Gas Chromatography-

Mass Spectrometric

Analysis of Flunitrazepam

Alexandra Uihlein

Presentation Outline1. Flunitrazepam

2. Method of flunitrazepam identification in blood and plasma

3. Method of flunitrazepam identification after UV exposure

4. How does this help forensic scientists?

Flunitrazepam• Common name: Rohypnol (a.k.a. “roofies”)• C16H12FN3O3

• Type of benzodiazepine: classification of drugs used to treat insomnia and anxiety (same family as alprazolam and diazepam)

• Medically used as a sleeping aid and to treat anxiety, muscles spasms, and epileptic seizures

• Common side effects are drowziness, dizziness, slurred speech, breathing problems, and rapid heartbeat

Sampson, et. al.

Rohypnol’s Potential for Abuse• Schedule IV drug, depressant• Commonly used as a date-rape drug• Manufactured as white tablet - tasteless,

colorless, odorless• In 1997, manufactured as green tablet with

blue dye inside – however generic versions may not contain dye

http://fa.oregonstate.edu/publicsafety/date-rape-drugs

http://addictionsearch.com/_media/addictionsearch/stock/290_200/Rohypnol_Flunitrazepam/rohypnol1.png

Rohypnol’s Potential for Abuse• When combined with alcohol, can produce

exaggerated intoxication – sedation, amnesia, loss of motor coordination, weakness, impaired mental judgement, confusion, and slurred speech

• About 10 times as strong as Valium• Also used by addicts to treat negative side

effects of other controlled substances• Not approved by FDA for medical use in US• Smuggled into the US• Used by other countries to treat insomniahttp://wedorecover.com/

addiction/addiction-types/rohypnol.html

Detection of Rohypnol in BloodFlunitrazepam and its major metabolite, 7-amino-flunitrazepam, were analyzed in both whole blood and blood plasma through gas chromatography–mass spectrometry (GC-MS)

1. Two subjects were dosed with 2 mg of flunitrazepam

2. Blood samples were collected from the subjects at times 0, 0.25, 0.5, 1, 2, 4, 8, and 12 hours after dosing

3. Half of the blood samples from the subjects were centrifuged for plasma separation – samples were prepared for analysisFlunitrazepam

7-amino-flunitrazepam

Sampson, et. al.

Sample Preparation

Plasma/Blood

7-amino-FN (1μg/mL)

HPLC-grade water

Concentrated HCl

10M KOH

Chloroform

Volume

1 mL 25 μL 3 mL 1 mL 1.5 mL 6 mL

• 7-amino-FN was added to plasma (or blood). HPLC-grade water was used to dilute mixture – then vortexed briefly.

• HCl was added to mixture, which was then placed in a 100°C oven for 1 hour.

• Sample was cooled to room temperature. KOH was added to sample, which was then vortexed again.

• Chloroform was added to the sample for extraction. Sample was shaken for 2 minutes, then top layer was removed as waste.

• The chloroform layer of the sample was washed with 1 mL of water twice, then poured into a test tube.

• The solvent was evaporated under N2 in a 50°C water bath.

• The sample residue was dissolved in chloroform containing 20 μg/mL 4-pyrrolidinopyridine, and heptafluorobutyric anhydride was added

• The sample was vortexed and allowed to sit at room temperature for 1 hour, followed by the addition of NaOH and carbonate buffer

• The sample was vortexed again for 30 seconds, and the aqueous layer was removed as waste

• One mL of deionized water was added to sample, which was vortexed for 30 seconds more, and the organic layer was extracted and deposited into a GC vial

• The solvent was evaporated under N2 at 50°C • Ethyl acetate was added to the residue to dissolve it

for analysis

Chloroform (with 4-pyrrolidinopyridine)

Heptafluorobutyric anhydride

2M NaOH

1.5 M carbonate buffer (pH 11)

Ethyl Acetate

Volume

0.5 mL 100 μL 0.2 mL 1 mL 50 μL

Detection of Rohypnol in Blood2 μL of each sample were injected into the GC-MS under the following conditions:

GC: Hewlett Packard 5890 model• Oven: Initial 180°C, held for 0.5 minutes, increased to 260°C at 20°C/min, held for 1 minute, increased to 280 °C at 30 °C/min, held for 8 minutes• Carrier Gas: Helium, 36.2 cm/s splitless flow

MS: Hewlett Packard 5970 mass selective detector

Column: DB-5 MS column, 25 m x 0.2 mm, 0.33 μm thickness

• Flunitrazepam is present in significantly lower amount than 7-amino flunitrazepam in whole blood, and completely absent in plasma

• 7-amino-flunitrazepam is more present in plasma than whole blood

• Flunitrazepam was only detectable until 4 hours after dosing, while 7-amino-flunitrazepam was detectable the full 12 hours

ElSohly, et. al.

Exposure to UV IrradiationAnalysis of flunitrazepam under UV conditions may provide insight into how the drug degrades under normal environmental conditions

1. Flunitrazepam (1 mg/mL in methanol) was prepared with HPLC-grade methanol in solutions of 0.10, 0.050, and 0.025 mg/mL in methanol

2. The flunitrazepam solutions were exposed to a UV lamp (100 W, 365 nm) for 0, 10, 20, 30, 40, 50, and 60 minutes, with a distance of 20 cm between the flunitrazepam and the lamp

3. A control using only methanol was prepared and exposed under the same conditions

Exposure to UV Irradiation1-3 μL of each sample were injected into the GC-MS under the following conditions:

GC: Thermal Fisher TRACE GC Ultra 2.2 gas chromatography• Injector: 250°C, split less mode• Carrier gas: Helium, 1 mL/min flow•Oven: Initial 150°C, held for 2 minutes, increased to 225°C at 15°C/min, held for 15 minutes

MS: Thermal Fisher ISQ 1.0 SP4 mass spectrometer

Column: 5.7 m x 0.32 mm x 0.25 μm

Sampson, et. al.

Before UV

After UV

After UV irradiation• 8 lower boiling point fragments: 1.84, 3.23, 4.50,

5.62, 6.63, 7.62, 8.98, and 10.98 • 3 higher boiling point fragments: 14.04, 18.72, and

20.23• Major peak: 13.08

Sampson, et. al.

At GC peak 13.08:• Major signals: 65, 75, 109, 119, 170, 183, 210, 238,

266, 286, 312, and 313• Identified as flunitrazepam by data library• Typical signals for flunitrazepam: 238, 266, 285, 312,

and 313

Sampson, et. al.

Results• Multiple fragments of flunitrazepam due to UV

exposure• Analysis of flunitrazepam over the 60 minute

UV exposure provided two significant GC peaks at times 13.5 and 14.1 on the chromatograph (data not shown)• The peak at 14.1 was identified as flunitrazepam, while the peak at 13.5 was identified as amino-flunitrazepam• The nitro group on flunitrazepam is most likely converted into an amine group by UV irradiationFlunitrazep

amAmino-flunitrazepam

Sampson, et. al.

Usefulness of Research• Flunitrazepam is converted into amino-

flunitrazepam not only by metabolism, but also by UV irradiation

• A reference spectra for the degradation of flunitrazepam due to UV exposure• This will be useful for identifying trace amounts of flunitrazepam in real crime scenes where the drug may have partially degraded

• More research can be conducted on how flunitrazepam degrades over time

http://www.ahoskiepd.com/information/narcotic-information/rohypnol/

Special Thanks To

Dr. Jennifer Firestine-Scanlon

For her education and guidance

Resources• ElSohly M, Feng S, Salamone S, Brenneisen R. GC-MS

Determination of Flunitrazepam and its Major Metabolite in Whole Blood and Plasma. J. Anal. Toxicol. 23: 486-489 (1999).

• "Public Safety - Date Rape Drugs." Office of Finance and Administration. Oregon State University, n.d. Web.

• “Rohypnol." Drugs of Abuse - A DEA Resource Guide. Drug Enforcement Administration, 2015. Web.

• Sampson L, Wilson B, Hou HJM. Gas Chromatography- Mass Spectrometric Analysis of Forensic Drug Flunitrazepam upon Exposure to UV Irradiation. J. Forensic Res. 4: 193 (2013).