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Discrimination of sibutramine and its analogues

based on surface-enhanced Raman spectroscopy
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Cite this: RSC Adv., 2015, 5, 5886

and chemometrics: toward the rapid detection of
synthetic anorexic drugs in natural slimming
products†
Hui Mao,ab Meihui Qi,a Yujie Zhou,a Xiaoyan Huang,a Liying Zhang,a Yang Jin,a
Yan Penga and Shuhu Du*a

Received 2nd September 2014
Accepted 11th December 2014
DOI: 10.1039/c4ra09636c
www.rsc.org/advances
Nowadays there is growing concern about the adulteration of synthetic anorexics in natural slimming
products (NSP) due to the increasing use of weight-reducing aids. Here, we report surface-enhanced
Raman spectroscopy (SERS) coupled with chemometrics for rapid discrimination and detection of
sibutramine (SIB) and its analogues (mono-desmethylsibutramine, MDS; di-desmethylsibutramine, DDS)
in konjac pressed candy (KPC), which is a NSP used in the treatment of obesity. It has been
demonstrated that the two characteristic peaks at 1060 cm
1 (in SERS spectrum of SIB) and 1407 cm
1
(in SERS spectrum of MDS) were used to discriminate between SIB, MDS and DDS. Moreover, the two
spectra for KPC with MDS and KPC with DDS only exhibited some slight differences. After the second
derivative transformation, the differences were much more clear, especially for the peak at 1407 cm
1 in
the SERS spectrum of KPC with MDS. Meanwhile, partial least-squares regression was used for
quantitative analysis of the samples. The limit of detection for SIB, MDS and DDS was 5.00  10
8 M,
5.00  10
7 M and 1.00  10
6 M, respectively. Compared with previously reported studies, the method
is simple (no pretreatment), less time-consuming and inexpensive, and can be applied to rapidly screen
SIB and its analogues in NSP.

2010, the European Medicines Agency (EMA) and the Chinese

1 Introduction
Obesity, which directly affects the quality of people's lives, is
one of the most serious public health problems of the 21st
century. The prevalence has increased at an alarming rate,
resulting in the search for a number of weight-reducing aids
including sibutramine (SIB), orlistat, phentermine, rimonabant
and especially natural slimming products (NSP) due to them
having no signicant side effects on the human body. However,
the weight-reducing effect of NSP is not usually obvious in the
short-term. As a result, some manufacturers illegally adulterate
synthetic anorexic drug in NSP in order to promote their efficacy
of weight loss. SIB, as a reuptake inhibitor of 5-hydroxytrypta-
mine and noradrenaline,1–3 which makes people lose weight by
stimulating thermogenesis,4 has been one of the most
commonly adulterated anorexic medicines found in NSP. In
a
School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China. E-mail:
shuhudu@njmu.edu.cn; Fax: +86 25 86868476; Tel: +86 25 86868476
b
Nuclear Medicine Department, Nanjing General Hospital of Nanjing Military
Command, Nanjing, 210002, China
† Electronic supplementary information (ESI) available.
10.1039/c4ra09636c
State Food and Drug Administration (SFDA) recommended the
market withdrawal of SIB owing to an association with
increased cardiovascular events and strokes.5 To avoid the SIB
examination of local authorities related to public health control
and surveillance, mono-desmethylsibutramine (MDS) or di-
desmethylsibutramine (DDS), which has the same function as
SIB,5 has been used as the substitute of SIB adulterated into NSP
by some illegal manufacturers. However, MDS and DDS, as
main desmethyl active metabolites of SIB, can still cause an
adverse reaction. As their chromatographic behaviors are not
similar to that of SIB in the routine detection using high
performance liquid chromatography (HPLC), the two metabo-
lites are easily missed, leading to false-negative judgments.
Without adequate quality control, the adulterated NSP may
cause consumers to suffer from several unpredictable distresses
ranging from headache, vertigo and numbness to serious
cardiovascular effects.6 Therefore, the identication and
measurement of SIB and its analogues illegally adulterated in
numerous brands of NSP available in the market are important
to protect consumers from risks.
See DOI:
Up to date, various conrmatory detection methods for the
screen of SIB and/or its analogues have been reported, such as

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Paper
HPLC,7 ultra-HPLC,8 liquid chromatography-mass spectrometry
(LC-MS),9 gas chromatography-mass spectrometry (GC-MS),10
ion mobility spectrometer (IMS),11 IMS-MS,12
chemiluminescence-molecularly imprinted sol–gel lms
(CL-MIFs),13 ow injection electrochemiluminescence (FI-
ECL),14 capillary electrophoresis,15 infrared spectroscopy,16 and
so on. Due to the structural similarity and the trace amount (0.1
mg per capsule11 or tablet) in pharmaceutical formulations, the
identication of SIB and its analogues usually requires
sophisticated sampling beforehand separation/extraction and
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pre-concentration procedures in above techniques, which are
highly expensive or time-consuming and also cause environ-
mental problems. The method for the rapid identication and
detection of trace SIB or its analogues in NSP still remains a
challenge.
Raman spectroscopy, as a vibrational spectroscopic
method, has been considered as a useful analytical technique
to evaluate food safety and quality.17,18 Although Raman
spectrum can provide spectroscopic ngerprint information
about various chemical and biochemical species19–21 in
complex matrix without sample pretreatment, the signal of
normal Raman spectroscopy is weak, and thus only suitable
for detecting target compounds present in high concentration.
Surface-enhanced Raman spectroscopy (SERS) technique
signicantly enhances the sensitivity of normal Raman spec-
troscopy detection. In the SERS technique, molecules are
absorbed onto the roughened surface of noble metals, which
results in signicant enhancement of the Raman signals by
many orders of magnitude in highly localized optical elds of
nanostructures. In the last decades, both gold (Au) and silver
(Ag) nanoparticles (NPs) are most frequently used nano-
materials for fabrication of SERS-active nanosubstrates.22–25
Although Au has some advantages, i.e., chemical stability and
better biocompatibility, Ag is a much more efficient optical
enhancer, and for the same particle size and shape it yields
enhancement factor 2 or 3 orders of magnitude larger than
that of Au.26 Nowadays, SERS technique has become one of the
most widely pursued spectroscopic tools for rapid discrimi-
nation and detection of various contaminants and adulter-
ants,27,28 including banned additives melamine and its
derivatives,29 banned food dyes Sudan I and its derivatives30
and banned seafood drugs crystal violet and malachite
green.31,32 It is thus expected that the use of simple and inex-
pensive SERS method can overcome the drawbacks of conr-
matory techniques and provide a direct spectroscopic
identication of trace analyte in complicated matrix.
In this work, we report the rapid identication and detection
of trace SIB and its analogues in NSP using SERS technique in
combination with chemometrics. Experimental SERS condi-
tions such as excitation wavelength, concentration of Ag NPs
colloid and type of aggregating agent were rst optimized.
Subsequently, konjac pressed candy (KPC), as one common type
of NSP, was selected as sample, because it could be the major
target of SIB or its analogues monitoring. Finally, multivariate
statistical methods including discriminant analysis (DA) and
partial least-squares (PLS) analysis were used to analyze SERS
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spectral data and develop qualitative and quantitative models
for SIB, MDS and DDS.
2 Experimental
2.1 Chemicals and materials
Sodium citrate (Na3C6H5O7$2H2O, 99.8%), silver nitrate
(AgNO3, 99%), KI, KNO3, KBr, K2CO3, KCl, ammonium
formate (HPLC grade) and formic acid (HPLC grade) were
purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). Sibutramine hydrochloride mono-
hydrate, mono-desmethylsibutramine hydrochloride, di-
desmethylsibutramine hydrochloride, genistein, daidzein,
salidroside, chlorogenic acid, caffeic acid and gallic acid were
purchased from National Institutes for Food and Drug Control
(Beijing, China). All of these reagents were used without
further purication. Konjac pressed candy (KPC), which
mainly contained konjac powders (tablet, 1 g), and the herbal
slimming tea (HST), which mainly contained Green tea,
Lonicerae japonicae os, Cassiae semen, Nelumbinis folium,
Gynostemmatis pentaphylli herba, Crataegi fructus, Sennae
folium and Mel (pack, 5 g), were purchased from a local
drugstore. Ultrapure water (18.2 MU cm) was produced using a
Milli-Q apparatus (Milford, MA, USA).
2.2 Apparatus and analytical methods
The morphologies and structures of NPs were examined by a
JEM-1010 transmission electron microscope (Tokyo, Japan).
The hydrodynamic sizes of NPs were determined using a Mal-
vern Zetasizer Nano-ZS90 particle size analyzer (Malvern
Instrument, UK). UV-vis absorption spectra were obtained by
UV-2501 spectrometer (Tokyo, Japan) in the range from 200 to
800 nm with 2 nm resolution, using 1 cm path length quartz
cuvettes. Raman measurements were conducted with Thermo
Fisher DXR Raman Microscope equipped with a CCD detector
in backscattered conguration using a 10 objective (Madison,
USA), and three types of lasers were used to produce different
excitation wavelengths (532 nm, 633 nm and 780 nm). Liquid
chromatography-mass spectrometry (LC-MS) analysis was per-
formed using an Agilent 6410B triple quad LC-MS/MS (Santa
Clara, USA). Please see the details of LC-MS analysis condition
in ESI.†
2.3 Preparation of Ag NPs
All glassware was thoroughly immersed overnight with freshly
prepared aqua regia (3 : 1 HCl–HNO3) and washed with ultra-
pure water before use. The preparation of Ag NPs was based on a
previous method.33 Briey, 25 mg of silver nitrate was dissolved
in 63 mL of ultrapure water and then heated to boiling under
magnetic stirring. To this solution, 1.25 mL of 2% trisodium
citrate was added and kept boiling for 1 h. Aer cooled to room
temperature, the Ag NPs colloid was obtained and stored at
4.0  C for further use. The concentration of Ag NPs was 0.2 nM,
which was calculated using Beer's law and the extinction coef-
cient (3) (42 nm Ag NPs is 3.7  1010 M
1 cm
1).34
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2.4 Theoretical calculation
The molecular vibrational spectra of the investigated
compounds (including SIB, MDS and DDS) were simulated by
density functional theory (DFT) within Gaussian 09 program
package. On the basis of the molecular geometries optimized by
B3LYP/6-31+G** method, the frequency calculations were
carried out at the same level to validate the minima and to
obtain the vibrational information. Meanwhile, the interaction
between Ag atom and investigated compounds was simulated
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through the basis sets of 6-31+G** and LanL2DZ, which per-
formed to non-metal (C, N and O) and Ag atoms. Based on the
optimized geometries, the Raman spectra calculations were
carried out at the same computational level. The assignments of
calculated vibrational modes were made with the help of Gauss
View 5.0. The calculated wavenumbers of all normal modes
were scaled by a factor of 0.987 to correct systematic errors of
the theoretical methodology.
2.5 SERS measurements of SIB and its analogues
In brief, 5.00 mM SIB solution was prepared and diluted with
ultrapure water to the predetermined concentrations. Then,
10 mL of the analyte solution was added into 90 mL of Ag NPs
colloid in a 1.5 mL centrifuge tube, followed by shaking for 1
min. The mixture was rst sucked into a capillary glass tube,
and then xed onto a glass slide. The Raman spectra were
recorded using 532 nm laser with 10 mW power and 10
objective. The collecting time was 10 s with 2 rounds of accu-
mulations and the pinhole aperture was 25 mm. The experi-
ments were repeated six times for every concentration. MDS and
DDS were also tested using the same method.
2.6 Spectral data analysis
Multivariate statistical tools (DA and PLS) were adopted to
analyze the spectral data with TQ Analyst soware (7.2.0.161
Release, Thermo Fisher Scientic). Through computing the
unknown's distance from each class center in Mahalanobis
distance units, DA classication technique can be used to
determine the class or classes of known materials which are
most similar to an unknown material. The closer each distance
value is to zero, the better is the match. Aer the material was
successfully classied by DA, PLS was applied to adulterated
samples (which only contain one synthetic anorexic per sample)
to obtain a calibration based on the level of adulteration. For
PLS analysis, spectra of 40 samples with ve different concen-
trations of constituent were used. Among these samples, thirty
samples were used for calibration and ten samples were used
for validation. The calibration data were used to create the
method calibration model, while the validation data were used
to validate the model. Evaluation of the models was made
through correlation coefficient (R) and root-mean-square error
of prediction (RMSEP). The higher the R value or the lower the
RMSEP value is, the better predictability the model has. Before
the analysis, spectral data were normalized using a standard
normal variate (SNV) algorithm and mean centered to correct
for instrumental effects on the data. To further discriminate
5888 | RSC Adv., 2015, 5, 5886–5894
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between three structural analogues in spiked sample, second
derivative transformation and smoothing were also applied
when necessary to capture a more signicant variance. The
spectral region used for the DA and PLS was from 1800 to 900
cm
1.
2.7 SERS detections of SIB, MDS and DDS in spiked samples
Briey, SIB with different amounts was rst spiked into one
gram of KPC powders, and then the mixture was dissolved in 10
mL ultrapure water by sonication, statically cooled down and
ltered. 10 mL of ltrate was added into 90 mL of Ag NPs colloid.
The Raman spectra were recorded with the same condition as
the detection of analytes in aqueous solution. Meanwhile,
accuracy of the method was determined by standard addition
technique. Known amounts of reference standard SIB in a range
of low, medium and high concentrations were added to the KPC
powders and analyzed under the above measuring conditions.
The experiments were replicated six times for each concentra-
tion and the accuracy was calculated as the % of analyte
recovered. MDS and DDS was also tested using the same
method, respectively.
3 Results and discussion
3.1 Characterizations of Ag NPs
UV-vis spectroscopy and transmission electron microscopy
(TEM) were applied to characterize the size and morphology of
the synthesized Ag NPs. The maximum absorption of the
measured UV-vis spectrum of Ag NPs colloid provides the
information on the diameter of particles, moreover, its full
width at half-maximum (FWHM) is used to estimate the NPs
dispersion.35 As shown in Fig. S1A,† the maximum UV-vis
absorption for Ag NPs colloid was at 409 nm, and the FWHM
was approximately 90 nm, which indicated that the size
dispersion of Ag NPs was narrow. Meanwhile, TEM image
clearly displayed that the diameter of Ag NPs was 42  9 nm
(Fig. S1B†). These results revealed that the size of Ag NPs was
mainly homogeneous and the shape of the most of NPs was
spherical.
3.2 Optimization of experimental conditions for SERS
The optimum of the experimental conditions for SERS detection
is related to various parameters including excitation wave-
length, aggregating agent, pH value, etc. In order to select the
optimal detection condition, a series of tests were performed in
this study.
Excitation wavelength. It is known that the basic mecha-
nisms of SERS mainly include charge-transfer (CT) mechanism
and electromagnetic mechanism. For the CT mechanism, it is
related to the chemical complexation of the analyte molecules
to the surface of the metallic NPs. In the case of surface
complexation, electronic absorption of the complexed analyte
may shi with respect to the free molecule, and even overlap the
excitation wavelength leading to resonant effect.36 Fig. 1A
showed the SERS spectra of SIB absorbed on Ag NPs for three
excitations (532, 633 and 780 nm). The intensity of peak at 1691
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cm
1 in the spectrum recorded with the 532 nm laser line was
stronger than those obtained by 633 and 780 nm laser. The
surprising differences on the peak intensities should be caused
by the charge-transfer Ag complex with an electronic absorption
in the spectral region of the 532 nm laser. The UV-vis spectrum
of SIB (Fig. S2a†) showed a strong absorption at 223 nm, very far
from the 633 and 780 nm to have any resonant effect. However,
the UV-vis spectrum of the SIB complexed with Ag NPs displayed
a broad shoulder that extended up to 500 nm region
(Fig. S2b†). Thus, the 532 nm laser was in pre-resonance with
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the absorption of the complex, and was used as excitation
source in the following tests.
Aggregating agent. The aggregating agent is crucial for the
application of SERS using metallic NPs. It is usually an elec-
trolyte, and can induce the NPs aggregation by increasing the
ionic concentration inside the metallic colloid and decreasing
the electrostatic barrier. Fig. S3† showed the UV-vis absorption
spectra of Ag NPs colloid blended with different aggregating
agents (the concentration of each aggregating agent is 10 mM).
The absorbance of Ag NPs colloid decreased aer mixed with KI,
KNO3, KBr, K2CO3 and KCl, respectively, and maximum
absorption at 409 nm shied to 415 nm, 415 nm, 414 nm, 407
nm and 406 nm, respectively. From the previous literature, the
evolution of the absorbance of Ag NPs colloid could be related to
the aggregation of colloidal NPs.41 When aggregating agents
containing anions are mixed with Ag NPs colloid, the energy
states on particles' surface are affected by chemical contribu-
tions of the anions and the inter-band transitions between NPs
through anions. The change of particles' surface energy states
caused by additional anions is regarded as energy hybridization
effect on particles' surface.42 For this reason, the maximum
absorption of Ag NPs colloid changed in the presence of addi-
tional anions compared with the absence of additional anions.
The different interactions of Ag NPs with aggregating agents
were also conrmed by TEM and dynamic light scattering
(Fig. S4†).
Fig. 1B displayed a series of SERS spectra of SIB absorbed on
Ag NPs colloid to which the same amount of KI, KNO3, KBr,
K2CO3 and KCl was added, respectively. Through comparison of
the six spectra (the feature peak at 1691 cm
1 was chosen as the
Fig. 1 (A) SERS spectra of 5.00  10
4 M SIB in Ag NPs colloid excited by different excitation wavelengths. (B) Effects of aggregating agents on
SERS intensity of 1.00  10
5 M SIB.
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indicator of particles-aggregating degree), it has been noted that
extra addition of anions can enhance the SERS signal of SIB to a
certain extent. The SERS activation induced by anions can be
ascribed to two effects: one is the aggregation of Ag NPs
producing “hot spots”; the other is modication of the Ag
surface chemistry, resulting from the addition of strongly
binding anion. As clearly presented in Fig. 1B, the Br
, as the
more strongly binding anion, gave the greatest enhancement
among different kinds of anions, while Cl
gave the least
enhancement for SIB. So, KBr was chosen as the optimal
aggregating agent to conduct the following optimization
experiments. Additionally, the stability of Ag NPs colloid with
addition of KBr was also examined, as shown in Fig. S5.†
Clearly, the absorbance of Ag NPs with KBr is very stable and the
Raman intensity of SIB adsorbed on Ag NPs is nearly unchanged
within an hour, which ensures the measurement reliability.
Substrate concentration. In order to maximize the Raman
signal enhancement, Ag NPs colloid at different concentrations
was obtained by the treatment of centrifugation or dilution.
Fig. S6† revealed that the Raman intensity of peak at 1691 cm
1
continuously increased with the Ag NPs concentration in range
of 0.007–0.2 nM. It could be ascribed to the increasing “hot
spots”, which could maximize the localized electric eld and
then greatly amplify Raman signals of SIB in the colloid. The
further increase of substrate concentration led to the decrease
of the Raman peak intensity. It was because that Ag NPs colloid
with high concentration was easily precipitated. Therefore,
optimal concentration of Ag NPs colloid was 0.2 nM.
pH value. When the Ag NPs colloid at different pH values was
used, it was found that considerable changes occurred in the
Raman spectra of SIB. As shown in Fig. S7,† the intensity of
band (at 1691 cm
1) obtained at pH 7.4 was stronger than that
at pH 2.5, but this band nearly vanished at pH 11.5. These
phenomena may be related to variations in molecular proton-
ated and deprotonated forms as pH values under ambient
conditions37–39 or slight differences on adsorption geometries of
target molecule on nano-roughened metallic surface.40 To ach-
ieve maximum SERS intensity, we chose Ag NPs colloid with pH
7.4 as substrate for the detection of SIB. Meanwhile, for the
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Fig. 2 Chemical structures of SIB, MDS and DDS.

other two target analytes (MDS and DDS), similar optimization ring, respectively. In comparison to MDS and DDS, the SIB
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results were also obtained (Fig. S8–11†).
3.3 SERS discrimination of SIB, MDS and DDS
Fig. 2 showed the molecular structures of SIB, MDS and DDS.
The structural difference between three molecules is only in the
amounts of substituent (methyl) of amino-group. To discrimi-
nate between three structural analogues, the Raman spectra of
the three analytes were determined. The observed Raman bands
with their assignment accomplished with the help of theoretical
calculations were listed in Table 1.
Fig. 3A displayed the Raman spectra of three analyte solids.
Their dominant bands appeared at 1595 cm
1, 1100 cm
1 and
629 cm
1, which was assigned to the stretching vibration of C–C
bonds from phenyl ring, deformation vibration of the CH2
groups and out-of-plane deformation vibration of the phenyl
showed the characteristic peak at 742 cm
1, corresponding to
the symmetry and asymmetry stretch vibrations of the N1–C2
bond. A close analysis of spectra of MDS and DDS solids
revealed no dramatic differences except the two weak feature
peaks at 1311 cm
1 (deformation vibration of the C2–N1–C7
bond) and 1390 cm
1 (deformation vibration of the N–H bond
and in-plane deformation vibration of C–H bond from phenyl
ring) for MDS.
Fig. 3B presented the normal Raman spectra of three analyte
solutions. Compared with the Raman spectra of solids (Fig. 3A),
it was observed that all bands of three analytes were too weak to
discriminate between three structural analogues.
Fig. 3C exhibited the SERS spectra of three analytes in Ag NPs
colloid. In comparison to the normal Raman spectra (Fig. 3B),
we found that the intensities of some bands were obviously

Table 1 Raman (experimental and calculated) wavenumbers (in cm
1) of SIB, MDS and DDS with their vibrational assignmenta

Raman

Analyte Solid (cm
1) Calc. (cm
1) SERS (cm
1) Vibrational assignment

SIB 1657 1621 1691 u(C7–N–C8) + n(C–C) (phenyl ring)

1595 1598 1593 n(C–C) (phenyl ring)
1461 1513 — d(CH3) + d(CH2)
1441 1491 1415 n(C–C) (phenyl ring) + d(CH3)
1100 1102 1095 s(CH2)
1061 1051 1060 n(C2–N1–C8)
1009 1003 1000 Phenyl ring breathing
742 741 — n(N1–C2) + d(CH2)
629 628 628 Phenyl ring out-of-plane def. + four-membered ring in-plane def.
MDS — 1685 1694 d(NH) + d(C7–N–H)
1595 1598 1595 n(C–C) (phenyl ring)
1460 1467 — d(CH2) + d(CH3)
1390 1410 1407 d(NH) + d(CH) (phenyl ring)
1311 1325 — d(C2–N1–C7) + n(C4–C5)
1095 1117 1092 s(CH2)
1012 1011 999 Phenyl ring breathing
723 749 726 s(CH2) + s(C–H) (phenyl ring)
631 645 622 Phenyl ring out-of-plane def. + four-membered ring in-plane def.
DDS — 1693 1690 d(NH2)
— 1620 1634 n(C–C) (phenyl ring) + d(C–H) (phenyl ring) + u(NH2)
1593 1598 1599 n(C–C) (phenyl ring)
1470 1470 1466 d(NH2)
1445 1389 1442 s(NH2) + n(C2–N1)
1098 1070 1092 s(CH2)
997 992 999 Phenyl ring breathing
726 735 722 s(C–H) (phenyl ring)
631 644 632 Phenyl ring out-of-plane def. + four-membered ring in-plane def.
a
r, rocking; d, scissoring; u, wagging; s, twisting; n, stretching; def., deformation.

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Fig. 3 The Raman spectra of SIB, MDS and DDS: (A) solid, (B) water
solution and (C) in Ag NPs colloid.
enhanced. The enhancement effects can be explained by two
mechanisms: (1) the electromagnetic mechanism based on the
coupling of the radiation eld with surface plasmons of Ag NPs;
(2) the chemical mechanism based on a change in the molec-
ular polarizability arising from the interaction with the metal
substrate. As compared to MDS and DDS, SIB displayed two
characteristic peaks including 1060 cm
1 (stretching vibration
of C2–N1–C8 bond) and 1415 cm
1 (stretching vibrations of C–C
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Fig. 4 (A) SERS spectra of KPC with SIB, KPC with MDS and KPC with
DDS (2.5 mg g
1), (B) second derivative transformations of above
spectra and (C) Cooman plot for the classification of KPC with SIB, KPC
with MDS and KPC with DDS.
bonds in phenyl ring and CH3 scissoring vibration). By further
inspecting spectra of MDS and DDS, we observed that there was
one obvious feature peak at 1407 cm
1 (deformation vibrations
of the NH bond and CH bonds in phenyl ring) in spectrum of
MDS.
In order to apply SERS spectroscopy to differentiate the three
analytes even in complicated matrix, we have to identify the
feature bands that allow an easy discrimination of SIB and its
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Fig. 5 (A) Schematic drawing for the detection of SIB and its analogues from the KPC by the SERS spectroscopy using Ag NPs as Raman sensor,
and the SERS spectra of KPC spiked with (B) SIB, (C) MDS and (D) DDS by the enhancement of Ag NPs colloid.

analogues. In general, a good discriminant peak will be rela- compared the SERS spectra of KPC (blank), KPC with SIB, KPC
tively intense and isolated from nearby peaks which can with MDS and KPC with DDS (Fig. 4A), and discriminant peaks
possibly interfere. Such peaks can serve as markers to identify of each target analyte in KPC were determined. For KPC with
the presence of each compound in mixtures. Herein, we SIB, the peaks at 1060 and 1415 cm
1 were relatively uncluttered

Table 2 Real detections and recovery tests of SIB, MDS and DDS spiked in KPC, respectively (n ¼ 6)

Detected concentration (mg g
1) Recovery (%)

Sample Spiked concentration (mg g
1) Raman method LC-MS method Raman method RSD (%) LC-MS method RSD (%)

SIB 0 nda nda — — — —

1.25 1.24 1.27 99.2 4.19 102 4.44
2.50 2.47 2.52 98.8 3.40 101 3.50
3.75 3.74 3.69 99.7 2.16 98.4 2.08
MDS 0 nda nda — — — —
1.25 1.22 1.24 97.6 4.60 99.2 4.92
2.50 2.43 2.41 97.2 3.03 96.4 4.68
3.75 3.70 3.72 98.7 2.47 99.2 2.50
DDS 0 nda nda — — — —
1.25 1.24 1.25 99.2 4.73 100 4.15
2.50 2.45 2.44 98.0 3.56 97.6 3.41
3.75 3.71 3.73 98.9 2.55 99.5 2.36
a
Not detected.

5892 | RSC Adv., 2015, 5, 5886–5894 This journal is © The Royal Society of Chemistry 2015

Paper
by nearby peaks, and served as discriminant peaks. For KPC
with MDS and KPC with DDS, the two spectra exhibited some
slight differences. Aer the second derivative transformation
(Fig. 4B), the differences looked much more clear, especially for
the peak at 1407 cm
1 which was used to discriminate between
MDS and DDS. These ndings demonstrated that SERS was able
to differentiate spectral patterns between the three analogues in
KPC samples. To be more intuitive, DA model, as a multivariate
statistical tool, was also introduced based on the spectra of KPC
spiked with SIB (n ¼ 50), KPC spiked with MDS (n ¼ 50) and KPC
Published on 12 December 2014. Downloaded by Carleton University on 12/05/2015 08:23:02.
spiked with DDS (n ¼ 50) without the second derivative trans-
formation. DA accurately classied 100% of all samples either
as the SIB or its two analogues. Results of DA were shown in a
Cooman plot (Fig. 4C), which was constructed by plotting the
Mahalanobis distance between each anorexic drug. Eigen
analysis, one of the diagnostic checks, denes how much vari-
ation of spectral information is explained by the use of principal
components. For this case, eigen analysis revealed that it was
possible to obtain 99.4% of desired information with only six
principal components. The obtained results illustrated that
SERS combined with DA could be used to differentiate the three
analytes intuitively.
3.4 SERS detections of SIB, MDS and DDS in spiked samples
Before the detection of trace SIB, MDS and DDS in KPC, the
detection sensitivity to analytes in water using the SERS method
has been tested by the addition of 10 mL analyte aqueous solu-
tion into 90 mL of Ag NPs colloid. Fig. S12A† showed SERS
spectra of SIB. With the increase of SIB concentration from 5.00
 10
8 M to 5.00  10
6 M, the Raman intensity gradually
increases. The intensity of the strongest peak at 1691 cm
1,
assigned to u(C7–N–C8) + n(C–C) (phenyl ring), was used for the
quantitative evaluation of SIB level and the limit of detection
(LOD) was determined to be 5.00  10
8 M (equal to 16.7 ng
mL
1) from three standard deviations above the background.
Meanwhile, similar tests were also performed for MDS and DDS.
As shown in Fig. S12B and C,† the LOD of MDS and DDS was
5.00  10
7 M (equal to 151 ng mL
1) and 1.00  10
6 M (equal
to 288 ng mL
1), respectively (herein, the intensity of the
stronger peak at 999 cm
1, assigned to phenyl ring breathing,
was used for the quantitative evaluation of MDS and DDS
levels). Subsequently, PLS analysis was applied on spectra of
three target analytes. RMSEP values obtained from the PLS
model of SIB with different latent variables were shown in
Fig. S13A.† The lowest RMSEP value was achieved when 4 latent
variables were used, suggesting that the optimal number of
latent variables to construct a PLS model was 4. The optimal
number for building PLS model was 3 for MDS and 4 for DDS
(Fig. S13B and C†). PLS prediction result for SIB was shown in
Fig. S14A† by plotting calculated SIB concentrations against
actual SIB concentrations (R ¼ 0.993; RMSEP ¼ 0.102 mM). For
MDS and DDS, R value was 0.989 and 0.981, and RMSEP value
was 1.08 mM and 1.22 mM, respectively (Fig. S14B and C†). With
R values all larger than 0.980, results of PLS analysis suggested
that PLS could serve as a reliable method to quantify SIB, MDS
and DDS.
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View Article Online
RSC Advances
In order to verify the usefulness of the proposed method
for the identication and detection of three anorexics in
practical applications, the selectivity of the SERS method for
the analytes was rst measured using the structure analogues
(including genistein, daidzein, salidroside, chlorogenic acid,
caffeic acid and gallic acid) because these aromatic
compounds (from herbs) might affect the detection of target
analytes in real sample analysis. As shown in Fig. S15,† it is
clearly seen that there is the peak at 999 cm
1 in few SERS
spectra of aromatic compounds, but there is not the peak in
the SERS spectra of KPC (blank) (Fig. 4A). The result indicates
that a highly selective SERS strategy is attained with an
impressive specicity for three target analytes. Subsequently,
the analysis of three anorexics in real samples was demon-
strated here by the use of the SERS sensor (Fig. 5). Similar to
three anorexics in water solution, PLS was also applied for
quantitative analysis of three anorexics spiked in KPC
(Fig. S16 and S17†). The relative data of recoveries were listed
in Table 2, which indicated that the obtained results were
highly consistent with those from the LC-MS method.
However, the SERS method does not need to pretreat samples
before assay, and the whole test can be accomplished within
3 min. These conrm that the SERS-based Ag NPs sensor
has a high accuracy and reliability to meet the requirements
of practical application.
4 Conclusion
We have developed a rapid and effective method which can
clearly discriminate between SIB and its analogues adulterated
in KPC based on SERS data, DA and second derivative trans-
formation. Citrate-reduced Ag NPs were used as the SERS
substrate. Optimal excitation wavelength (532 nm), substrate
concentration (0.2 nM), pH value (7.4) and aggregating agent
(KBr) for high Raman enhancement were obtained. The SERS
discrimination of SIB and its analogues was achieved by DA
method, mainly relying on the differences in Raman spectral
window between 1000 and 1600 cm
1. SIB, MDS and DDS was
successfully quantied using PLS model, respectively, and the
LOD for SIB, MDS and DDS was 5.00  10
8 M (equal to 16.7 ng
mL
1), 5.00  10
7 M (equal to 151 ng mL
1) and 1.00  10
6 M
(equal to 288 ng mL
1), respectively. Moreover, the recoveries of
SIB, MDS and DDS in the spiked KPC were in the ranges of 98.8–
99.7%, 97.2–98.7% and 98.0–99.2%, respectively. Compared
with LC-MS method, this new technique was simple, rapid and
inexpensive (each test only needed a droplet of 10 mL sample
and was accomplished within 3 min), and thus can be suitable
for high throughput screening analysis, which is performed
with a motorized sample holder stage together with a 96 wells
plate to make the method automatic and reduce the total
analysis time of batch samples. Although this work is mainly
focused on the SERS analysis of SIB, MDS and DDS in KPC, we
have demonstrated that the SERS technique is generally appli-
cable to detect SIB, MDS and DDS of other NSP such as HST
(Fig. S18–20 and Table S1†). Importantly, the monitoring and
tracking of synthetic anorexic drugs illegally adulterated in NSP
RSC Adv., 2015, 5, 5886–5894 | 5893

RSC Advances
may greatly reduce the possibility of occurrence of negative
effects on public health.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (no. 21275075) and the Specialized Research
Fund for the Doctoral Program of Higher Education of China
(no. 20113234110001).
Published on 12 December 2014. Downloaded by Carleton University on 12/05/2015 08:23:02.
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