Improved GC/MS Derivatization Techniques for Analysis of New Designer Drugs: Methylone, Ethylone, Butylone, Mephedrone, and Methedrone

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

Introduction

As new “bath salt” drugs such as methylone, ethylone, mephedrone, butylone and other methcathinone analogs increase in popularity, toxicologists require native and labeled certified reference materials to accurately identify and quantify the new compounds in patient samples. Internal standards must have quantitation ions that do not interfere with native quantitation ions. There have been problems with use of PFPA and BSTFA derivatives of deuterated internal standards of these methcathinones due to loss of label in the GC/MS fragmentation. An alternate derivatization method is presented in this study.

Derivative Method

Native and deuterated reference materials of methylone, ethylone, butylone, mephedrone, and methedrone were synthesized at Cerilliant as HCl salts and were used to develop the derivatization method with trifluoroacetic anhydride (TFAA). The HCl salts were converted to free base with 0.1M sodium bicarbonate and heated at 60°C for five minutes with TFAA and ethyl acetate to acyalte the amino group. The free up procedure is sensitive to choice of base due to instability of α-amino ketones. Derivatization time is critical, as decomposition occurs with excessive heating.

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.

chromatographic-data

 

  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

 

Mass Spectra and Isotopic Distribution

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.

Compound MW Pair Q1 Pair Q2 Pair
Mephedrone / Mephedrone-D3 HCl 273.1 / 276.1 154.1 / 157.1 110.1 / 113.1
Methylone / Methylone-D3 HCl 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-D5 HCl 317.1 / 322.1 168.0 / 173.0 NA

 

MASS SPECTRA AND ISOTOPIC DISTRIBUTION

 



Methylone-D3 HCl
MW Pair Q1 Pair
D3 98.01% 98.42%
D2 1.84% 1.04%
D1 0.10% 0.51%
D0 0.05% 0.03%
D0/D3 0.05% 0.03%

 

MASS SPECTRA AND ISOTOPIC DISTRIBUTION

 



Butylone-D3 HCl
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%

 

MASS SPECTRA AND ISOTOPIC DISTRIBUTION

For Methedrone and Mephedrone, the molecular ion abundance is low. The fragment ion is used for quantiation.

MASS SPECTRA AND ISOTOPIC DISTRIBUTION

 

  Mephedrone-D3 HCl
  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%

 

MASS SPECTRA AND ISOTOPIC DISTRIBUTION

 

  Ethylone-D5 HCl
  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%

 

Butylone /Ethylone Comparison

Ethylone and butylone are distinguished by the response of fragment ions 110 and 140 relative to 121 amu.

Butylone Expansion

Ethylone Expansion

 

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

 

Advantages of TFAA as Derivatizing Reagent

  • Thermally stable, repeatable derivatization.
  • Increased mass spectral abundances for molecular and fragment ions.
  • Labeled analogs retain deuterium label in the fragment ions.
  • The deuterium labeled analogs are suitable for use as internal standards to quantitate the native compounds.
  • Butylone and ethylone are readily distinguished by the different relative abundance of two common fragment ions.

Conclusions

  • Deuterium labeled internal standards were developed for quantitation of new methcathinone analogs, “bath salts”. These standards are suitable for both GC/MS and LC/MS applications.
  • A method of derivatization of these analogs with TFAA was successfully developed with retention of label in the GC/MS fragment ions.

Materials

     
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