Simultaneous Detection of
GHB, Ketamine and
Flunitrazepam Using Hair
Samples
Samantha Olson
CHEM 4101
December 13,2010
The Problem
I was at a bar one night where it was
suspected that a friend of mine was drugged
with a “date rape drug.” She made it home
safely without an assault, but she did not go to
the police until a few days later. I would like to
attempt to qualitatively and quantitatively
detect three date rape drugs in her system.
The Hypothesis
Based on her behavior, I hypothesize that
GHB was placed in her drink, the dosage of
which is between endogenous and lethal
levels.
The Analytes
Since I do not know what was put in the drink, three
major date rape drugs will be analyzed.
O
OH
N
Cl
O2N
O
N
F
HO
NH
O
GHB
flunitrazepam
(Rohypnol)
ketamine
Effects of these drugs include: drowsiness, loss of
consciousness, impaired judgment, impaired motor
skills and the inability to remember events that
occurred while under the influence. This is why they
6
Analyte Complications
•
•
•
•
These drugs are quickly Metabolized.5
Endogenous levels of GHB exist.6
It’s usually a low, one time dosage.5
Drug facilitated sexual assault cases
are often not reported right away. 5
Hair was chosen for sample collection,
because drugs linger in the hair much
longer than blood and urine and
endogenous levels of GHB can be
detected by analyzing several
segments of the hair.5
Techniques Considered
Method
Advantages
Disadvantages
FTIR
Fast Runs, fairly
inexpensive
Need extensive
separation technique,
overlap in simultaneous
measurements, poor
for quantification
MEKC-UVvis
Good LODs : 50pg/mL
- 1.8µg/mL, faster run
times (15 mins), good
resolution
Different pH’s are
preferred for the
different analytes, UV
detection is poor with
GHB
GC-MS
Good LOD (pg-ng),
instruments currently
used in labs, good
separation,
Derivitization must be
done to increase
volatility
More sensitive than
Drugs must be
derivatized ($), and
Method Chosen:
HPLC/MS/MS
• There is less sample preparation
• Quantification and Identification are achieved
simultaneously.
• The reproducibility is good.
• The limit of detection is good.
• These instruments are already used in many labs,
so it’s cost effective.
Qu ickTime™ and a
TIFF (Uncompressed) deco mpressor
are nee ded to see this picture.
Specific HPLC and MS/MS
Instruments
Waters 2795 Alliance HT
HPLC1
• Fairly affordable model
• Autosampler provides
more accurate, faster
injections
• Common brand used
throughout labs
• An Atlantis dC18
column may be
adequate for the drugs
9
•
•
•
•
Micromass Quattro Ultima
MS 7
Can use CID to get
parent/daughter ion
pairs10
Sensitive enough to
detect low concentrations
of this problem’s analytes
Good mass resolution
Uses ESI
Proposed Sample Preparation
• 4-5 Weeks after being drugged, hair samples taken from the
vertex posterior (back of the head)
• Cut hair strands into 2 cm segments and place in separate
containers and decontaminated
• Cut into small pieces and incubate roughly 20 mg overnight in
phosphate buffer (pH 7) in presence of diazepam-d5 (internal
standard)
• Extract with methylene chloride/diethyl ether (90/10 v/v)
Proposed LC/MS/MS Method
• Calibration standards must be prepared and run
• Internal standard is added and a 20 µL sample is injected and
elution is run
• Gradient is started at 5:95 acetonitrile:formic acid and
increased to 95:5 acetonitrile:formic acid after 15 minutes with
eluent flow of 0.2mL/min
• Mass analysis is done on the positive ion mode
Results
Qu ickTime™ and a
TIFF (Uncompressed) decompressor
are need ed to see this picture .
Anticipated Figures of Merit
Flunitrazepa
m
10
LOQ
2 pg/mg
LOD
0.5 pg/mg
8
GHB
Ketamine
0.4 ng/mg
9
0.1ng/mg
0.2 ng/mg
9
0.18 ng/mg
3
3
Precisio
n
Intra-day CV of
3.58 8
CV of 5.2% at
2 ng/mg and
7.4% at 10
ng/mg 9
Intra-day % CV
of 4 8
Accurac
y
Within batch
precision: 12.1
(n=6, 10pg/mg 10
97% for
2ng/mg and
94% for 10
ng/mg 9
% Relative
Accuracy of 7.2
8
Conclusions
• Hair samples were chosen due to the longer
retention of drugs and the ability to determine
endogenous GHB
• HPLC/MS/MS can be used to separate, identify and
quantify all three analytes.
• Instrumentation was chosen for its affordability,
accuracy and its ability to solve the problem.
• Seeing as victims of DFSA don’t always come
forward in the time allotted for traditional sample
analysis (i.e.: urine), this technique would be useful
to prove that date rape drugs were consumed.
References
1. Alliance HPLC High Thoroughput System. Waters Corporation, Oct 2008. Web.
<http://www.waters.com/webassets/cms/library/docs/720000781en.pdf>.
2. Cheng, Pai-Sheng, Chien-Yu Fu, Choung-Huei Lee, Chiareiy Liu, and Chun-Sheng Chien. "GCﾐMS
Quantification of Ketamine, Norketamine, and Dehydronorketamine in Urine Specimens and Comparative
Study Using ELISA as the Preliminary Test Methodology." Journal of Chromatography B 852.1-2 (2007):
443-49. Print.
3. Harun, Norlinda, and Robert A. Anderson. "Analysis of Ketamine and Norketamine in Hair Samples Using
Molecularly Imprinted Solid-phase Extraction (MISPE) and Liquid Chromatography-Tandem Mass
Spectrometry (LC-MS/MS)." Anal Bioanal Chem 396 (2010): 2249-459. Web.
4. Hsin-Yu, Bai. "Characterization and Evaluation of Two-Dimensional Microfluidic Chip-HPLC Coupled to
Tandem Mass Spectrometry for the Quantitative Analysis of 7-aminoflunitrazepam in Human Urine." Analyst
135 (2010): 2737-742. Web.
5. Kintz, Pascal, Marion Villain, and Vincent Cirimele. "Chemical Abuse in the Elderly: Evidence From Hair
Analysis." Ther Drug Monit 30.2 (2008): 207-11.
6. Maxwell, James Carlisle. "Party Drugs: Properties, Prevalence, Patterns and Problems." Substance Use &
Misuse 40 (2005): 1203-240. Print
7. ."Micromass Quattro Ultima Pt Mass Spectrometer Operator's Guide." Waers, 2003. Web.
<http://www.waters.com/webassets/cms/support/docs/quattro_ultima_pt_guide_reva.pdf>.
8. Negrusz, Adam. "Detection of "Date-Rape" Drugs in Hair and Urine, Final Report." (2003). Print.
9.Stout, Phillip A., Kelsie D. Simons, and Sarah Kerrigan. "Quantitative Analysis of Gamma-Hydroxybutyrate at
Endogenous Concentrations in Hair Using Liquid Chromatography Tandem Mass Spectrometry." Journal of
Forensic Sciences 55.2: 531-37. Web.
10.Villain et al, Marion. "Screening Method for Benzodiazepines and Hypnotics in Hair at Pg/mg Level By Liquid
Chromatography-Mass Spectrometry/Mass Spectrometry." Journal of Chromatography B 825 (2005): 72-78.