Ng Song Peng, Matthew

Chan Xu Rui, Isaac

Pan Yanqi, Quince

1. LITERATURE REVIEW
2014 saw the worst obesity rates ever recorded in history, with over 2.1 billion people
suffering from obesity, a stark contrast from a mere 875 million in 1980 [1]. This means
approximately 1 in 3 people are obese. Obesity can potentially cause diabetes, high blood
pressure, cardiovascular disease and even cancer, and obesity is one of the leading causes of
death in the world. The most common method of preventing obesity is through regular
exercise and eating in moderation [2]. However, some might find it extremely difficult to lose
a significant amount of weight through exercise and resort to consuming weight loss
products. This have led to a variety of weight-loss products. Examples include NutriFirst
CUTS!, BPI Sports B4 and Natrol AcaiBerry Diet [3]. While some are medically certified
safe, other weight loss products may contain unlisted drugs or adulterants. These adulterants
are added into these weight loss products to enhance their effectiveness and thus improve
sales. One common adulterant is sibutramine. Sibutramine regulates chemicals in the brain
that reduces hunger or craving for food, hence reducing obesity. However, sibutramine also
increases the risk of cardiovascular diseases such as heart attacks, cardiac arrhythmias,
seizures, jaundice and in rare cases, death [4]. As reported by the US Food and Drug
Administration (FDA), just this year, over 15 weight loss products have been tainted by this
drug [5], including Zero Xtreme Capsules [6], Akttive Capsules [7] and Slim Forte Slimming
Capsules [8]. Therefore it is necessary to detect sibutramine in tainted weight loss products
effectively and efficiently so as to prevent accidental consumption of sibutramine among
obese patients.
With reference to Figure 1 [9], Sibutramine is a tertiary amine, and according to He, et al.,
nitrogen atoms in amines have high affinity to AuNP [10]. The lone pair of electrons in the
amine group allows the Sibutramine molecule to datively bond to a gold atom in the AuNP,
forming a coordination complex. This increases the size of the nanoparticles, which decreases
the frequency of light absorbed by electrons on the surface of the nanoparticle, thus changing
the colour of the AuNP colloid from red to violet. This will allow for colorimetric detection
of sibutramine in tainted weight loss products. Therefore the objective of this research is to
design a suitable detection method using AuNP for detecting sibutramine in tainted weight
loss products.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

2. MATERIALS AND METHODS

2.0 Overall hypothesis
Sibutramine molecules within the weight loss product will bind to AuNP, resulting in a
visible colorimetric change in the suspension.
2.1 Synthesis of AuNP
2.1.1 Materials
HAuCl43H2O (99.999%), Na3C6H5O7 (99.0%), NaCl, ethanol (96%), isopropanol (99.5%),
tetrahydrofuran anhydrous (99.0%), hexane (99.8%), dichloromethane (99.8%) and
sodium sulfate anhydrous (99.0%), from Sigma-Aldrich, DSC from Guardian Singapore,
and Sibutramine hydrochloride monohydrate (>98.0%) from Tokyo Chemical Industry Co.
Ltd. were used as received.
2.1.2 Synthesis of AuNP
The synthesis of AuNP was adapted from the procedure outlined by McFarland, et al. [11].
150 ml of 1.0 mM HAuCl43H2O solution was poured into a round bottom flask and refluxed
at high heat. With rapid stirring using the magnetic stir bar, 15 ml of 38.8 mM Na3C6H5O7
solution was quickly poured into the flask through its side neck. After ten minutes, the flask
containing AuNP was then taken off heat and cooled in an ice bath.
2.2 Plotting a calibration curve of the absorbance of AuNP colloid at = 519.5 nm
against the concentration of Sibutramine using water as a solvent
2.2.1 Preparation of Sibutramine solutions
25 ml aqueous solutions of Sibutramine concentrations ranging from 10 mg/l to 60 mg/l, in
10 mg/l intervals, were prepared. 3 glass vials each containing 5 ml of the above
concentrations were prepared.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

2.2.2 Preparation of AuNP control

2 ml of AuNP was added to an empty glass container, followed by 5 ml of deionised water.
The glass container was then vortexed at 2500 rpm for 3 s, before a UV-Vis
Spectrophotometer was used to determine the wavelength at which maximum absorbance
occurs. The wavelength was at =519.5 nm.
2.2.3 Plotting of calibration curve
2 ml of AuNP was added to one of the glass vials from subsection 2.2.1. The glass vial was
vortexed at 2500 rpm for 3 s. 1 min after the addition of AuNP, the mixture in the glass vial
was analysed using a UV-Vis Spectrophotometer to measure its absorbance at =519.5 nm.
The process was then repeated for all the other glass vials. The absorbance readings obtained
were subtracted from the absorbance of the control at =519.5 nm to obtain the change in
absorbance. The average of the three measurements were taken to obtain the average change
in absorbance for a single concentration of sibutramine. The values were plotted on a graph
of change in absorbance at =519.5 nm against concentration of sibutramine, as seen in
Figure 4.
2.3 Determining a suitable solvent that can separate electrolytes from Detox Slim
Cleanse for effective detection of Sibutramine
2.3.1 Rationale
DSC, a popular weight loss product, was used for subsequent tests. DSC has been tested by
the Health Sciences Authority of Singapore to contain no Sibutramine. However, the
presence of strong electrolytes in DSC will cause AuNP to aggregate, decreasing the
absorbance of AuNP.
2.3.2 Test for electrical conductivity of solvents after addition of DSC
A list of 4 potential solvents, namely de-ionised water, isopropanol, ethanol and
tetrahydrofuran (THF), was created. An ideal solvent is able to dissolve sibutramine readily
while unable to allow electrolytes present in DSC to dissolve. Electrolytes interfere with the
detection process by causing AuNP to aggregate. One DSC capsule was dissolved into 25 ml
of each solvent. The suspension was then vacuum-filtered and the filtrate was collected.
Using a conductivity meter, the conductivity of the solution was measured to determine the

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

concentration of electrolytes present. THF had the lowest value of 0.61, indicating that it has
the lowest conductivity among all the solvents. Thus THF was selected to dissolve DSC so
that a minimal amount of electrolytes will be present in the solution.
2.4 Plotting a calibration curve of the absorbance of AuNP colloid at = 525 nm against
the concentration of Sibutramine using THF as a solvent
2.4.1 Preparations of Sibutramine solutions
The procedure described in subsection 2.2.1 was repeated using THF as a solvent instead of
deionised water.
2.4.2 Preparation of AuNP control with THF as a solvent
2 ml of AuNP was added to an empty glass vial, followed by 5 ml of THF. The glass vial was
then vortexed at 2500 rpm for 3 s. A UV-Vis Spectrophotometer was used to determine the
wavelength at which maximum absorbance occurs. Deionised water and THF was mixed in
the ratio of 2:5 to serve as our blank. The maximum absorbance of the control was at =525
nm.
2.4.3 Plotting of calibration curve
The procedure described in subsection 2.2.3 was repeated using the glass vials prepared in
subsection 2.4.1. The absorbances were measured at 525 nm instead of 519.5 nm, as stated in
subsection 2.4.2. Refer to Figure 5 for the graph.
2.5 Determining whether DSC will affect the detection process of Sibutramine
2.5.1 Spiking DSC with Sibutramine
25 ml of 40 mg/l Sibutramine solution was prepared the same way as described in subsection
2.4.1. 1 capsule of DSC was then dissolved into the Sibutramine solution, and the mixture
was stirred vigorously for 1 minute. The resultant suspension was then vacuum filtered and
the filtrate was collected. 3 glass vials each containing 5 ml of the filtrate were then prepared.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

2.5.2 Testing if DSC will affect the detection process of Sibutramine

For each of the 3 glass vials from subsection 2.5.1, the same procedure was repeated, as
mentioned in subsection 2.4.3. The average of the absorbance readings was then compared
with Figure 4 at 40 mg/l Sibutramine concentration.
2.6 Phase transfer of AuNP into hexane
2.6.1 Rationale
As stated in section 3.3, despite the low dielectric constant of THF, electrolytes from DSC
are still affecting the detection process of Sibutramine. Thus, an even more nonpolar solvent
such as dichloromethane (DCM) has to be used in order to completely separate the
electrolytes from DSC. Unfortunately, DCM is immiscible in water. Thus the aqueous AuNP
needs to be phase transferred into a solvent that is miscible with DCM.
2.6.2 Phase transfer of AuNP into hexane
This procedure was adapted from Yang's method of phase transferring AuNP [12]. 25 ml of
saturated hexadecylamine solution in 96% ethanol was added to an equal amount of aqueous
AuNP. The mixture was stirred vigorously for 3 minutes. 12.5 ml of hexane was added to the
mixture and stirred for another 2 minutes. The mixture was allowed to settle into two layers
and the hexane layer containing the organic AuNP was collected using a separatory funnel,
before being dried with anhydrous sodium sulfate.
3. RESULTS AND DISCUSSION
3.1 Validation of AuNP
3.1.1 Confirmatory tests for the presence of aqueous AuNP
To confirm that aqueous AuNP has been successfully synthesised, 2 ml of AuNP was added
into a glass vial labelled A, followed by 5 ml of water. Another 2 ml of AuNP was added
into another glass vial labelled B, followed by 5 ml of saturated NaCl solution. The AuNP
in glass vial A retained its characteristic red colour, while the AuNP in glass vial B
immediately turned from red to violet. This proves that AuNP has been successfully
synthesised.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

The AuNP in glass vial A was then transferred into a plastic cuvette and scanned using the
Shimadzu UV-1800 UV-Vis Spectrophotometer to determine the wavelength at which
maximum absorbance occurs. A single absorbance peak at 519.5 nm was observed, which
closely matches the ideal wavelength of 520 nm, as stated in the procedure outlined by
McFarland et al. [11]
3.1.2 SEM images of AuNP
Refer to figures 2 and 3 for SEM images of the synthesised AuNP. The average AuNP size is
14.5 nm.
3.2 Calibration curves of the absorbance of AuNP colloid against the concentration of
Sibutramine using water/THF as solvents
Figure 4 shows the relationship between the concentration of Sibutramine and the absorbance
of AuNP colloid at = 519.5 nm. The graph shows a strong linear trend with an R2 value of
0.997.
Figure 5 shows the relationship between the concentration of Sibutramine and the absorbance
of AuNP colloid at = 519.5 nm. The graph shows a strong linear trend with an R2 value of
0.981. Unfortunately the large error bars suggests that the results are inconsistent and not
reliable.
3.3 Determining whether DSC will affect the detection process of Sibutramine
The difference in absorbance reading obtained from section 2.5 was -0.064. This is vastly
different from the 0.635 reading obtained in Figure 4 that corresponds to a Sibutramine
concentration of 40 mg/l. This shows that DSC has significantly affected the detection
process of Sibutramine, and that THF is not a suitable solvent to separate the electrolytes
from DSC.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

3.4 Phase transfer of AuNP into hexane

The confirmatory tests for the presence of aqueous AuNP, as described in 3.1.1, was repeated
to test the presence of organic AuNP, using hexane instead of water as the solvent, and 1 g/L
Sibutramine solution in DCM instead of saturated NaCl solution in water. A peak absorbance
of 523 nm was obtained, which was very close to the peak of the original AuNP at 519.5 nm.
Furthermore, TEM images has shown reasonably dispersed AuNP, as seen in figure 6. This
shows that AuNP has been successfully phase transferred into hexane. However, when
Sibutramine solution in DCM was added into AuNP, a white precipitate likely to be excess
hexadecylamine was formed and the colloid turned colourless. Therefore a calibration curve
using organic AuNP could not be plotted.
4. CONCLUSION
In conclusion, AuNP can be used to detect and quantify Sibutramine in tainted weight loss
products. However, more experimental research has to be done in order to to separate the
chemicals present in weight loss products so that the detection process of Sibutramine is not
affected.
Analytical tests will be conducted in order to confirm the identity of the unknown white
precipitate to be hexadecylamine, before calculating the stoichiometric amount of
hexadecylamine needed for the phase transfer of AuNP.

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

REFERENCES
[1]

British Broadcasting Corporation. (2014). Overweight total tops 2.1bn globally.

Retrieved July 13, 2015, from http://www.bbc.com/news/health-27586365

[2]

World Health Organisation. (2015). Obesity and overweight. Retrieved July 13, 2015,

from http://www.who.int/mediacentre/factsheets/fs311/en/
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Nutrifirst. (2015). Thermogenics / Fat Loss. Retrieved July 13, 2015, from

http://www.nutrifirst.net/view_products.asp?catid=48&categoryname=Thermogenics%20/%
20Fat%20Loss&gclid=CPq8r5qT_8MCFQURjgod4Z4AFw
[4]

Drugs.com. (n.d.). Sibutramine Side Effects. Retrieved July 13, 2015, from

http://www.drugs.com/sfx/sibutramine-side-effects.html
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Capsules

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contain

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[8]

US Food and Drug Administration. (2015). Public Notification: Slim Forte Slimming

Capsule

contains

hidden

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ingredient.

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Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

http://www.fda.gov/Drugs/ResourcesForYou/Consumers/BuyingUsingMedicineSafely/Medi
cationHealthFraud/ucm445213.htm
[9]

Wikipedia (2015). Sibutramine's 2D molecular structure in ACS style. Retrieved 26

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He, S., Liu, D. B., Wang, Z., Cai, K. Y., Jiang, X. Y. (2011). Utilization of

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(2004). Journal of Chemical Education, 81(4), 544A. DOI: 10.1021/ed081p544A. Retrieved

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[12] Yang, J. (2007). Phase Transfer of Monometallic Nanoparticles. 94-112. Retrieved
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Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

Appendix A: Figures

Figure 1: Structure of Sibutramine

Figures 2 and 3: SEM images of AuNP

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

Figure 4: Calibration curve of the absorbance of AuNP colloid at = 519.5 nm against the
concentration of Sibutramine using deionised water as a solvent

Figure 5: Calibration curve of the absorbance of AuNP colloid at = 525 nm against the
concentration of Sibutramine using THF as a solvent

Ng Song Peng, Matthew

Chan Xu Rui, Isaac
Pan Yanqi, Quince

Figure 6: TEM image of organic AuNP