GHB Detection Using Visual Methods
GHB Detection Using Visual Methods
GHB Detection Using Visual Methods
FIGURE 2 Supramolecular GHB detection paradigm. Images of a plate in the absence of analyte taken under white light or monochromatic UV
(355 nm) light. Left: original image; right: digitalized image. Two views of CB[7] capsule are shown on the top right. Adapted with permission from
Baumes et al. (2010). Copyright 2000 Wiley-VCH.
showed a fluorescent quenching response in the presence of GHB In this work, the interaction mechanism between GHB
(Figure 3(B)). With addition of GHB, the intrinsic bright orange Orange and GHB was explored by nuclear magnetic resonance
fluorescent color could be quenched dramatically to a dark state. (NMR) spectroscopy, which showed that protons in GHB Orange
Proper optimization of the sensing event was also conducted by shifted up-field upon increasing equivalents of GHB. This indi-
the researchers and the working condition was confirmed to be cated the formation of a hydrogen bond between GHB Orange and
50% dimethyl sulfoxide (DMSO) aqueous solution with a 5.5- GHB, which enhanced photo-induced electron transfer effect of
fold fluorescence decrease at 10 mg/ml GHB, which is the nor- the fluorophore and quenched its fluorescence.
mal GHB spiking amount. More importantly, GHB Orange was A protocol of this sensor is listed below.
further applied to test GHB in a large variety of beverages rep-
resenting alcoholic, nonalcoholic, colored, and colorless drinks.
The detection process was optimized as a simple mix-and-see Materials:
procedure. Explicit intensity change was observed under the irra- GHB Orange DMSO solution (100 μM)
diation of a handheld 365 nm lamp (Figure 3(D)). This is a clear Protocol:
sign of application possibility because 365 nm UV light is readily 1. Mix the GHB Orange solution with GHB analyte
available in most of the bars where DFSA takes place. Thus one solution in a 1:1 ratio by volume.
plausible usage of the sensor was to develop it into nail polish 2. Visualize the mixture with a 365 nm UV lamp or green
or similar styles of carriable stuff that one can readily apply to laser.
drink testing.
532 PART | II Club Drugs
FO/ F
F F 4 3 mg/mL
8000
y = 0.063x + 1 5 mg/mL
DMSO,ΦF 0.86 7000 2
R2 = 0.976
Br λabs max 564 nm 0 10 mg/mL
6000
λflu max 587 nm 0 20 40 60 80 100
HO GHB Orange 5000 25 mg/mL
GHB Concentration / mg/mL
4000 50 mg/mL
(B) without with 3000 100 mg/mL
GHB GHB 2000
1000
0
560 580 600 620 640
(D) Wavelength / nm
water beer red wine vodca cola Korean soju apple juice whiskey
FIGURE 3 The sensing paradigm of GHB Orange and fluorescence responses. (A) Structure of GHB Orange. (B) Picture of GHB Orange solution
with and without GHB taken from the screening camera box. (C) Fluorescent spectra of GHB Orange after incubation with different concentrations of
GHB (inner). Linear correlation of fold change of fluorescence versus concentration of GHB. (D) Pictures of beverage samples with (right) and without
(left) GHB containing GHB Orange under irradiation using a handheld 365 nm lamp. Adapted with permission from Zhai et al. (2014). Copyright 2014
Royal Society of Chemistry.
COMMERCIAL KITS FOR THE DETECTION matrix, high concentration of the drugs required, and extensive
time required for ketamine analysis (Meyers & Almirall, 2004).
OF GHB
Studies also showed that this product performed much better in
There is an increasing public awareness and attention to DFSA. the laboratory than it did in the untrained hands of consumers
Fear of DFSA has generated a growing market for products that in the field. In fact, there is a possibility that consumers would
claim to be able to reduce potential victims’ vulnerability. These misinterpret a result and record a false-negative outcome (Quest
products allow customers to do in vitro field test for beverages & Horsley, 2007).
suspected of being drugged.
EZ Test Kits
Drink Safe Coaster EZ Test Kits is a company founded in the United Kingdom,
Drink Safe Technology (http://www.drinksafetech.com/) is the which sells test kits specifically for GHB (http://www.eztestkits.
best-known company launching products detecting GHB. Their com/en/ez-testing-kits/ghb-drug-testing-kit). The test kit uses
product, Drink Safe Coaster (http://store.drinksafetech.com/ a chemical reagent absorbed in silica gel that is held inside a
categories/), is an inexpensive color change test kit marketed glass ampoule. During the test, consumers were instructed to
internationally for customs to detect potentially incapacitating crack open the ampoule and drip in a small amount of the sam-
concentrations of GHB (complexed with either sodium or potas- ple. They then put the plastic lid on and shook it well to allow
sium ion) and another date rape drug, ketamine. Until now, mil- full reaction between the reagent and GHB. A color change took
lions of such coasters have been sold, which indicates the huge place if GHB was present in the sample. The customers could
market size. In the test, customers were directed to place one thus compare the changed color with the color chart provided
drop of beverage onto both spots of one test area, rub each spot on the instructions within the package. This product, however,
gently, and allow it to dry. If dark blue showed in either spot, has shown several disadvantages including complicated handling
it indicated detection of a possible date rape drug. However, process and a merely presumptive test result, as claimed by the
some beverages are not recommended for testing, including inventors themselves.
milk products, very acidic beverages, oily liquors, or any drinks
containing quinine. Research was conducted to study the effec-
tiveness of Drink Safe Coaster, which revealed that although
Scott Kit
Drink Safe Coaster was able to detect the presence of GHB and Scott Company has introduced a chemical colorimetric test for
ketamine, there were a series of limitations. Examples ranged GHB (http://www.scottcompany.com/∼shop/ghb-test/259375/).
from hindrance of the reaction due to complicated beverage During the test, a cotton bud is soaked in the GHB sample and
Assay Sensor for GHB Chapter | 49 533
then placed in a small vial containing their chemical reagents. Date, thus reducing the static quenching effect of Green Date and
Should the drink contain GHB, the color changes from yellow restore the fluorescence (Figure 4(C)).
to brown, which is derived from a ferric chloride reagent. How- A protocol of this sensor is listed below.
ever, this test lacks sensitivity as analysis of a series of GHB
amounts using Scott Kit demonstrates that concentration as high
as 300 mM GHB only gives a weak positive test, yet this amount Materials:
of GHB is enough to cause death. Normal GHB amounts within Green Date aqueous solution (50 μM), dichloromethane.
a cup of drink only give negative result with Scott Kit (Parsons, Protocol:
Harris, & Bravo, 2004). 1. Add 2 ml of dichloromethane to 2 ml GHB analyte
solution.
2. Shake the mixture well.
A FLUORESCENT SENSOR FOR THE 3. Wait until two layers separate and collect the downside
PRODRUG OF GHB–GBL organic layer.
4. Dry the organic layer under reduced pressure.
GBL is a pharmacologically inactive compound. However, it is 5. After drying, add 100 μl Green Date solution and mix
easily metabolized to GHB in blood stream and stomach in the well.
presence of peripheral lactonases. Therefore, GHB and GBL 6. Visualize with green laser.
have similar psychopharmacological effects after ingestion. Due
to its wide usage and easy accessibility as an industrial solvent
and a chemical reagent, sexual predators have turned to GBL
as a result of the much more stringent regulations on GHB. CONCLUSION
Compared with GHB, GBL has higher bioavailability due to the
There is no doubt that the involvement of GHB and some other
fact that it is more lipophilic and can be absorbed through oral
date rape drugs in sexual assaults is becoming a serious social
administration more rapidly (Palatini et al., 1993). In view of
problem. Since their first introduction in 1960, GHB’s fate has
its adverse effects as a date rape drug, Zhai et al. (2013) devel-
been changed from a medicine that heals patients to a notori-
oped the first fluorescent sensor for GBL. Similar to the GHB
ous date rape drug that brings nothing more than pain and loss.
sensor, the researchers screened thousands of fluorescent dyes
Although the legal status of GHB has become harsher than before,
generated from a large variety of fluorescent scaffolds. The
it is still a popular date rape drug for sexual predators due to the
high-throughput screening followed an image-based process
easy availability of its precursors. Therefore, the necessity to
with the pictures of the fluorescent probes taken before and after
detect GHB in the body fluids of presumed victims and test kits
addition of GBL (Figure 4(D)). Researchers could then compare
that could conduct multisample field tests prior to ingestion has
the observed fluorescence intensities of the images and identify
dramatically increased. Over the past 20 years significant efforts
the probes that exhibit clear differences. With such a method,
have been made to develop useful tools for the practical and rapid
one compound named Green Date (Figure 4(A)) was selected
detection of GHB. However, the number of such tools is still lim-
as the final GBL fluorescent sensor. Upon excitation using
ited whereas their properties in terms of efficiency and selectivity
530 nm green light, Green Date emits an orange fluorescence
are not satisfying. We hope with this discussion of currently avail-
when interacting with GBL. Because the GBL detection limit of
able GHB detection methods, new sensing tools could be designed
Green Date (3 mg/ml) in water is above the allowed amount in
with improved methodology and performances. It should be noted
drinks (Figure 4(B)), an extraction method that can help remove
that such sensing tools cannot only help to prevent DFSA but will
the interfering components and preconcentrate GBL was devel-
also assist in understanding GHB-related neural activities.
oped by the researchers in order to construct a visual detection
kit of GBL. As a brief introduction, a certain amount of drink
samples was extracted with same volume of dichloromethane. APPLICATIONS TO OTHER ADDICTIONS
The organic layer was collected, dried, and resuspended in a
AND SUBSTANCE MISUSE
small portion of Green Date aqueous solution (50 μM) prepared
beforehand. After mixing, the fluorescence could be readily GHB and GBL are key CNS depressants that exist in almost all liv-
visualized with a green laser pointer. If samples do not contain ing bodies but their neural functions still remain blurred. Unravel-
GBL, green light can pass through the clear sample solution and ing these mysteries could help to understand the most fundamental
customers observe a green color; when the drink samples con- aspects of brain activity. On the other hand, optical methods pro-
tain GBL, orange fluorescence will be clearly seen and caution vide a feasible and convenient way to detect these species not only
has to be taken. within the range of cells and tissues but also in vivo. Thus optical
The mechanism of the interaction between GBL and Green detection of GHB and GBL should enlighten the biological studies
Date was further explored by dynamic light scattering and 19F of these neurodepressants. Furthermore, among the various neuro-
NMR experiments. It was proved that Green Date molecules logical species, GHB and GBL are especially difficult to design
stacked together and formed aggregates in aqueous solution sensors due to their simplified structure and lack of response sites.
to minimize contact with water, and hence their fluorescence However, with the help of a diversity-oriented fluorescent library
is diminished (aggregation caused quenching). With increas- approach, for the first time, researchers could successfully develop
ing amounts of GBL which is relatively more hydrophobic than fluorescent sensors for these species. It should encourage and
water molecules, they could enter the stacked aggregates of Green inspire the practitioners who are exploring this field.
534 PART | II Club Drugs
FIGURE 4 Fluorescence detection of GBL using Green Date. (A) Structure of Green Date. (B) Fluorescent spectra of Green Date after incubation
with different concentrations of GBL (inner). Linear correlation of fold change in fluorescence versus concentration of GBL. (C) Schematic explanation
of the proposed mechanism of interaction between Green Date and GBL (star: Green Date molecule; black ball: GBL molecule). (D) Schematic work
flow of screening platform in GBL sensor development. Adapted with permission from Zhai et al. (2013). Copyright 2013, Royal Society of Chemistry.
l Optical detection, especially fluorescence sensing of GHB, is Mamelak, M. (1989). Gammahydroxybutyrate: an endogenous regulator
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l Green Date and GHB Orange are both applicable to various Meyers, J. E., & Almirall, J. R. (2004). A study of the effectiveness of
drinks. The next step would be optimizing these sensors and commercially available drink test coasters for the detection of “date
developing them into product version. rape” drugs in beverages. Journal of Analytical Toxicology, 28(8),
685–688.
Nemeth, Z., Kun, B., & Demetrovics, Z. (2010). The involvement of
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