11. The standard stock solutions of each designer drug in powder form were prepared in methanol at different concentrations depending on the amount of standards: 1000, 2000,
and 2500 μg/mL. These stock solutions were diluted to 10 μg/mL in methanol in order to prepare the 4 mixtures of working standard solutions. GC-MS/MS The qualitative analysis of 34 designer drugs was performed using Agilent 7890A GC system (Santa Clara, CA) with a DB-5MS column (30 m × 250 μm × 0.25 μm, J&W, Agilent) coupled with Agilent 7000 GC-MS Triple Quad (Santa Clara, CA, USA). Retention time was locked for proadifen at 20.765 min in order to provide
Figure 1. EI full scan mass spectra for (a) JWH-251, (b) JWH-250, (c) JWH-249, (d) CPE, (e) AM2233, and (f) AM1220.
3
Figure 2. CI full scan mass spectra for (a) JWH-251, (b) JWH-250, (c) JWH-249, (d) CPE, (e) AM2233, and (f) AM1220 with methane as a reagent gas.
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
4
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
30 (100) 42 (74) 56 (41) 30 (100) 42 (74) 56 (40) 30 (100) 42 (73) 56 (39) 69 (100) 97 (98) 84 (96) 81 (100) 147 (33) 215 (16) 144 (100) 247 (2) 314 (1.6) 81 (100) 147 (33) 215 (14) 144 (100) 116 (45) 214 (7) 144 (100) 116 (45) 214 (4)
30 (100) 42 (74) 56 (37) 30 (100) 42 (74) 56 (40) 44 (100) 29 (15)
Product Ion (Relative Abundance, %)
8 20 12 8 20 12 8 20 12 20 12 12 16 12 12 12 8 12 16 12 12 12 28 4 12 28 4
8 20 12 8 20 12 8 16
CE (V) 214 214 335 214 214 335 321 321 321 214 214 383 214 214 232 232 435 200 200 327 215 217 365 155 339 339 214 341 341 228 355 355
AM694 AM694 AM694 JWH-073 JWH-073 JWH-073 APINACA APINACA APINACA JWH-022 JWH-022 JWH-022 JWH-018 JWH-018 JWH-018 JWH-019 JWH-019 JWH-019
Precursor Ion
JWH-250 JWH-250 JWH-250 JWH-302 JWH-302 JWH-302 RCS-4 RCS-4 RCS-4 JWH-249 JWH-249 JWH-249 JWH-201 JWH-201
Analyte
144 (100) 116 (54) 232 (44) 144 (100) 116 (48) 310 (40) 145 (100) 90 (42) 215 (5) 127 (100) 155 (9) 184 (8.5) 144 (100) 284 (48) 167 (27) 144 (100) 284 (51) 338 (43)
144 (100) 116 (44) 214 (5) 144 (100) 116 (46) 214 (5) 264 (100) 135 (74) 186 (62) 144 (100) 116 (45) 214 (2) 144 (100) 116 (45)
Product Ion (Relative Abundance, %)
12 32 4 8 24 8 12 36 24 12 20 4 8 8 20 8 12 8
12 28 4 12 28 4 12 32 12 12 28 4 12 28
CE (eV) AM2201 AM2201 AM2201 CPE CPE CPE JWH-122 JWH-122 JWH-122 RCS-8 RCS-8 RCS-8 JWH-398 JWH-398 JWH-398 AM2233 AM2233 AM2233 CB-13 CB-13 CB-13 APICA APICA APICA JWH-081 JWH-081 JWH-081 AM1220 AM1220 AM1220 JWH-200 JWH-200 JWH-200
Analyte 232 359 359 98 98 98 298 355 355 254 254 254 214 375 375 98 98 98 171 171 368 214 214 364 214 371 371 98 98 98 100 100 100
Precursor Ion
* Average relative abundance was calculated based on the 3 replicates and the relative standard deviation (%RSD) for each transition were less than 2.4%.
58 58 58 58 58 58 58 58 58 126 126 126 215 215 318 232 329 329 215 215 332 214 214 319 214 214 339
58 58 58 58 58 58 72 72
3-FMC 3-FMC 3-FMC 4-MMC 4-MMC 4-MMC 4-MEC 4-MEC
3,4-DMMC 3,4-DMMC 3,4-DMMC Methedrone Methedrone Methedrone Methylone Methylone Methylone MDPV MDPV MDPV CP-47,497 CP-47,497 CP-47,497 XLR-11 XLR-11 XLR-11 CP-47,497 C8 CP-47,497 C8 CP-47,497 C8 JWH-251 JWH-251 JWH-251 JWH-203 JWH-203 JWH-203
Precursor Ion
Analyte 144 (100) 284 (42) 232 (13) 70 (100) 42 (81) 68 (12) 181 (100) 298 (62) 181 (53) 69 (100) 144 (90) 158 (32) 144 (100) 318 (33) 358 (21) 70 (100) 42 (82) 68 (12) 115 (100) 143 (53) 171 (17) 144 (100) 116 (43) 307 (11) 144 (100) 354 (43) 214 (10) 70 (100) 42 (82) 68 (12) 56 (100) 70 (52) 44 (17)
Product Ion (Relative Abundance, %)
12 12 20 12 24 24 8 8 20 20 12 12 8 8 12 12 28 24 24 8 24 12 28 8 8 12 28 12 28 24 12 12 12
CE (eV)
Table 2. Optimized EI MRM transitions for the analytes of interest with the average relative abundance* (%) and selected collision energy (CE, eV). The most abundant ions in bold are used for quantification in generating the calibration curves
Drug Testing and Analysis S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
5
Drug Test. Analysis (2014)
182 182 182 178 178 178 192 192 192 192 192 192 194 194 194 208 208 208 276 276 276 301 301 301 330 330 330 315 315 315 320 320 320 340 340 340
Precursor Ion
164 (100) 149 (61) 103 (20) 160 (100) 145 (58) 119 (11) 174 (100) 145 (36) 131 (15) 174 (100) 159 (73) 144 (18) 176 (100) 161 (36) 135 (9) 160 (100) 190 (67) 132 (48) 126 (100) 205 (58) 175 (68) 233 (100) 216 (7) 121 (33) 125 (100) 232 (46) 97 (23) 247 (100) 149 (16) 121 (57) 105 (100) 119 (9) 214 (63) 125 (100) 214 (15) 188 (15)
Product Ion (Relative Abundance, %) 8 20 32 8 20 24 8 20 28 12 20 36 8 20 20 16 8 32 24 16 20 8 12 32 20 20 28 8 24 24 24 24 24 32 24 16
CE (V) 336 336 336 336 336 336 322 322 322 384 384 384 336 336 336 436 436 328 328 366 366 340 340 342 342 342 356 356 356
JWH-073 JWH-073 APINACA APINACA JWH-022 JWH-022 JWH-018 JWH-018 JWH-018 JWH-019 JWH-019 JWH-019
Precursor Ion
JWH-250 JWH-250 JWH-250 JWH-302 JWH-302 JWH-302 RCS-4 RCS-4 RCS-4 JWH-249 JWH-249 JWH-249 JWH-201 JWH-201 JWH-201 AM694 AM694
Analyte
155 (100) 214 (25) 127 (93) 155 (100) 228 (21) 127 (92)
155 (100) 212 (24)
135 (100) 215 (11)
155 (100) 200 (28)
121 (100) 200 (8) 188 (6) 121 (100) 214 (62) 144 (29) 135 (100) 214 (7) 107 (20) 169 (100) 214 (31) 188 (19) 121 (100) 149 (24) 135 (31) 231 (100) 309 (6)
Product Ion (Relative Abundance, %)
24 20 56 24 24 56
24 20
16 20
24 20
20 20 12 20 24 44 24 24 44 32 24 16 28 20 28 28 16
CE (eV)
JWH-081 JWH-081 JWH-081 AM1220 AM1220 AM1220 JWH-200 JWH-200 JWH-200
APICA APICA
AM2201 AM2201 AM2201 CPE CPE CPE JWH-122 JWH-122 JWH-122 RCS-8 RCS-8 RCS-8 JWH-398 JWH-398 JWH-398 AM2233 AM2233 AM2233 CB-13 CB-13
Analyte
372 372 372 383 383 383 385 385 385
365 365
360 360 360 377 377 377 356 356 356 376 376 376 376 376 376 459 459 459 369 369
Precursor Ion
* Average relative abundance was calculated based on the 3 replicates and the relative standard deviation (%RSD) for each transition were less than 1.3%.
3-FMC 3-FMC 3-FMC 4-MMC 4-MMC 4-MMC 4-MEC 4-MEC 4-MEC 3,4-DMMC 3,4-DMMC 3,4-DMMC Methedrone Methedrone Methedrone Methylone Methylone Methylone MDPV MDPV MDPV CP-47,497 CP-47,497 CP-47,497 XLR-11 XLR-11 XLR-11 CP-47,497 C8 CP-47,497 C8 CP-47,497 C8 JWH-251 JWH-251 JWH-251 JWH-203 JWH-203 JWH-203
Analyte
185 (100) 214 (34) 157 (24) 98 (100) 155 (25) 112 (82) 155 (100) 114 (76) 127 (50)
214 (100) 188 (17)
155 (100) 232 (25) 127 (95) 112 (100) 229 (17) 121 (50) 169 (100) 214 (33) 141 (62) 121 (100) 228 (5) 144 (9) 189 (100) 214 (12) 161 (50) 98 (100) 362 (7) 231 (17) 299 (100) 241 (36)
Product Ion (Relative Abundance, %)
24 20 44 32 24 20 20 28 60
20 20
24 24 56 20 12 24 24 20 48 24 12 40 24 24 52 36 16 32 12 12
CE (eV)
Table 3. Optimized CI MRM transitions for the analytes of interest with the average relative abundance* (%) and selected collision energy (CE, eV). The most abundant ions in bold are used for quantification in generating the calibration curves
Analysis of Cannabinoids and Cathinones
Drug Testing and Analysis
Drug Testing and Analysis
S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
Figure 3. Overlaid MRM total ion chromatograms of the 4 standard mixtures including all 34 analytes for (a) EI and (b) CI with potential co-elutions observed for some of the compounds. Numbers for each peak correspond to the numbers in Table 1 and a Peak #12 is beyond the scale shown in both EI and CI chromatograms.
6 Figure 4. EI and CI MRM mass spectra for JWH-250 and AM1220 as representative compounds at different collision energies for each transition.
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Analysis of Cannabinoids and Cathinones consistent retention times for the same analytes over time. GC parameters for full scan mode were as follows: injection volume of 5 μL, split ratio of 1:10, injection temperature of 280 °C, transfer line temperature of 300 °C, helium as a carrier gas at different flow rate depending on the pressure for RTL. Oven temperature was programmed with initial temperature of 60 °C held for 1 min and 10 °C/min to 325 °C held for 8 min. The operation parameters for MS in the full scan mode were as follows: gain factor of 1, source temperature at 300 °C, quadrupole temperature at 150 °C, solvent delay of 3.75 min, ionization energy of 70 eV, and acquisition mass range of m/z 45–570. Methane was used as the reagent gas for the CI mode. In the product ion scan and multiple reaction monitoring (MRM) mode, the flow rate of nitrogen as the collision gas and helium as the quench gas was 1.0 mL/min. The acquisition mass range was varied in product ion scan mode during the MRM optimization starting from m/z 35 up to the mass of the selected precursor ion. Other MS operating conditions remained the same. The optimization of the MRM mode was performed by selecting an appropriate precursor ion for each analyte of interest and selecting 2 to 3 transitions to product ions from the precursor ion. The ideal collision energy and dwell time were optimized for each transition during this process using the Design Experiments Assistant, Analyze Experiments Assistant, and Dynamic MRM Assistant software provided by Agilent Technologies. The MRM method was then optimized and validated by determining the limits of detection (LODs) for each analytes prepared in methanol at different concentration from 50 to 1200 ng/mL. The gain factor was increased to 50 from 1 and the flow rate of helium quench gas was set to 2.0 mL/min with pulsed splitless injection mode for the LODs study with both EI and CI sources.
Results and discussion Prior to the analysis of the 34 designer drugs, the gas chromatographic system was locked for proadifen in order to obtain consistent results over time with the use of retention-time locking (RTL) feature. The pressure of the capillary column was optimized based on the RTL calibration and the flow rate of carrier gas (He) was adjusted in accordance with the optimized pressure for the target compound (proadifen). Detailed information on RTL is explained elsewhere.[29] Table 1 summarizes the EI full scan mass spectra for 34 designer drugs of interest presenting a base peak and two of the most abundant peaks along with their relative abundance as a preliminary study. The molecular ions were present for most of the synthetic cannabinoids (n = 16), whereas extensive fragmentation was observed for all of the synthetic cathinones and some of the synthetic cannabinoids (n = 18). Figure 1 shows the actual EI full scan mass spectra for some of the phenylacetylindoles, including JWH-251, JWH-250, and JWH249. The common fragment ions at m/z 214, 144, and 116 were observed with significant relative abundance, but extremely low relative intensity for a molecular ion (
Research article Received: 11 December 2013
Revised: 27 March 2014
Accepted: 12 April 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1667
Qualitative analysis of seized synthetic cannabinoids and synthetic cathinones by gas chromatography triple quadrupole tandem mass spectrometry Seongshin Gwak, Luis E. Arroyo-Mora and José R. Almirall* Designer drugs are analogues or derivatives of illicit drugs with a modification of their chemical structure in order to circumvent current legislation for controlled substances. Designer drugs of abuse have increased dramatically in popularity all over the world for the past couple of years. Currently, the qualitative seized-drug analysis is mainly performed by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) in which most of these emerging designer drug derivatives are extensively fragmented not presenting a molecular ion in their mass spectra. The absence of molecular ion and/or similar fragmentation pattern among these derivatives may cause the equivocal identification of unknown seized-substances. In this study, the qualitative identification of 34 designer drugs, mainly synthetic cannabinoids and synthetic cathinones, were performed by gas chromatography-triple quadrupole-tandem mass spectrometry with two different ionization techniques, including electron ionization (EI) and chemical ionization (CI) only focusing on qualitative seized-drug analysis, not from the toxicological point of view. The implementation of CI source facilitates the determination of molecular mass and the identification of seized designer drugs. Developed multiple reaction monitoring (MRM) mode may increase sensitivity and selectivity in the analysis of seized designer drugs. In addition, CI mass spectra and MRM mass spectra of these designer drug derivatives can be used as a potential supplemental database along with EI mass spectral database. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: designer drugs; synthetic cathinones; synthetic cannabinoids; GC-MS/MS
Introduction
Drug Test. Analysis (2014)
* Correspondence to: José R. Almirall, Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, Miami, FL 33199, USA. E-mail: almirall@fiu.edu Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, Miami, Florida, 33199, USA
Copyright © 2014 John Wiley & Sons, Ltd.
1
Designer drugs are structurally and chemically modified substances manufactured illicitly to circumvent the current legislation for controlled substances.[1] Some early examples of drugs that could today be considered as designer drugs are 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxy-amphetamine (MDA), first synthesized in the 1910s; they have been widely abused since the beginning of 1970s. Recently, the abuse of designer drugs has proliferated with the ease of purchase over the Internet and in head shops at relatively low cost, especially for the synthetic cannabinoid and synthetic cathinone classes of designer drugs.[2–4] Synthetic cannabinoids are the main components used to produce herbal marijuana alternatives (HMAs), also called synthetic marijuana.[5] One or more synthetic cannabinoids are dissolved in solution and sprayed onto herbs or plants for a natural appearance resembling an herbal product.[6] ‘K2’ and ‘Spice’ are the most common street names for HMAs, which are known to be more potent than delta-9-tetrahydrocannabinol (Δ9-THC), an active component in marijuana. Although there is no structural similarity with Δ9-THC, these synthetic cannabinoids act as cannabinoid receptor agonists.[7] Synthetic cathinones are derivatives of cathinone, an active component found in the Khat leaves.[8] The individual synthetic cathinones or the mixture of different synthetic cathinones are called ‘bath salts’ and generally sold in the form of a white powder, but may be found in other forms as well.[9] As a betaketone amphetamine analogue, the effects of synthetic cathinones
are similar to illicit stimulants such as amphetamine, cocaine, and MDMA (ecstasy).[9] In the United States, the number of emergency response calls related to synthetic cannabinoid exposures has dramatically increased from 2906 (2010) to 5205 (2012) and from 304 (2010) to 2656 (2012) related to synthetic cathinone exposures, according to the American Association of Poison Control Centers (AAPCC).[10,11] Despite the fact that some of these classes of designer drugs are currently regulated by the Federal Controlled Substances Act, new compounds with similar structures and effects continue to appear on the streets, replacing currently controlled substances as of May 2013.[12] This recent prevalence of seized designer drugs, especially of synthetic cannabinoids and cathinones, has led to research interest at various forensic laboratories across the world focusing on the identification and characterization of these drugs, including the clinical case series of synthetic cathinones.[13–21] As the gold standard technique that is most widely available in the forensic laboratory, gas chromatographymass spectrometry (GC-MS) has been widely implemented for the analysis of designer drugs. The identification of synthetic
Drug Testing and Analysis
S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
cathinones was reported by several research groups using GC-MS with the electron ionization (EI) source and with gas chromatography-ion trap-mass spectrometry (GC-IT-MS) with the EI and chemical ionization (CI) sources.[13–16] Extensive fragmentation with the EI source resulted in the absence of molecular ions (M•+), whereas the protonated molecular ions ([M + H]+) were determined with the use of CI source for those synthetic cathinone derivatives, such as 4-fluoromethcathinone, 4-methyl-Nethylcathinone, mephedrone, MDPV, butylone, and naphyrone. A number of synthetic cannabinoids have also been studied in various research groups using GC-MS with an EI source.[17–21] Gas chromatography-tandem mass spectrometry (GC-MS/MS) has been applied to the identification and quantitation of traditional drugs of abuse, especially for the presence of GHB, opioids, cocaine, and amphetamine derivatives in human hair where a method of high sensitivity is required.[22–24] More recently, GC-MS/MS has been used for the mass spectrometric differentiation of designer drug regioisomers.[25–28] In this study, the qualitative identification of 34 designer drugs was performed by gas chromatography-triple quadrupole-tandem mass spectrometry (GC-MS/MS) with two different ionization
techniques, including EI and CI, only focusing on qualitative seized-drug analysis. The advantage of using the soft CI source is the formation of molecular ions in order to assist with the easier identification for emerging designer drug derivatives. We report a sensitive and selective method using GC-MS/MS that can be used for the positive identification of these increasingly important classes of designer drugs at the level of qualitative seized-drug analysis and also report the corresponding mass spectra expected for these designer drugs.
Material and methods Chemicals Methanol (Optima®, LC/MS grade) and proadifen were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA), respectively. Proadifen was prepared in methanol at 10 μg/mL and used to lock the retention time in GC system due to the structural similarity to the target analytes. Reference materials of 34 designer drugs were provided by Cayman Chemical (Ann Arbor, MI, USA), including AKB48 (APINACA), AM694, AM1220,
Table 1. Summary of major peaks in EI and CI full scan MS for the analytes of interest with the molecular ions, shown in bold. Molecular ions are absent for the highlighted analytes in EI full scan MS No.
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Analyte 3-FMC 4-MMC 4-MEC 3,4-DMMC Methedrone Methylone MDPV CP-47,497 XLR-11 CP-47,497 C8 JWH-251 JWH-203 JWH-250 JWH-302 RCS-4 JWH-249 JWH-201 AM694 JWH-073 APINACA JWH-022 JWH-018 JWH-019 AM2201 CPE JWH-122 RCS-8 JWH-398 AM2233 CB-13 APICA JWH-081 AM1220 JWH-200
Retention Time (min)
Molecular Weight (amu)
Major Peaks in EI Full Scan MS in order of Relative Abundance (%)
Major Peaks in CI Full Scan MS in order of Relative Abundance (%)
9.715 11.584 12.307 13.143 13.634 14.639 19.101 22.897 22.970 23.648 24.777 25.376 25.461 25.732 25.862 26.070 26.093 26.562 26.590 26.923 27.144 27.155 27.759 27.855 28.002 28.030 28.256 28.369 28.408 28.425 28.442 29.115 30.035 30.431
181.09 177.12 191.13 191.13 193.11 207.09 275.15 318.26 329.22 332.27 319.19 339.14 335.19 335.19 321.17 383.10 335.19 435.05 327.16 365.25 339.16 341.18 355.19 359.17 376.22 355.19 375.22 375.14 458.09 368.18 364.25 371.19 382.20 384.18
58 (100), 95 (12.28), 123 (6.10) 58 (100), 91 (8.68), 119 (5.79) 72 (100), 91 (8.27), 119 (5.48) 58 (100), 133 (5.47) 58 (100), 135 (8.26), 77 (6.24) 58 (100), 149 (6.17) 126 (100), 149 (5.52) 215 (100), 233 (72.85), 318 (5.90) 232 (100), 144 (24.86), 329 (7.17) 215 (100), 233 (75.07), 332 (6.25) 214 (100), 144 (23.47), 116 (6.47) 214 (100), 144 (24.60), 116(6.93) 214 (100), 144 (24.31), 116 (6.01) 214 (100), 144 (23.92), 116 (5.88) 135 (100), 321 (87.28), 264 (78.89) 214 (100), 144 (21.44), 116 (5.36) 214 (100), 144 (22.53) 232 (100), 435 (51.84), 220 (49.53) 200 (100), 327 (92.61), 284 (66.56) 215 (100), 294 (29.79), 365 (16.27) 155 (100), 127 (72.01), 339 (58.51) 214 (100), 284 (82.99), 341 (75.16) 355 (100), 284 (96.12), 228(94.36) 359 (100), 232 (98.72), 284 (93.74) 98 (100), 70 (5.87) 355 (100), 214 (83.82), 298 (74.97) 254 (100), 144 (21.02), 55 (9.19) 214 (100), 375 (67.11), 318 (63.38) 98 (100), 70 (6.11) 171 (100), 368 (47.68), 297 (44.77) 214 (100), 307 (28.30), 364 (21.64) 371 (100), 214 (71.12), 314 (64.96) 98 (100), 70 (6.32) 100 (100), 127 (5.89), 56 (5.53)
182 (100), 164 (36.02), 58 (7.69) 160 (100), 178 (72.11), 58 (17.83) 192 (100), 174 (69.25), 72 (13.96) 174 (100), 192 (82.34), 163 (23.95) 176 (100), 194 (60.63), 165 (29.20) 190 (100), 208 (99.96), 160 (41.26) 276 (100), 126 (54.93), 207 (42.39) 301 (100), 233 (39.33), 318 (5.76) 330 (100), 310 (67.56), 125 (18.59) 315 (100), 247 (25.72), 332 (5.45) 320 (100), 214 (22.84), 304 (22.07) 340 (100), 214 (24.05), 127 (13.09) 336 (100), 214 (30.53), 320 (18.77) 336 (100), 214 (19.18), 320 (5.01) 322 (100), 188 (34.07), 135 (21.50) 384 (100), 288 (66.76), 214 (46.49) 336 (100), 188 (36.23), 214 (27.01) 436 (100), 310 (23.68), 231 (12.30) 328 (100), 200 (9.92) 135 (100), 366 (23.65), 214 (14.09) 340 (100), 212 (10.16) 342 (100), 214 (14.42), 155 (9.62) 356 (100), 202 (20.88), 157 (8.84) 360 (100), 340 (11.75), 232 (10.71) 377 (100), 98 (65.24), 359 (7.33) 356 (100), 214 (14.17), 169 (8.45) 376 (100), 360 (33.56), 254 (29.15) 376 (100), 214 (16.50), 189 (8.41) 459 (100), 98 (64.98), 331 (8.20) 369 (100), 241 (10.73), 155 (8.29) 365 (100), 135 (43.38), 213 (22.34) 372 (100), 188 (34.54), 355 (17.62) 383 (100), 98 (96.57), 286 (14.48) 385 (100), 100 (14.98), 281 (10.31)
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Analysis of Cannabinoids and Cathinones AM2201, AM2233, Cannabipiperidiethanone (CPE), CB-13, CP-47,497, CP-47,497 C8 homolog, 3,4-dimethylmethcathinone (3,4-DMMC), 3-fluoromethcathinone (3-FMC), 4-methoxymethcathinone (methedrone), 3,4-methylenedioxy-N-methcathinone (methylone), 3,4-methylenedi oxypyrovalerone (MDPV), 4-methylethcathinone (4-MEC), 4-methyl methcathinone (4-MMC), JWH-018, JWH-018 adamantyl carboxamide (APICA), JWH-019, JWH-022, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-249, JWH-250, JWH-251, JWH-302, JWH-398, RCS-4, RCS-8, and XLR-11. The standard stock solutions of each designer drug in powder form were prepared in methanol at different concentrations depending on the amount of standards: 1000, 2000,
and 2500 μg/mL. These stock solutions were diluted to 10 μg/mL in methanol in order to prepare the 4 mixtures of working standard solutions. GC-MS/MS The qualitative analysis of 34 designer drugs was performed using Agilent 7890A GC system (Santa Clara, CA) with a DB-5MS column (30 m × 250 μm × 0.25 μm, J&W, Agilent) coupled with Agilent 7000 GC-MS Triple Quad (Santa Clara, CA, USA). Retention time was locked for proadifen at 20.765 min in order to provide
Figure 1. EI full scan mass spectra for (a) JWH-251, (b) JWH-250, (c) JWH-249, (d) CPE, (e) AM2233, and (f) AM1220.
3
Figure 2. CI full scan mass spectra for (a) JWH-251, (b) JWH-250, (c) JWH-249, (d) CPE, (e) AM2233, and (f) AM1220 with methane as a reagent gas.
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
4
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
30 (100) 42 (74) 56 (41) 30 (100) 42 (74) 56 (40) 30 (100) 42 (73) 56 (39) 69 (100) 97 (98) 84 (96) 81 (100) 147 (33) 215 (16) 144 (100) 247 (2) 314 (1.6) 81 (100) 147 (33) 215 (14) 144 (100) 116 (45) 214 (7) 144 (100) 116 (45) 214 (4)
30 (100) 42 (74) 56 (37) 30 (100) 42 (74) 56 (40) 44 (100) 29 (15)
Product Ion (Relative Abundance, %)
8 20 12 8 20 12 8 20 12 20 12 12 16 12 12 12 8 12 16 12 12 12 28 4 12 28 4
8 20 12 8 20 12 8 16
CE (V) 214 214 335 214 214 335 321 321 321 214 214 383 214 214 232 232 435 200 200 327 215 217 365 155 339 339 214 341 341 228 355 355
AM694 AM694 AM694 JWH-073 JWH-073 JWH-073 APINACA APINACA APINACA JWH-022 JWH-022 JWH-022 JWH-018 JWH-018 JWH-018 JWH-019 JWH-019 JWH-019
Precursor Ion
JWH-250 JWH-250 JWH-250 JWH-302 JWH-302 JWH-302 RCS-4 RCS-4 RCS-4 JWH-249 JWH-249 JWH-249 JWH-201 JWH-201
Analyte
144 (100) 116 (54) 232 (44) 144 (100) 116 (48) 310 (40) 145 (100) 90 (42) 215 (5) 127 (100) 155 (9) 184 (8.5) 144 (100) 284 (48) 167 (27) 144 (100) 284 (51) 338 (43)
144 (100) 116 (44) 214 (5) 144 (100) 116 (46) 214 (5) 264 (100) 135 (74) 186 (62) 144 (100) 116 (45) 214 (2) 144 (100) 116 (45)
Product Ion (Relative Abundance, %)
12 32 4 8 24 8 12 36 24 12 20 4 8 8 20 8 12 8
12 28 4 12 28 4 12 32 12 12 28 4 12 28
CE (eV) AM2201 AM2201 AM2201 CPE CPE CPE JWH-122 JWH-122 JWH-122 RCS-8 RCS-8 RCS-8 JWH-398 JWH-398 JWH-398 AM2233 AM2233 AM2233 CB-13 CB-13 CB-13 APICA APICA APICA JWH-081 JWH-081 JWH-081 AM1220 AM1220 AM1220 JWH-200 JWH-200 JWH-200
Analyte 232 359 359 98 98 98 298 355 355 254 254 254 214 375 375 98 98 98 171 171 368 214 214 364 214 371 371 98 98 98 100 100 100
Precursor Ion
* Average relative abundance was calculated based on the 3 replicates and the relative standard deviation (%RSD) for each transition were less than 2.4%.
58 58 58 58 58 58 58 58 58 126 126 126 215 215 318 232 329 329 215 215 332 214 214 319 214 214 339
58 58 58 58 58 58 72 72
3-FMC 3-FMC 3-FMC 4-MMC 4-MMC 4-MMC 4-MEC 4-MEC
3,4-DMMC 3,4-DMMC 3,4-DMMC Methedrone Methedrone Methedrone Methylone Methylone Methylone MDPV MDPV MDPV CP-47,497 CP-47,497 CP-47,497 XLR-11 XLR-11 XLR-11 CP-47,497 C8 CP-47,497 C8 CP-47,497 C8 JWH-251 JWH-251 JWH-251 JWH-203 JWH-203 JWH-203
Precursor Ion
Analyte 144 (100) 284 (42) 232 (13) 70 (100) 42 (81) 68 (12) 181 (100) 298 (62) 181 (53) 69 (100) 144 (90) 158 (32) 144 (100) 318 (33) 358 (21) 70 (100) 42 (82) 68 (12) 115 (100) 143 (53) 171 (17) 144 (100) 116 (43) 307 (11) 144 (100) 354 (43) 214 (10) 70 (100) 42 (82) 68 (12) 56 (100) 70 (52) 44 (17)
Product Ion (Relative Abundance, %)
12 12 20 12 24 24 8 8 20 20 12 12 8 8 12 12 28 24 24 8 24 12 28 8 8 12 28 12 28 24 12 12 12
CE (eV)
Table 2. Optimized EI MRM transitions for the analytes of interest with the average relative abundance* (%) and selected collision energy (CE, eV). The most abundant ions in bold are used for quantification in generating the calibration curves
Drug Testing and Analysis S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
5
Drug Test. Analysis (2014)
182 182 182 178 178 178 192 192 192 192 192 192 194 194 194 208 208 208 276 276 276 301 301 301 330 330 330 315 315 315 320 320 320 340 340 340
Precursor Ion
164 (100) 149 (61) 103 (20) 160 (100) 145 (58) 119 (11) 174 (100) 145 (36) 131 (15) 174 (100) 159 (73) 144 (18) 176 (100) 161 (36) 135 (9) 160 (100) 190 (67) 132 (48) 126 (100) 205 (58) 175 (68) 233 (100) 216 (7) 121 (33) 125 (100) 232 (46) 97 (23) 247 (100) 149 (16) 121 (57) 105 (100) 119 (9) 214 (63) 125 (100) 214 (15) 188 (15)
Product Ion (Relative Abundance, %) 8 20 32 8 20 24 8 20 28 12 20 36 8 20 20 16 8 32 24 16 20 8 12 32 20 20 28 8 24 24 24 24 24 32 24 16
CE (V) 336 336 336 336 336 336 322 322 322 384 384 384 336 336 336 436 436 328 328 366 366 340 340 342 342 342 356 356 356
JWH-073 JWH-073 APINACA APINACA JWH-022 JWH-022 JWH-018 JWH-018 JWH-018 JWH-019 JWH-019 JWH-019
Precursor Ion
JWH-250 JWH-250 JWH-250 JWH-302 JWH-302 JWH-302 RCS-4 RCS-4 RCS-4 JWH-249 JWH-249 JWH-249 JWH-201 JWH-201 JWH-201 AM694 AM694
Analyte
155 (100) 214 (25) 127 (93) 155 (100) 228 (21) 127 (92)
155 (100) 212 (24)
135 (100) 215 (11)
155 (100) 200 (28)
121 (100) 200 (8) 188 (6) 121 (100) 214 (62) 144 (29) 135 (100) 214 (7) 107 (20) 169 (100) 214 (31) 188 (19) 121 (100) 149 (24) 135 (31) 231 (100) 309 (6)
Product Ion (Relative Abundance, %)
24 20 56 24 24 56
24 20
16 20
24 20
20 20 12 20 24 44 24 24 44 32 24 16 28 20 28 28 16
CE (eV)
JWH-081 JWH-081 JWH-081 AM1220 AM1220 AM1220 JWH-200 JWH-200 JWH-200
APICA APICA
AM2201 AM2201 AM2201 CPE CPE CPE JWH-122 JWH-122 JWH-122 RCS-8 RCS-8 RCS-8 JWH-398 JWH-398 JWH-398 AM2233 AM2233 AM2233 CB-13 CB-13
Analyte
372 372 372 383 383 383 385 385 385
365 365
360 360 360 377 377 377 356 356 356 376 376 376 376 376 376 459 459 459 369 369
Precursor Ion
* Average relative abundance was calculated based on the 3 replicates and the relative standard deviation (%RSD) for each transition were less than 1.3%.
3-FMC 3-FMC 3-FMC 4-MMC 4-MMC 4-MMC 4-MEC 4-MEC 4-MEC 3,4-DMMC 3,4-DMMC 3,4-DMMC Methedrone Methedrone Methedrone Methylone Methylone Methylone MDPV MDPV MDPV CP-47,497 CP-47,497 CP-47,497 XLR-11 XLR-11 XLR-11 CP-47,497 C8 CP-47,497 C8 CP-47,497 C8 JWH-251 JWH-251 JWH-251 JWH-203 JWH-203 JWH-203
Analyte
185 (100) 214 (34) 157 (24) 98 (100) 155 (25) 112 (82) 155 (100) 114 (76) 127 (50)
214 (100) 188 (17)
155 (100) 232 (25) 127 (95) 112 (100) 229 (17) 121 (50) 169 (100) 214 (33) 141 (62) 121 (100) 228 (5) 144 (9) 189 (100) 214 (12) 161 (50) 98 (100) 362 (7) 231 (17) 299 (100) 241 (36)
Product Ion (Relative Abundance, %)
24 20 44 32 24 20 20 28 60
20 20
24 24 56 20 12 24 24 20 48 24 12 40 24 24 52 36 16 32 12 12
CE (eV)
Table 3. Optimized CI MRM transitions for the analytes of interest with the average relative abundance* (%) and selected collision energy (CE, eV). The most abundant ions in bold are used for quantification in generating the calibration curves
Analysis of Cannabinoids and Cathinones
Drug Testing and Analysis
Drug Testing and Analysis
S. Gwak, L. E. Arroyo-Mora and J. R. Almirall
Figure 3. Overlaid MRM total ion chromatograms of the 4 standard mixtures including all 34 analytes for (a) EI and (b) CI with potential co-elutions observed for some of the compounds. Numbers for each peak correspond to the numbers in Table 1 and a Peak #12 is beyond the scale shown in both EI and CI chromatograms.
6 Figure 4. EI and CI MRM mass spectra for JWH-250 and AM1220 as representative compounds at different collision energies for each transition.
wileyonlinelibrary.com/journal/dta
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Analysis of Cannabinoids and Cathinones consistent retention times for the same analytes over time. GC parameters for full scan mode were as follows: injection volume of 5 μL, split ratio of 1:10, injection temperature of 280 °C, transfer line temperature of 300 °C, helium as a carrier gas at different flow rate depending on the pressure for RTL. Oven temperature was programmed with initial temperature of 60 °C held for 1 min and 10 °C/min to 325 °C held for 8 min. The operation parameters for MS in the full scan mode were as follows: gain factor of 1, source temperature at 300 °C, quadrupole temperature at 150 °C, solvent delay of 3.75 min, ionization energy of 70 eV, and acquisition mass range of m/z 45–570. Methane was used as the reagent gas for the CI mode. In the product ion scan and multiple reaction monitoring (MRM) mode, the flow rate of nitrogen as the collision gas and helium as the quench gas was 1.0 mL/min. The acquisition mass range was varied in product ion scan mode during the MRM optimization starting from m/z 35 up to the mass of the selected precursor ion. Other MS operating conditions remained the same. The optimization of the MRM mode was performed by selecting an appropriate precursor ion for each analyte of interest and selecting 2 to 3 transitions to product ions from the precursor ion. The ideal collision energy and dwell time were optimized for each transition during this process using the Design Experiments Assistant, Analyze Experiments Assistant, and Dynamic MRM Assistant software provided by Agilent Technologies. The MRM method was then optimized and validated by determining the limits of detection (LODs) for each analytes prepared in methanol at different concentration from 50 to 1200 ng/mL. The gain factor was increased to 50 from 1 and the flow rate of helium quench gas was set to 2.0 mL/min with pulsed splitless injection mode for the LODs study with both EI and CI sources.
Results and discussion Prior to the analysis of the 34 designer drugs, the gas chromatographic system was locked for proadifen in order to obtain consistent results over time with the use of retention-time locking (RTL) feature. The pressure of the capillary column was optimized based on the RTL calibration and the flow rate of carrier gas (He) was adjusted in accordance with the optimized pressure for the target compound (proadifen). Detailed information on RTL is explained elsewhere.[29] Table 1 summarizes the EI full scan mass spectra for 34 designer drugs of interest presenting a base peak and two of the most abundant peaks along with their relative abundance as a preliminary study. The molecular ions were present for most of the synthetic cannabinoids (n = 16), whereas extensive fragmentation was observed for all of the synthetic cathinones and some of the synthetic cannabinoids (n = 18). Figure 1 shows the actual EI full scan mass spectra for some of the phenylacetylindoles, including JWH-251, JWH-250, and JWH249. The common fragment ions at m/z 214, 144, and 116 were observed with significant relative abundance, but extremely low relative intensity for a molecular ion (
