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Analytical Challenges with MS Analysis of Bath Salts and Spice Cannabinoid Metabolites

By: Joshua Cooper, Ryan Carrell, Ning Chang, Yunming Ying, Greg Kirkovits, Isil Dilek, Uma Sreenivasan,

Introduction

Mass spectrometry (MS) is the tool of choice for detection and quantitationof new illicit drugs like Spice cannabinoidsand bath salt cathinones. While GC/MS and LC/MS provide numerous benefits for these purposes, they also offer challenges unique to the particular technique.

GC/MS methods, for example, require use of derivatizationreagents for analysis of cathinone-based analogs like bath salts. The use of PFPA and BSTFA derivatives with deuterium-labeled internal standards have been reported to cause loss of label in the GC/MS fragmentation.

Analysis of matrix-based samples by LC/MS can suffer from interferences or lower ionization efficiency due to matrix effects. While deuterium-labeled internal standards are most commonly used to compensate for matrix effects in LC-MS/MS applications, some labeled compounds may exhibit hydrogen-deuterium scrambling/exchange in the collision cell which necessitates careful selection of MS/MS transitions.

Materials & GC/MS Derivatization Method

Materials:

3,4-MDPV HCl, Cerilliant Cat# M146
3,4-MDPV-D8 HCl, Cerilliant Cat# M150
Ethylone HCl, Cerilliant Cat# E071
Ethylone-D5HCl, Cerilliant Cat# E072
ButyloneHCl, Cerilliant Cat# B045
Butylone-D3HCl, Cerilliant Cat# B046
MephedroneHCl, Cerilliant Cat# M138
Mephedrone-D3HCl, Cerilliant Cat# M139
MethyloneHCl, Cerilliant Cat# M140
Methylone-D3HCl, Cerilliant Cat# M141
MethedroneHCl, Cerilliant Cat# M147
JWH-018 4-Hydroxypentyl metabolite, Cerilliant Cat# S035
JWH-018 4-Hydroxypentyl metabolite-D5, Cerilliant Cat# S039
JWH-073 3-Hydroxybutyl metabolite, Cerilliant Cat# S037
JWH-073 3-Hydroxybutyl metabolite-D5, Cerilliant Cat# S040

GC/MS Derivatization Method

Native and deuteratedreference materials of the above bath salts were used to develop the derivatizationmethod with trifluoroaceticanhydride (TFAA). The HClsalts were converted to free base with 0.1M sodium bicarbonate and heated to 60°C for five minutes with TFAA and ethyl acetate to acylatethe amino group. The free up procedure is sensitive to choice of base due to instability of α-amino ketones. Optimization of derivatizationtime is critical, as decomposition occurs with excessive heating.

GC/MS Chromatographic Data

Derivatives were analyzed directly by GC/MS with cool-on-column injection on a DB-5ms narrow-bore (30m x 0.25mm x 0.25μm) column.

Temperature ramp: 3 min at 150°C, 150°C to 200°C at 10°C/min, 200°C to 210°C at 2°C/min.

Temperature ramp: 3 min at 150°C, 150°C to 200°C at 10°C/min, 200°C to 210°C at 2°C/min

 

  Compound Peak Width Resolution Tailing RRT vs. Methylone
1 Mephedrone 0.046 NA 0.67 0.691
2 Methedrone 0.063 20.08 0.64 0.876
3 Methylone 0.065 11.50 0.63 1.000
4 Butylone 0.072 7.47 0.63 1.087
5 Ethylone 0.080 3.03 0.63 1.126

 

GC Mass Spectra And Isotopic Distribution Of TFA-Derivatized Bath Salts

The labeled compounds retain deuterium label from the molecular ion to one or two fragmentations. Quant ion pairs were selected based on ion abundance. Isotopic distribution was evaluated to ensure the majority of the label was on the quant ion.

The labeled compounds retain deuterium label from the molecular ion to one or two fragmentations

 

  Butylone-D3HC
  MW Pair Q1 Pair
D3 98.96% 99.16%
D2 0.97% 0.39%
D1 0.04% 0.42%
D0 0.02% 0.02%
D0/D3 0.03% 0.02%

 

 

Compound (TFA) MW Pair Q1 Pair Q2 Pair
Mephedrone/ Mephedrone-D3 HCl 273.1 / 276.1 154.1 / 157.1 110.1 / 113.1
Methylone/ Methylone-D3HCl 303.1 / 306.1 154.1 / 157.1 NA
Butylone/ Butylone-D3 HCl 317.1 / 320.1 168.0 / 171.0 110.0 / 113.0
Ethylone/ Ethylone-D5HCl 317.1 / 322.1 168.0 / 173.0 NA

 

The labeled compounds retain deuterium label from the molecular ion to one or two fragmentations

 

  Ethylone-D5HCl
  MW Pair Q1 Pair
D5 96.69% 97.65%
D4 3.09% 2.02%
D3 0.15% 0.21%
D2 0.06% 0.04%
D1 0.01% 0.03%
D0 0.01% 0.05%
D0/D5 0.01% 0.04%

For Methedroneand Mephedrone, the molecular ion abundance is low. The fragment ion is used for quantitation.

Abundance Scan

The labeled compounds retain deuterium label from the molecular ion to one or two fragmentations

 

  Mephedrone-D3HCl
  Q1 Pair Q2 Pair
D3 97.51% 98.74%
D2 1.62% 0.81%
D1 0.84% 0.21%
D0 0.03% 0.24%
D0/D3 0.03% 0.24%

 

Butylone & Ethylone Comparison By GC/MS

 

Ratio Butylone/TFA Ethylone/TFA
121/110 58.11% 0.27%
140/121 14.99% 34.05%

 

Butylone Expansion

Ethylone Expansion

Ethyloneand Butylonehave identical molecular and primary ion fragmentations of 168 and 149. Differentiation between Ethyloneand Butyloneis achieved by monitoring the response of fragment ions 121/110 and 140/121.

Investigation Of Scrambling In LC-MS/MS Analysis Of Bath Salts & Spice Cannabinoid Metabolites

In this study, we investigated several variables that potentially contribute to scrambling in LC-MS/MS applications in order to ascertain reproducibility and impact on scrambling ratios: collision energies and deuterium placement in the internal standard.

LCMS System:
Waters Alliance UPLC-XevoG2 Q-Tof

JWH-018 4-Hydroxypentyl Metabolite Scrambling at m/z 340

JWH-018 4-Hydroxypentyl Metabolite Scrambling at m/z 340

JWH-073 3-Hydroxybutyl Metabolite Scrambling at m/z 216

JWH-073 3-Hydroxybutyl Metabolite Scrambling at m/z 216

Scrambling was observed for the deuterium-labeled Spice metabolites but not for the bath salts. Scrambling was eliminated with selection of a different transition.

Scrambling Comparison using XevoG2 Q-Tof

Compound Label Polarity Collision Energy Transition(s) dn Scrambling
% dn-1/dn
JWH-018 4-Hydroxypentyl metabolite D5 pos 20 363→345 22.83
Native pos 20 358→340 0
JWH-018 4-Hydroxypentyl metabolite D5 pos 20 363→155 0
Native pos 20 358→155 0
JWH-073 3-Hydroxybutyl metabolite D5 pos 20 349→221 25.44
Native pos 20 344→216 0
JWH-073 3-Hydroxybutyl metabolite D5 pos 20 363→155 0
Native pos 20 358→155 0
3,4-MDPV HCl D8 pos 15 284→134 0
Native pos 15 284→126 0
Ethylone HCl D5 pos 15 227→209 0
Native pos 15 222→204 0
ButyloneHCl D3 pos 15 225→209 0
Native pos 15 222→204 0
MephedroneHCl D3 pos 10 181→163 0
Native pos 10 178→160 0
Methylone HCl D3 pos 10 211→163 0
Native pos 10 208→160 0
Methylone HCl D3 pos 10 211→135 0
Native pos 10 208→132 0

 

Conclusions

  • TFAA derivatizationis an effective method for analysis of bath salt cathinonesby GC/MS. The derivatizationmethod using TFAA prevented loss of label in the GC/MS fragmentation of deuterium-labeled bath salts.
  • Increased mass spectral abundances for the molecular and fragment ions were also observed using TFAA derivatization. Butyloneand ethyloneare readily distinguished by the different relative abundance of two common fragment ions.
  • Scrambling was observed at select transitions in the LC-MS/MS analysis of deuterium-labeled omega-1 hydroxySpice metabolites. In each case, scrambling was eliminated by optimizing instrument conditions and transition selection.
  • Scrambling may be mitigated or eliminated by altering instrument conditions and transition selection. The impact of scrambling must be considered in choice of transitions for optimal selectivity.
  • Direct infusion can provide rapid and accurate determination of scrambling ratios.
  • Awareness of potential scrambling is important for proper internal standard selection.

Deuterium-labeled internal standards of bath salt cathinonesand Spice cannabinoidmetabolites are suitable for GC/MS and LC/MS applications when consideration is given to choice of derivatizationreagent and transition selection.

Materials

     
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