Development of a fluorescent sensor for illicit date rape drug GHB

Article abstract of DOI:10.1039/c3cc49603a

The first fluorescent sensor (GHB Orange) for date rape drug GHB was developed. It exhibits the fluorescence quenching property for GHB and allows its detection in various drinks. The interaction mechanism was elucidated as intramolecular charge transfer induced by a hydrogen bond. This discovery will help in solving the drug facilitated sexual assault problems.

Full text of DOI:10.1039/c3cc49603a

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Development of a fluorescent sensor for illicit
date rape drug GHB†
Cite this: Chem. Commun., 2014,
0, 2904
5
a
a
a
ab
Duanting Zhai, Yong Qiao Elton Tan, Wang Xu and Young-Tae Chang*
Received 19th December 2013,
Accepted 20th January 2014
DOI: 10.1039/c3cc49603a
www.rsc.org/chemcomm
The first fluorescent sensor (GHB Orange) for date rape drug GHB GHB is the most commonly used date rape drug in DFSA, likely
was developed. It exhibits the fluorescence quenching property for because it is much more easily available, is cheaper and leaves
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GHB and allows its detection in various drinks. The interaction the body more quickly than other drugs. Therefore, develop-
mechanism was elucidated as intramolecular charge transfer ment of a real time detection method for GBL would be a great
induced by a hydrogen bond. This discovery will help in solving contribution to prevent DFSA.
the drug facilitated sexual assault problems.
Several test kits for detection of GHB have been developed,
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such as ‘‘DrinkSafet’’ cards, ‘‘DrinkSafet’’ coasters, ‘‘Drink
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Drug facilitated sexual assault (DFSA) has been defined as Detectivet’’ and ‘‘drug detection straw’’. However, there has
a sexual assault carried out after the victim has become been no fluorescent sensor reported for GHB detection so far.
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incapacitated due to consumption of alcohol or drugs. These Fluorescent sensors are attractive tools for analytical sensing
drugs are referred to as date rape drugs, including gamma- because of their high sensitivity, fast response time, and
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hydroxybutyric acid (GHB), Rohypnol, Ketamine and Soma.
technical simplicity. The Diversity-Oriented Fluorescence Library
Among them, GHB is a central nervous system depressant, and Approach (DOFLA) has proved its ability in fast and unique
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was used as a general anaesthetic in the 1960s and 1970s.
fluorescence sensor development during the last decade.
Subsequently, GHB was employed for a variety of purposes Through this approach, our group has recently reported the
including the treatment of sleep disorders and depression, and development of the first fluorescent sensor for GBL (gamma-
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the promotion of fat reduction and muscle development, until butyrolactone), the pro-drug of GHB. As a systematic extension
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being banned for sale as a supplement in US by FDA in 1990.
Over the past two decades, it has gained media notoriety as a fluorescent sensor for the illicit date rape drug GHB.
drug allegedly used in instances of drink spiking which leads to A similar high throughput image based screening system was
of our previous work, here we report the development of the first
DFSA. Small amount (o1 g) of GHB acts as a relaxant, causing applied for novel GHB sensor discovery. In our previous report, a
loss of muscle tone and reduced inhibitions. At 1 to 2 g, it slows GBL sensor (Green Date) requires an extraction method to
down the heart rate and respiration. At 2 to 4 g, it interferes eliminate alcohol effects for GBL detection in real drinks, which
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with motor and speech control, and may induce a coma-like is an obstacle for its practical usage. Taking this fact into
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sleep.
GHB takes effect in 15 to 30 minutes, and the effect account, here we set the screening medium as 50% EtOH in
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lasts for 3 to 6 hours. It is only detectable in urine for 6 to water instead of pure water, to simulate the working conditions.
2 hours after ingestion. Due to the fact that GHB is odourless, The sedative dosage of GHB is between 2 and 4 g per ingestion.
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colourless, and slightly salty, it is almost undetectable when Therefore we set the screening concentration of GHB to
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mixed in a drink, thus making it desirable to sexual predators.
10 mg mL , assuming the average volume of a drink to be
between 150 and 200 mL. After screening 5500 dyes generated
from different fluorescent scaffolds, 17 compounds (data not
shown) were selected as primary hits due to their fluorescence
intensity change from the pictures. Secondary screening was
then carried out on a fluorescent microplate reader for a wide
a
Department of Chemistry and MedChem Program, Life Sciences Institude,
National University of Singapore, 3 Science Drive 3, Singapore 117543.
E-mail: chmcyt@nus.edu.sg
b
Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium
(
SBIC), Agency for Science, Technology and Research (A*STAR), #02-02 Helios,
1 Biopolis Way, Biopolis, Singapore 138667
Electronic supplementary information (ESI) available: Detailed synthetic and
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range of concentrations of GHB (i.e., 10, 20, 40 mg mL ) to
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render one best hit compound, named GHB Orange (Fig. 1a).
GHB Orange belongs to our previous reported BODIPY
†
screening procedure, characterization data, additional spectroscopic informa-
tion. See DOI: 10.1039/c3cc49603a
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library BD. It has an absorption and an emission maximum
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Table 1 Concentrations of GHB to cause 1.3-fold of fluorescence
decrease of GHB Orange in different beverages
À1
Beverage (mg mL
)
Water Beer
Red wine
2.3
Vodka
o1
o1
4.7
À1
Beverage (mg mL
)
Cola
Korean soju Apple juice Whiskey
o1 6.1 10.1
5.5
Fig. 1 (a) Structure of GHB Orange. (b) Picture of GHB Orange solution
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(
20 mM in 50% EtOH) with and without GHB (10 mg mL ) taken from the
screening camera box.
Fig. 3 Pictures of beverage samples with and without GHB containing GHB
Orange under irradiation using a hand-held 365 nm lamp. (final concentra-
tions: DMSO 50% by volume, GHB Orange 50 mM, GHB 5 mg mL ).
Fig. 2 Fluorescent spectra of GHB Orange (20 mM) after incubation with
different concentrations of GHB. (inner) Linear correlation of fold change
of fluorescence versus concentration of GHB.
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from this experiment (Table 1). On the other hand, the effi-
at 557 and 574 nm in the testing medium (i.e., 50% ethanol in ciency of GHB Orange as a visual detection kit was also
À1
water) respectively. It exhibited a 2.2-fold fluorescent decrease explored. GHB-free and GHB spiked beverages (10 mg mL
with 10 mg mL
)
À1
GHB in 50% ethanol aqueous solution were mixed with GHB Orange DMSO solution (100 mM) in a 1 : 1
(
Fig. 2), with an obvious fluorescence intensity decrease that ratio, and the fluorescence intensity differences were directly
can be observed by the naked eye (Fig. 1b). The fluorescence observed under the irradiation of a hand-held 365 nm lamp. As
intensity fold change (F
/F) of GHB Orange showed a linear shown in Fig. 3, the fluorescence intensity differences between
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decrease with respect to the concentration of GHB within the GHB-free and GHB-spiked beverages are distinguishable by the
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to 100 mg mL range (Fig. 2 inner).
naked eye. These results illustrated that GHB Orange can
In order to examine the efficiency of GHB Orange as a
convenient GHB detection kit, we tested various real drink
samples spiked with GHB. First, we tried to optimize the
working conditions of GHB Orange. Several solvents which
are miscible with water were tested, including DMSO, methanol,
ethanol, acetone and acetonitrile. They were mixed with water
in a 1 : 1 ratio (v/v) containing different concentrations of GHB,
and their fluorescence responses to GHB Orange were
measured (Fig. S2, ESI†). Among the different solvent systems,
GHB Orange showed the best quenching response in 50%
DMSO aqueous solution with a 5.5-fold fluorescence decrease
À1
at 10 mg mL GHB. Hence, DMSO was selected as the working
solvent for developing a GHB detection kit. Several beverages
representing alcoholic, non-alcoholic, coloured and colourless
drinks were selected for this test. GHB Orange was first
dissolved in DMSO, and then mixed with beverage samples
which were spiked with different concentrations of GHB
(1 : 1 ratio by volume). The concentrations of GHB that caused
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a 1.3-fold fluorescence decrease (which were distinguishable by
Fig. 4 Partial H NMR spectra of GHB Orange (30 mM) upon addition of
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the naked eye) of GHB Orange in the beverages were calculated different equivalents of GHB in DMSO-d and D O (9 : 1).
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enable visual detection of GHB in various drinks. Remarkably, Ministry of Education Academic Research Fund Tier
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this detection can be done through a simple mix-and-see (MOE2010-T2-2-030).
process, which takes less than 30 seconds.
Notes and references
The mechanism of the interaction between GHB Orange and
GHB was further explored. The hydroxyl group in the structure
of GHB Orange suggests that a hydrogen bond may form
between GHB Orange and GHB. To confirm the assumption,
NMR experiments were performed with different GHB:GHB
Orange ratios (Fig. 4). Upon increasing equivalents of GHB,
the proton in GHB Orange shifted up-field, especially for H8
and H9 (0.39 and 0.51 ppm respectively). This result illustrated
the formation of a hydrogen bond between GHB Orange and
GHB. Due to the formation of the hydrogen bond, the electron
intensity of GHB Orange was increased, and photo-induced
electron transfer (PET) effect was enhanced, which quenched
the fluorescence of GHB Orange.
1
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simple mix-and-see process, GHB Orange is capable of detect-
ing the presence of GHB in different kinds of beverages with
explicit intensity change under the irradiation of a hand-held
365 nm lamp. NMR experiments confirmed the formation of a
hydrogen bond between GHB Orange and GHB. This discovery
will improve the protection against DFSA.
This study was supported by an intramural funding
from A*STAR (Agency for Science, Technology and Research,
Singapore) Biomedical Research Council and a Singapore
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2906 | Chem. Commun., 2014, 50, 2904--2906
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