Injection technique for

On-column derivatization Sandwich

injection

Reaction

zone at

Sandwich

injection

Reaction

zone at

On-column

derivatization

Pause between 

sample and 

reagent injection

Development of a GC-MS method for determination of Benzodiazepine Series Drugs

Kirill Tretyakov

1

, Aviv Amirav

2

, Anzor Mikaia

1

1National Institute of Standards and Technology, USA; 2Tel Aviv University, Tel Aviv, Israel.

A GC-MS method for determination of drugs of

benzodiazepine series is developed. Thermal and EI

induced decompositions of these compounds are studied

by varying sample inlet configurations and ion source

systems. Reliable GC retention index (GCRI) values and

mass spectral data are acquired.

Benzodiazepines are widely prescribed drugs. They are

sensitive to elevated temperature. Detection of

benzodiazepines by various mass spectrometry methods,

including GC-MS, involves preliminary chemical

modification of these compounds yielding stable

derivatives; often the spectra of derivatives exhibit

fragmentation pathways useful for structure

determination. In this study synthesis, measurements

and examination of GCRI and EI mass spectra of

trimethylsilyl derivatives of these drugs of abuse are

conducted. Acquired data will be included in the 2017

release of the NIST/EPA/NIH library.

• Lorazepam and Oxazepam molecules undergo dehydrating processes in a GC system and in the ion source;

• Information on stability of Lorazepam and Oxazepam molecules and their transformation at different stages of

GC-MS analysis is obtained and optimal trimethylsilylation conditions for their GC-MS analysis are established;

• An aromatic ring at C-5 is a major source for the elimination of hydrogen and halogen radicals;

• The Influence of a nature of N(1) on dissociative ionization of benzodiazepines is evaluated.

Chemicals: Lorazepam (1), Lormetazepam (2), 

N-Desmethylflunitrazepam (3), Clonazepam (4), Oxazepam (5), 

Temazepam (6) are commercially available. 

(1) R1,R4= Cl; R2= H; R3= OH. (2) R1,R4= Cl; R2= CH3; R3= OH.

(3) R1= NO2; R2,R3= H; R4= F. (4) R1= NO2; R2,R3= H; R4= Cl.

(5) R1= Cl; R2= H; R3= OH; R4= H.

(6) R1= Cl; R2= CH3; R3= OH; R4= H.

NOVELTY RESULTS and DISCUSSION

EXPERIMENTAL

CONCLUSIONS

Substituents at 5-phenyl are the major donors of eliminating radicals under EI: 

Figure 3. EI spectra of Clonazepam (a), N-Desmethylflunitrazepam (b) and TMS ethers of Oxazepam (c) and D5-Oxazepam (d).

EI mass spectral data for the standard GC experiment, Open Probe and Cold EI

Figure 2. Mass spectra of Quinazoline aldehyde (a), mixture of Lorazepam and Quinazoline aldehyde (b), Lorazepam (c).

R

1

N

O

N

R

4

R

2

R

3 Figure 1. Total ion currents (TIC) obtained for Oxazepam (a), products of its silylation carried out prior (b) and online GC-MS analysis(c, d, e), 

and for dehydration product of Oxazepam at 200oC (f). 

Oxazepam (R4=H) and Lorazepam (R4=Cl) under EI do not show peaks of molecular ions while in the spectra of their N(1)-methyl analogs

these peaks are present. Peaks of ions due to loss of water can be used for determination of M+· of Oxazepam and Lorazepam.

However, one can suggest that these molecules are converted to quinazolines in a GC injection port, and GC retention indices and the

spectra recorded correspond to dehydration products. It also has been established earlier

[1]

that toluene or xylene solutions of

Oxazepam and Lorazepam at 110°C decompose with the formation of corresponding quinazoline aldehydes; they are the products of

water elimination.

GC retention index values and mass spectra indicate degradation most of Oxazepam and Lorazepam molecules in a GC injection port

and further water elimination in a GC column, and probably in EI source (Figure 1). Additional experiments on “Open Probe Fast GC-MS”

have confirmed degradation of Oxazepam and Lorazepam in a GC system (injection port and column), and experiments conducted on

“Fast GC-MS with Cold EI” demonstrated sample degradations in the ion source as well.

a) Inlet temperature 200oC, standard injection of the 

Oxazepam solution.

b) Inlet temperature 200oC, standard injection of the 

Oxazepam bisTMS derivative synthesized prior 

GC-MS experiment.

c) Inlet temperature 150oC, sequential injection of the 

Oxazepam sample and silylating reagent (MSTFA) -

On-column derivatization.

d) Inlet temperature 200oC, sequential injection of the 

Oxazepam sample and silylating reagent (MSTFA) -

On-column derivatization.

e) Inlet temperature 200oC, Sandwich injection of the 

Oxazepam sample (1mg/mL in acetonitrile) and 

silylating reagent (MSTFA). 

f ) Inlet temperature 200oC, standard injection of the 

Oxazepam sample preheated at 200oC followed by 

silylation with the MSTFA reagent.

100%

100%

0

0

0

0

100%

100%

100%

Time, min

a

b

c

d

e

0

“Oxazepam” Oxazepam

bis TMS derivative

0

100%

f

Trimethylsilylation was carried out at 60°C with the use of

N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1%

trimethylchlorosilane (TMCS) or N-Methyl-N-(trime

thylsilyl)trifluoroacetamide (MSTFA) as silylating reagents. Oncolumn derivatization of Lorazepam and Oxazepam was

performed to identify any thermal decomposition products

(Figure 1).

Instrumentation and measurements

Standard GC-MS method: EI mass spectra were measured

on GC-MS systems with quadrupole analyzers (ionization

energy 70 eV; ion source temperature 230oC), with GC

conditions: VF-5ms capillary column (30 m, 0.25 mm, 0.25 μm,

1 mL/min helium (He) flow); oven temperature program – from

60°C to 270°C at 10°C/min; inlet and transfer line at 270°C.

Open Probe Fast GC-MS: MS conditions were used as in the

standard method, GC conditions: Rx1-5HT capillary column

(2.0 m, 0.25 mm, 0.1 μm,  2.0 mL/min He flow); oven

temperature program – from 60°C to 320°C at 200°C/min; inlet

and transfer line 220°C.

Fast GC-MS with Cold EI: GC-MS with Cold EI is based on a

GC and MS interface with a supersonic molecular beam (SMB)

and on the EI of sample compounds while they are vibrationally

cold in the SMB (hence the name Cold EI) in a fly-through ion

source. GC conditions: Rxi-1HT capillary column (15 m, 0.32

mm, 0.1 μm, 24 mL/min He flow); oven temperature program –

from 80°C to 160°C at 40°C/min then to 300°C at 20°C/min;

inlet temperature 240°C.

R1 = Cl, NO2; R2 = H, SiMe3; R3 = H,OSiMe3; R4 = Cl, D, F, H 

N

O

N

R

1

R

2

R

4

R

3

N

O

+

N

R

1

R

2

CH

R

4

R

3

N

O

+

N

R

1

R

2

CH

R

4

R

3

N

O

+

N

R

1

R

2

CH R

3

- R4

Mass Spectrometry Data Center

Mass spectral data acquired for Lorazepam on the traditional GC-MS (Figure 2a), on Open Probe Fast GC-MS (Figure 2b) and on Fast

GC-MS with Cold EI (Figure 2c) demonstrate that we actually observe a spectrum of Quinazoline aldehyde (not Lorazepam) when

spectrum is obtained on a traditional GC-MS instrument. A replacement of a regular GC with an Open Probe Fast GC-MS produces a

spectrum that contains spectra of Quinazoline aldehyde and Lorazepam; a spectrum of an unseparated mixture of compounds is

acquired. A spectrum of individual Lorazepam is obtained only on Fast GC-MS with Cold EI instrument. However, this spectrum cannot

be considered as a traditional EI spectrum since low mass peaks are discriminated and intensities of molecular ions are elevated in Cold

EI ion source spectra. The results presented in Figures 2 a-c establish that thermal decomposition of Lorazepam occurs in the inlet

system and in the ion source.

(Spec. Edit) Component at scan 1153 (5.455 min) [Model = +214u] in F:\_GCMS_KIRILL\OPEN PROBE\LORAZEPAM AND OXAZEPAM COLD EI\LORAZEPAM COLD EI 884.D\DATA.MS

2 0 4 0 6 0 8 0 100 120 140 160 180 200 220 240 260 280 300 320

0

5 0

100

6 3 7 5 8 9 107

125 138 152 163

181

214 242

257 277 292

320

(Spec. Edit) Component at scan 2167 (4.338 min) [Model = +111u] in F:\_GCMS_KIRILL\OPEN PROBE\2016-08\2016-08-30\LORAZEPAM_B.D\DATA.MS

2 0 4 0 6 0 8 0 100 120 140 160 180 200 220 240 260 280 300 320

0

5 0

100

5 1 6 3

7 5

8 8

111

126

138

151

163

177

202 213 229

239

263

274

291

302

320

(imsc-2014) Lorazepam

2 0 4 0 6 0 8 0 100 120 140 160 180 200 220 240 260 280 300 320

0

5 0

100

2 9

3 9

5 0

6 2

7 5

8 5

102

111

119

138

150 163

177

186

203

239

274

302 N

O

N

H

Cl

OH

Cl

M+·

Relative Intensity, % Relative Intensity, % Relative Intensity, %

m/z

a

b

c

M+·

M+·

N

N CH

Cl

Cl

O

(Q)

(L)

(Q)+(L)

REFERENCES

[1] M.S. Siddegowda, et al. Indian Journal of Chemistry. 2012, V. 51B, P. 1628

(imsc-2014) Oxazepam-D5, bis(trimethylsilyl) derivative

30 60 90 120 150 180 210 240 270 300 330 360 390 420 450

0

50

100

15 27

45

59

73

82 100 131

147

155 184 210 243 256 269 282

318

345 358 376 389

406 420

433

(imsc-2014) Oxazepam, bis(trimethylsilyl) derivative

30 60 90 120 150 180 210 240 270 300 330 360 390 420 450

0

50

100

15 27

45

59

73

87 100 113 133

147

163 179 205 239 253 267 281

313

324 340

353 371 384

401 415

429

(imsc-2014) N-Desmethylflunitrazepam

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320

0

50

100

18 28 39

51 63

75

85

102

122 134 144 157

170

185

197

205

224

234

252

272

280

298

(imsc-2014) Clonazepam

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320

0

50

100

15

28 39

51 63

75

89 102

111

125

138

151

164

177

190

205

213

234

252

268

280

286

298

314

a

b

c

d

Relative Intensity, % Relative Intensity, %

N

O

N

H

N

O

O

Cl

N

O

N

O

Cl

Si

Si

N

O

N

O

Cl

Si

Si

D

D

D

D

D

m/z

N

O

O

N

O

N

H

F

315 M+·

433

Relative Intensity, % Relative Intensity, %

299 M+·

430 M+·

435 M+·

m/z

m/z

m/z

INTRODUCTION