Food Chemistry 229 (2017) 517526

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

A rapid ATR-FTIR spectroscopic method for detection of sibutramine

adulteration in tea and coffee based on hierarchical cluster and principal
component analyses
Nur Cebi, Mustafa Tahsin Yilmaz , Osman Sagdic
_
Yldz Technical University, Chemical and Metallurgical Engineering Faculty, Food Engineering Department, 34210 Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Sibutramine may be illicitly included in herbal slimming foods and supplements marketed as 100% nat-
Received 8 July 2016 ural to enhance weight loss. Considering public health and legal regulations, there is an urgent need for
Received in revised form 14 February 2017 effective, rapid and reliable techniques to detect sibutramine in dietetic herbal foods, teas and dietary
Accepted 15 February 2017
supplements. This research comprehensively explored, for the first time, detection of sibutramine in
Available online 22 February 2017
green tea, green coffee and mixed herbal tea using ATR-FTIR spectroscopic technique combined with
chemometrics. Hierarchical cluster analysis and PCA principle component analysis techniques were
Keywords:
employed in spectral range (27462656 cm 1) for classification and discrimination through Euclidian
Sibutramine
ATR-FTIR
distance and Wards algorithm. Unadulterated and adulterated samples were classified and discriminated
Chemometrics with respect to their sibutramine contents with perfect accuracy without any false prediction. The results
Adulteration suggest that existence of the active substance could be successfully determined at the levels in the range
Slimming tea of 0.37512 mg in totally 1.75 g of green tea, green coffee and mixed herbal tea by using FTIR-ATR tech-
nique combined with chemometrics.
2017 Elsevier Ltd. All rights reserved.

1. Introduction
Obesity is one of the most epidemic and common diseases,
resulting from environmental and genetic factors and has damag-
ing effects on health and quality of life during whole lifetime of
individuals (Chandrasekaran, 2012). Reports of world health orga-
nization have manifested that obesity is rising at alarming rate,
thus risks become more threatening with respect to diseases such
as diabetes, neurological disorders, cardiovascular diseases and
cancer. Considering this situation, preventing obesity and over-
weight increasingly comes into prominence (Al-salafi & Irshad,
2014). Findings from earlier studies demonstrate that regulations
in diets in the way of healthy eating plan and physical exercise
helps weight management and help to prevent obesity (World
Health Organization, 2003). At the same time, people are always
in search of helper foods or dietary supplements to prevent the
obesity (Carvalho et al., 2012).
Recently, herbal alternative slimming intakes and dietary sup-
plements are significantly valued by the people who are trying to
struggle with obesity and weight loss. Public usually tend to prefer
herbal products which are from natural sources such as green tea,
Corresponding author.
E-mail address: mtyilmaz@yildiz.edu.tr (M.T. Yilmaz).
green coffee and mixed herbal tea. These products are proclaimed
as being not harmful since they have been natural as hundred per-
cent. Also, they are marketed as being helpful for efficient and
rapid weight loss due to the natural phytochemicals inside of them
(Carvalho et al., 2012; Popescu & Radu, 2015). In very deed, natural
foods or herbal slimming food supplements affect the human body
slower as compared to synthetic slimming drugs. As a result, sibu-
tramine (sibutramine hydrochloride monohydrate) may be illicitly
included in herbal slimming foods and supplements in order to
ensure claimed effectiveness of products (Liang, Qu, Luo, & Wang,
2006).
Sibutramine is an oral anorexiant and usually present in the
form of the hydrochloride monohydrate salt. Sibutramine is a
centrally-acting serotonin-norepinephrine reuptake inhibitor
(SNRI), structurally related to amphetamines. Sibutramine, a
neuro-transmitter reuptake inhibitor, is used in the management
of obesity and have impacts such as heart attack and strokes on
the human body. For this reason, US Food and Drug Administration
announced that sibutramine was withdrawn from the market in
2010 (Deconinck et al., 2014). Although sibutramine have been
withdrawn from the markets, herbal supplements or foods are
intentionally adulterated with these compounds. Furthermore,
people have access to buy sibutramine and sibutramine containing
products through illegal systems like internet and black market

http://dx.doi.org/10.1016/j.foodchem.2017.02.072
0308-8146/ 2017 Elsevier Ltd. All rights reserved.
518 N. Cebi et al. / Food Chemistry 229 (2017) 517526
(Champagne & Emmel, 2011). Sibutramine may be deliberately
involved in energy drinks, green tea, green coffee and mixed herbal
tea dietetic formulations (Carvalho et al., 2012). This situation
impairs the legal decisions and prohibitions of the countries and
gives birth to quality and safety problems worldwide (Liang
et al., 2006). Furthermore, people have been exploited physically
and morally with adulterated, supposed to be natural products.
In Turkey, Republic of Turkey Ministry of Food, Agriculture and
Livestock regularly reports the adulterated foods and food supple-
ments. Considering recent announcements, form-teas that contain
active ingredients keep crucial place in the reported list. According
to the 2016 January report, sibutramine hydrochloride monohy-
drate was detected in mixed herbal tea and the producer company
was revealed by the ministry. In addition to this, U.S Food and Drug
administration warned the public about a life-threatening green
coffee product that contains sibutramine. Considering these prob-
lems, there is an urgent need for development of effective, rapid
and reliable techniques to detect sibutramine in dietetic herbal
foods, teas and dietary supplements.
In literature, several studies are dedicated to detect sibutramine
in dietary supplements, foods, beverages and pharmaceutical for-
mulations. Most of studies include chromatographic techniques
such as liquid and gas chromatography. GCMS (gas
chromatography-mass spectroscopy) and FTIR (Fourier transform
infrared spectroscopy) techniques were investigated for detection
of sibutramine in herbal slimming food supplements (Popescu &
Radu, 2015). Differential pulse voltammetry based method was
established to detect sibutramine in beverages and pharmeceutical
formulations (Carvalho et al., 2012). Portable ion mobility spec-
trometer was succesfully used in order to detect sibutramine in
dietary supplements (Dunn, Gryniewicz-Ruzicka, Kauffman,
Westenberger, & Buhse, 2011). In great detail, HPLC (high perfor-
mance chromatography) and HPTLC (high performance thin layer
chromatography) densitometry techniques were evaluated for
quantification of sibutramine in natural slimming products
(Ariburnu, Uludag, Yalcinkaya, & Yesilada, 2012). In another study,
HPLC-UV-ESI-MS (HPLC coupled with electrospray ionization mass
spectrometry) chromatographic method was succesfully adopted
to detect sibutramine in natural dietary supplements (Huang,
Xiao, Luo, Chen, & Yao, 2008). As an alternative method, UPLC
(ultra high pressure liquid chromatography) system was succes-
fully used in detection and quantification of hazardous active mat-
ters in adulterated slimming formulations (Rebiere, Guinot, Civade,
Bonnet, & Nicolas, 2012). Multi-step analytical method composed
of color tests, TLC, HPLC-DAD (HPLC coupled with diode array
detector), MS (mass spectroscopy) and NMR (nuclear magnetic res-
onance) was developed for reliable detection of sibutramin in slim-
ming formulations (Csupor et al., 2013). A simple method has been
established for rapid detection of sibutramine by using capillary
electrophoresis (Wang et al., 2016). LC-TQMS (liquid chromatogra-
phy triple quadrupole mass spectrometry) and TOF-MS (time-of-
flight mass spectrometry) were used for detection of sibutramine
in herbal products (Jackson & Attalla, 2010).
These methods are reliable and have been reported to be suc-
cessful; on the other hand, they are generally costly, time consum-
ing, destructive and require high skilled operator. Because of these
reasons, a demand exists for development of rapid, accurate, inex-
pensive and effective techniques for testing of adulteration in food
and food ingredients. FTIR spectroscopy can be used non-
destructively and rapidly to obtain biochemical fingerprints that
provide information about molecular structure and composition
(Sivakesava & Irudayaraj, 2001). This spectroscopic technique has
been used as an effective and successful tool in a wide range of
adulteration problems in food products. Recent studies have
shown that FTIR spectroscopy may have potential in detection of
sibutramine in natural herbal products, foods and dietary supple-
ments. Up to now, a very limited number of studies have investi-
gated to detect sibutramine as an active substance by using FTIR
spectroscopy technique. In one of these studies, FTIR spectroscopy
was used in order to detect adulterants in herbal slimming dietary
supplements (Popescu & Radu, 2015). In another study, detection
of sibutramin in adulterated dietary supplements was accom-
plished with minimum false prediction by using FTIR technique
combined chemometrics (Deconinck et al., 2014). However, in
these studies, there are some limitations in terms of robust, accu-
rate and reliable detection of sibutramine. In the former one, the
study was limited by the absence of any useful and robust chemo-
metric technique, which would also limit the robustness and accu-
racy of the detection. The main weakness of the latter study was
the false predictions obtained by the method; namely, three sam-
ples were misclassified by the preferred k-NN (k-Nearest Neigh-
bors) chemometric method as false positive. This means that,
although their method would not allow adulterated samples to
pass the initial ATR-IR screening, the false positive results would
lead some amount of legal (unadulterated) products to be unde-
servedly confiscated and seized by the inspectors or customs
(Deconinck et al., 2014), which proves that the developed method
based on the chemometrics used was not robust enough to get per-
fect results without any false prediction. In addition, in their study,
no attempt was made to define absorbtion bands in this region and
to assign the vibrational bands with functional groups which are
specific to the sibutramine hydrochloride monohydrate; thus, their
study suffers from chemical insight of the developed FTIR spectro-
scopic technique. Such reasons emerge an urgent need to develop
technique combined with a robust chemometrics based on chem-
ical insight of vibrational spectroscopy.
The primary aim of this paper was to develop a new, rapid,
effective, non-destructive and cost-effective method for detection
of sibutramine hydrochloride monohydrate which is so far the
most common adulterant in dietary supplements intended for
weight loss like green tea, green coffee and mixed herbal tea. Our
research comprehensively explores, for the first time, detection
of sibutramine in herbal dietary supplements by FTIR spectro-
scopic technique combined with robust chemometrics. In this
regard, we have used a combination of FTIR spectroscopy and
chemometrics including hierarchical cluster analysis (HCA) and
principal component analysis (PCA) for detection of sibutramine
in the dietary supplements. Favorably, ATR-IR spectra were
obtained for unadulterated and sibutramine adulterated samples
for test and validation steps. All of the samples could be perfectly
classified without any false prediction in terms of their sibu-
tramine contents by using chemometrics.
2. Materials and methods
2.1. Apparatus, reagents and materials
Bruker Tensor 27 spectrometer (Bremen-Germany) with a KBr
beamsplitter and a DLaTGS detector was employed in this study.
All measurements were performed with a diamond single-
bounce ATR accessory. Instrument control and data acquisition
were accomplished by using OPUS Version 7.2 for Windows from
Bruker Gmbh. Monohydrate sibutramine hydrochloride (molecular
formula: C17H26ClN.HCl
H2O), whose two and three dimensional
chemical structures are shown in Fig. 1(a) and (b) was provided
from U.S. Pharmacopeia, Rockville, USA. In this research, two sets
of samples were used to test and validate the methodology devel-
oped to determine sibutramine adulteration. For preparation of the
test group samples, green tea from two different brands (coded as
GT1 and GT2), mixed herbal tea from two different brands (coded
as MHT1 and MHT2) and green coffee (coded as GC1) were pro-
 N. Cebi et al. / Food Chemistry 229 (2017) 517526 519

Fig. 1. (a) Two dimensional chemical structure of sibutramine hydrochloride monohydrate, (b) three dimensional structure of sibutramine hydrochloride monohydrate. (c)
ATR-FTIR spectrum of sibutramine hydrochloride monohydrate at mid infrared region (4000650 cm 1).

vided from Turkish local supermarkets. For preparation of valida-
tion group samples, green tea (coded as GT3 and GT4), mixed her-
bal tea (coded as MHT3 and MHT4) and green coffee (coded as
GC2) from other different brands were also provided Turkish local
supermarkets. As declared in the label of the products, the mixed
herbal tea samples (MHT1 and MHT2) were composed of senna
leaves, cherry stalks, rosehip fruit, fennel fruit, anise fruit, heather
leaves, St Johns Wort, rosemary, sage, yarrow herb and juniper
fruit. The other mixed herbal tea samples (MHT3 and MHT3) were
in combination of senna leaves, frangula bark, fennel, nettle leaves,
birch bark, juniper fruit, yarrow, rosehip and hawthorn.
2.2. Sample preparation
ated with sibutramine at the levels reported in Table 1, also keep-
ing the tea and coffee quantities at the constant value (1.75 g). In
this study, solid tea and coffee samples were thoroughly mixed
with Sibutramine hydrochloride monohydrate salt in 50-mL glass
beakers at defined levels (Table 1) by using a steel laboratory spat-
ula during 15 min. Afterwards, beakers were tightly covered with
aluminum foil and mixtures were shaken by hand for 5 min.
Hand-mixing operations were implemented during totally twenty
minutes for each sample with the aim of achieving perfectly
homogenous mixtures of adulterated samples. Sibutramine pow-
der was punctiliously dispersed in solid tea and coffee samples in
other words all of the samples were mixed diligently to obtain
homogenous mixture prior to analysis.

In order to prepare the test samples, the samples were adulter-
ated with sibutramine at different levels (Table 1). Tea and coffee
quantities were hold at a constant value (1.75 g) for each sample
while amount of adulteration changed between 0.375 and 12 mg.
In this study, three samples were prepared for each concentration,
so a total of 225 samples were prepared (Table 1). For example,
GT1 is composed of three samples (GT11, GT12, GT13). For method
development, average FTIR spectra of three samples were obtained
thus sample number is mentioned as 75 in the text. Regarding the
preparation of the validation samples, the samples were adulter-
2.3. Infrared spectroscopy measurements
ATR-FTIR spectra of all samples were recorded with a resolution
of 4 cm 1, accumulating 16 scans per spectra. The spectra were
recorded at mid infrared region within range of 4000650 cm 1.
Spectrum acquisition of each sample repeated three times in the
same condition and an average spectrum was obtained. A back-
ground air spectrum was scanned before each measurement using
the same instrumental conditions. Spectra were subtracted against
background air spectrum. All of the samples were directly com-
520 N. Cebi et al. / Food Chemistry 229 (2017) 517526

Table 1
Test and validation samples unadulterated/adulterated with different amounts of sibutramine.

Adulteration amount (mg)

Test samples 0.375 mg 0.43 mg 0.6 mg 0.75 mg 1 mg 1.5 mg 3 mg 4.5 mg 6 mg 7.5 mg 9 mg 12 mg 0 mg
Green Tea (GT1) 2 0.375 0.43 0.6 0.75 1 1.5 3.0 4.5 6.0 7.5 9.0 12.0 0.0
Green Tea (GT2) 0.375 0.43 0.6 0.75 1 1.5 3.0 4.5 6.0 7.5 9.0 12.0 0.0
Mixed Herbal Tea (MHT1) 0.375 0.43 0.6 0.75 1 1.5 3.0 4.5 6.0 7.5 9.0 12.0 0.0
Mixed Herbal Tea (MHT2) 0.375 0.43 0.6 0.75 1 1.5 3.0 4.5 6.0 7.5 9.0 12.0 0.0
Green Coffee (GC1) 0.375 0.43 0.6 0.75 1 1.5 3.0 4.5 6.0 7.5 9.0 12.0 0.0
Validation samples 3 6 9 0
Green Tea (GT3)2 3.0 mg 0 mg
Green Tea (GT4) 9 mg 0 mg
Mixed Herbal Tea (MHT3) 3.0 mg 0 mg
Mixed Herbal Tea (MHT4) 6 mg 0 mg
Green Coffee (GC2) 3.0 mg 0 mg

pressed on ATR crystal prior to measurements. Cleanliness was
obtained by distilled water and ethanol.
2.4. Chemometric analysis
FTIR spectroscopy combined multivariate methods such as
principle component analysis and hierarchical cluster analysis
are used for arranging inspected elements into groups on the basis
of their similarity. Briefly, main topic is building a hierarchy of
clusters and both hierarchical cluster analysis (HCA) and principle
component analysis (PCA) are involved with arrangement of spec-
tral data into inherent groupings based on the resemblances. Che-
mometrics could be used effectively and efficiently to detect
hidden relationships between variables. As chemometrics, HCA
was performed using the software OPUS Version 7.2 (Bruker, Ger-
many). HCA was performed in order to evaluate the spectral differ-
ences between unadulterated and adulterated green tea, green
coffee and mixed herbal tea samples by using their ATR-FTIR spec-
tra as well as to monitor the related clusters and sub-clusters in
which the samples could be scattered. Discrimination and classifi-
cation of samples unadulterated and adulterated with sibutramine
were ensured by using the OPUS Version 7.2 software. In HCA, all
spectra were first derivatized with 9 smoothing points and
vector-normalized. Another chemometrics technique, the PCA
analysis was performed to evaluate whether the unadulterated
and adulterated samples could be discriminated with respect to
their sibutramine content. PCA was performed to monitor the clas-
sification as a graphical display which contains clustering patterns
of unadulterated and adulterated samples. The first derivatized
and vector normalized (9 smoothing points) versions of all spectra
were included in classification model and factorization algorithm
was employed for calculating the spectral distances. Two and
three-dimensional cluster analysis results were plotted by using
identity test method of software OPUS Version 7.2 (Bruker,
Germany). In this work, 27462656 cm 1 spectral range was
determined to perform PCA analysis.
3. Results
3.1. Characterization of FTIR spectrum of sibutramine
The IR spectrum of sibutramine is shown in Fig. 1(c). Spectrum
had significant vibrational bands at 3418, 2963, 2865, 2698, 1491,
1428, 1407, 1370, 1091, 1010, 833 and 822 cm 1. These bands
were associated with the chemical groups of components present
in the chemical structure of sibutramine. The widest and strongest
band was observed with the peak point at 3418 cm 1 and resulted
from the asymmetric and symmetric stretching vibrations of OAH
groups arising from water (Coates, 2000; Pajderska, Chudoba,
Mielcarek, & Wasicki, 2010). Second important region was located
between 3000 and 2500 cm 1. Three bands were observed in this
range at 2963, 2865 and 2698 cm 1. The bands with peak points
at 2963 and 2865 cm 1 corresponded to asymmetric and symmet-
ric stretching vibrations of CH3, CH2 and CH functional groups
(Coates, 2000). The peak observed at 2698 cm 1 resulted from
stretching vibrations of NAH groups (Maluf & Pontarolo, 2010).
In study, the band with peak point at 2698 cm 1 in the 2700
2600 cm 1 spectral range, was considered to be the most impor-
tant spectral characteristic which was further utilized for detection
of sibutramine in the adulterated samples since it is specific to the
hydrochloride salts of tertiary amines (Blachut, Siwinska,
Kobylecka, & Czarnocki, 2006). Tertiary amine hydrochloride salts
had strong absorbtion bands in the 27002330 spectral region
and these bands resulted from vibrations of NH+ stretching and
overtones or combination bands in Fermi resonance. This spectral
range is unique since only several compounds create significant
absorbtion bands in this region (Lin-Wien, Colthup, Fataley, &
Grasselli, 1991). Considering valuable knowledge in literature, it
is possible to mention that, our developed solid method utilized
the unique fingerprinting capabilities of FTIR spectroscopy tech-
nique since the method is based on the spectral features specific
to the monohydrate sibutramine hydrochloride.
As shown in Fig. 1(c), significant peaks were also observed at
1491 cm 1, corresponding to the aromatic C@C aromatic ring
stretching vibrations, at 1428 and 1407 cm 1 related to the NCH3
bending vibrations, at 1370 cm 1 due to isobutyl CH3 bending
vibrations, at 1091 cm 1, corresponding to the p-substituted aro-
matic vibrations, at 1010 cm 1 between 1225 and 950 cm 1 due
to the aromatic CAH in-plane bending vibrations and lastly at
833 cm 1 and 822 cm 1, resulting from aromatic CAH out-of-
plane bending vibrations (Coates, 2000; Blachut et al., 2006;
Maluf & Pontarolo, 2010; Popescu & Radu, 2015).
3.2. Discrimination and clusterization of unadulterated and
adulterated samples
The center thesis of this paper was to develop a method for dis-
crimination of adulterated and unadulterated tea and coffee sam-
ples by using FTIR-ATR technique combined with chemometrics.
Comparison of spectra of all samples in the infrared region at
4000650 cm 1 is shown in Fig. 2 where spectral differences can
be observed clearly. Characteristic vibrational bands of sibu-
tramine were broadly explained in previous section. This section
of study attempts to define significant spectral bands and chemo-
metrics for discrimination and classification of unadulterated and
adulterated samples. As seen in Fig. 2, spectral differences are
observed between samples while sibutramine hydrochloride
monohydrate active substance shows significant spectral distinc-
tion in comparison to adulterated tea and coffee samples in the
spectral bands at 1418 cm 1 arising from OAH stretching vibra-
 N. Cebi et al. / Food Chemistry 229 (2017) 517526
Fig. 2. Overlapped ATR-FTIR spectra of sibutramine and sibutramine adulterated samples at different amounts at mid infrared region (4000650 cm
tions and at 2698 cm 1 related to NAH stretching vibrations. The
present investigation evaluated two spectral regions and it was
seen that both of them were suitable for accurate and favorable
classification of samples. Although the band at 1418 cm 1 due to
asymmetric and symmetric stretching vibrations of water could
be used to classify the samples, this may not be always ideal for
establishing a robust and effective model since OH functional
group can also be originated and interfered from food or dietary
supplements own matrixes in further studies. Therefore, in this
study, this was why the band with a peak point at 2698 cm 1
related to NAH stretching vibrations was preferred to establish
the chemometric method since it is specific to the hydrochloride
salts of tertiary amines (Blachut et al., 2006) in the absorbtion
bands arising from vibrations NH+ stretching and overtones or
combination bands in Fermi resonance (Lin-Wien et al., 1991).
Based on this, the main goal of this research was to establish a
method built on unique spectral features specific to the sibu-
tramine hydrochloride monohydrate, thus we utilized unique fin-
gerprinting capabilities of the IR spectra.
This paper proposes a new method based on hierarchical cluster
analysis and principal component analysis for discrimination of
unadulterated and adulterated samples. Hierarchical cluster analy-
sis (HCA) is an algorithmic approach that aims to construct a hier-
archy of clusters. In HCA, clusters and sub-clusters are visualized
definitely in dendrogram graphs. Known as the minimum variance
method, the Wards method joins at each stage of the cluster pair
whose merger minimizes the increase in the total within-group
error sum of squares, based on the Euclidean distance between
centroids. Homogeneous clusters and a symmetric hierarchy are
tended to be produced with its definition of a cluster center of
gravity which provide a useful way of representing a cluster.
Although it was demonstrated to be good at recovering cluster
structure, it was also reported to be sensitive to outliers and poor
at recovering elongated clusters (Lorr, 1983).
In this regard, this study provides the first comprehensive
assessment of adulteration of sibutramine which was the active
substance in green tea, green coffee, and mixed herbal tea samples
using FTIR technique based on the chemometrics. For this purpose,
hierarchical cluster analysis (HCA) and principle component anal-
521
1
).
ysis (PCA) were successfully applied to process spectral data for
discrimination of unadulterated and adulterated samples. Discrim-
ination and classification of the samples was performed at two
steps: the first step involves classification of the test samples
(Table 1) and the second step includes classification of all samples;
namely, the validation samples (Table 1) along with the test sam-
ples in terms of their sibutramine content. As the first step, HCA for
discrimination of unadulterated and adulterated test samples is
presented in Fig. 3(A). One can conclude from the dendrogram that
unadulterated and adulterated test samples were clearly distin-
guished from each other with respect to their sibutramine
hydrochloride monohydrate content. In accordance with the HCA
dendrogram, two well-separated clusters were observed with high
heterogeneity value. All of the unadulterated test samples were
heaped together in one cluster at the left side of the HCA dendro-
gram in Fig. 3(A) while adulterated test samples were clustered at
the right side, which were numbered as 1 and 2 on the dendro-
gram, respectively. In conclusion, the obtained HCA dendrogram
revealed that the unadulterated test samples could be readily dis-
tinguished from the adulterated test samples without any false
prediction. Furthermore, principle component analysis (PCA) was
performed for all test samples and three dimensional (3-D) PCA
plot is presented in Fig. 3(B). PCA results also showed that success-
ful and explicit discrimination of unadulterated and adulterated
test samples were provided with no false prediction. In the same
way, all of the spectra were scattered into two main clusters corre-
sponding to unadulterated and adulterated test samples. The
observed results from two different chemometrics classification
techniques (HCA and PCA) were quite compatible and verify each
other without any fault or error.
At the second step, the validation samples along with the test
samples were also subjected to HCA and PCA analysis. All of the
unadulterated test and validation samples were aggregated
together in the first cluster at the left side of the HCA dendrogram,
which was numbered as 1 in Fig. 4(A). The unadulterated valida-
tion samples are circled and marked with white color on the den-
drogram and these were favorably located between unadulterated
test samples at the left side of the dendrogram. Regarding the adul-
terated samples, they were scattered together in the second cluster
522 N. Cebi et al. / Food Chemistry 229 (2017) 517526

Fig. 3. (A) Dendrogram of HCA (Wards Algorithm) of test samples unadulterated and adulterated with different amounts of sibutramine (refer to Table 1). (B) 3D (three
dimensional) PCA map of these samples.

at the right side of the HCA dendrogram, which was numbered as 2
in Fig. 4(A). Similarly, the adulterated validation samples are cir-
cled and marked with white color on the dendrogram and these
were favorably located between adulterated test samples at the
right side of the dendrogram. As a result, two main clusters for test
and validation samples were also observed and illustrated with
number as 1 and 2 on the dendrogram revealing that the results
from test samples were also validated with those from validation
samples. In addition, the PCA was performed for test and validation
samples and 3-D PCA plot is presented in Fig. 4(B). HCA results
show that successful and explicit discrimination of unadulterated
and adulterated test and validation samples were provided with
no false prediction and with high heterogeneity around 12.
Furthermore diagnosis results of the chemometric analysis;
HCA and PCA are presented in Table 2. As seen in the table, two
diagnosis results were obtained, the first one was obtained from
the HCA and PCA analysis of totally 35 samples which were
unadulterated and adulterated test samples. Second diagnosis
results were obtained from totally 45 samples which were con-
sisted of 35 unadulterated and adulterated test samples along with
10 ten unadulterated and adulterated validation samples. When
the results were evaluated, it was clearly seen that two different
 N. Cebi et al. / Food Chemistry 229 (2017) 517526 523

Fig. 4. (A) Dendrogram of HCA (Wards Algorithm) of test and validation (VAL) samples unadulterated and adulterated with different amounts of sibutramine (refer to
Table 1). (B) 3D (three dimensional) PCA map of these samples.

classes were determined by the chemometric analysis (HCA and
PCA) in both diagnosis cases. Satisfactorily, classification of all
samples with respect to their sibutramine content was verified
with one hundred percent accuracy without any disarrangements
or false prediction. Finally, one can conclude from these results
that the established FTIR-ATR method based on chemometric
model would work perfectly for discrimination of unadulterated
samples from adulterated ones. According to the literature, sibu-
tramine containing samples include concentrations between 3
and 20 mg per dosage unit (Deconinck et al., 2014). This study pri-
marily used 312 mg sibutramine/1.75 g tea concentration for
method development. For this study, a total of forty-five samples
were used as mentioned above. Furthermore method was further
optimized and minimum sibutramine concentration that can be
detected using developed method was determined. The lowest
sibutramine concentration was 0.375 mg/1.75 g for tea and coffee
samples in this study. For further optimization, a total of 65 sam-
ples (test samples in Table 1) were used. HCA results are presented
in Supplementary File. Hierarchical cluster analysis (HCA) provides
better understanding for evaluation of relation between clusters.
As seen on dendrograms in Figs. 3 and 4, perfect discrimination
of adulterated and unadulterated samples was obtained when
sibutramine adulteration kept between 3 mg and 12 mg. Discrimi-
nation power of the method related to the sibutramine content is
clearly presented in Supplementary File. Fig. S1 (see Supplemen-
tary Information) presents HCA dendrogram when minimum adul-
teration amount is 0.75 mg sibutramine/1.75 g sample and perfect
discrimination was obtained with two well separated adulterated
and unadulterated sample group. Unadulterated samples are on
the right arm of dendrogram with number 1. Fig. S2 (see Supple-
mentary Information) presents HCA dendrogram when minimum
adulteration amount is 0.43 mg sibutramine/1.75 g. It can be seen
524 N. Cebi et al. / Food Chemistry 229 (2017) 517526

Table 2
Diagnosis results of chemometric analysis (Hierarchical cluster analysis and principle component analysis).

Diagnosis number 1. Class 2. Class

9 9
Diagnosis 1 (for test samples) Data preprocessing: First derivative + Vector normalization GC1 > > GC1-3 mg >
>
> >
Wards algorithm, Frequency Ranges = 27462656 cm 1 Stabdart (Euclidian Distance) GT1 > = GC1-9 mg >
>
>
MHT2-12 mg >
unadulterated >
MHT1 >
>
> test samples MHT2-3 mg >
GT2 > >
>
>
>
>
;
MHT2 MHT1-12 mg >>
>
>
>
GT2-12 mg > >
>
>
MHT1-6 mg > >
>
>
MHT1-9 mg > >
>
>
GT1-7:5 mg >>
>
>
GT1-12 mg > >
>
>
>
GT2-9 mg >
>
>
>
GT1-9 mg >
>
>
>
GC1-6 mg >
>
>
>
GC1-12 mg > >
>
=
GC1-4:5 mg adulterated test
GC1-7:5 mg >> samples
>
GT1-4:5 mg >>
>
>
MHT1-7:5 mg >
>
>
>
>
GT1-6 mg >
>
>
MHT2-6 mg > >
>
>
MHT2-9 mg > >
>
>
MHT1-4:5 mg >
>
>
>
>
>
GT1-3 mg >
>
>
>
GT2-6 mg >
>
>
GT2-7:5 mg >>
>
>
MHT1-3 mg > >
>
>
>
GT2-3 mg >
>
>
>
GT2-4:5 mg >>
>
MHT2-4:5 mg >
>
>
;
MHT2-7:5 mg
9 9
Diagnosis 2 (for test and validation samples) Data preprocessing: First derivative + Vector GC1 >> GC1-3 mg >
>
> >
normalization Wards algorithm, Frequency Ranges = 27462656 cm 1 Standart GT1 >= GC1-9 mg >
>
>
MHT2-12 mg >
unadulterated >
(Euclidian Distance) MHT1 >
>
> test samples MHT2-3 mg >
GT2 >>
>
>
>
>
;
MHT2 MHT1-12 mg >>
>
>
9 >
GT4-VALIDATION > GT2-12 mg > >
>
> >
GT3-VALIDATION >
>
= unadulterated MHT1-6 mg > >
>
>
MHT3-VALIDATION validation MHT1-9 mg > >
>
>
GC2 VALIDATION >
>
>
> samples GT1-7:5 mg >>
>
; >
>
MHT4-VALIDATION GT1-12 mg > >
>
>
GT2-9 mg >
>
>
>
GT1-9 mg >
>
>
>
GC1-6 mg >
>
>
>
GC1-12 mg > >
>
=
GC1-4:5 mg adulterated test
GC1-7:5 mg >> samples
>
>
GT1-4:5 mg >>
>
MHT1-7:5 mg >
>
>
>
>
GT1-6 mg >
>
>
>
MHT2-6 mg > >
>
MHT2-9 mg > >
>
>
MHT1-4:5 mg >
>
>
>
>
>
GT1-3 mg >
>
>
>
GT2-6 mg >
>
>
>
GT2-7:5 mg >>
>
MHT1-3 mg > >
>
>
>
GT2-3 mg >
>
>
GT2-4:5 mg >>
>
>
MHT2-4:5 mg >
>
>
;
MHT2-7:5 mg
9
MHT3-VALIDATION >>
>
GT4-VALIDATION > = adulterated
GC2-VALIDATION validation
>
MHT4-VALIDATION >> samples
>
;
GT3-VALIDATION

that unadulterated samples were discriminated clearly but dis-
crimination power of method is lower than first one. Fig. S3 (see
Supplementary Information) presents HCA dendrogram when min-
imum adulteration amount is 0.375 mg sibutramine/1.75 g. Obvi-
ously, unadulterated samples are discriminated from other
samples but heterogeneity value is lower and adulterated samples
are seemed closely related with unadulterated ones. When, lower
than 0.375 mg sibutramine concentrations were tested using
method, it was seen that unadulterated samples cannot be dis-
criminated from adulterated ones. As a result, detection limit of
the method was as low as 0.375 mg sibutramine/1.75 g.
4. Discussion
The results of this study show that ATR-FTIR spectroscopy tech-
nique combined with the chemometric techniques such as hierar-
chical cluster analysis and principle component analysis had high
 N. Cebi et al. / Food Chemistry 229 (2017) 517526
potential and capability for detection of sibutramine hydrochloride
monohydrate in green tea, green coffee and mixed herbal tea.
Results have shown that there was a strong compatibility between
HCA and PCA findings. When we compare two chemometric clus-
tering technique (HCA, PCA), it is possible to mention that HCA
dendrogram provided a deeper insight into cluster analysis since
it clearly illustrated the diversity or contiguity between clusters
and each integral part in a dendrogram. Accordingly, Ward
(1963) expressed that hierarchical grouping procedure is based
on greatest amount of information, thus not limited to classifica-
tion problems and finds applications such as genetic background
based taxonomy in building of plants and animals, organizing of
materials, storage of documents in libraries (Ward, 1963). In the
present research, spectral range (27462656 cm 1) was used to
build up the dendrogram through Euclidian distance and the
Wards algorithm. Several reports have shown that Wards algo-
rithm was successfully used for evaluation of crucial issues and
favorably, accurate and significant classification results were
obtained in these studies by using Wards algorithm (Cebi, Durak,
Toker, Sagdic, & Arici, 2016; Gok, Severcan, Goormaghtigh,
Kandemir, & Severcan, 2015; Helm, Labischinski, Schallehn, &
Naumann, 1991). Several attempts have been made to detect sibu-
tramine hydrochloride monohydrate active substance in dietary
supplements and pharmaceutical formulations. Popescu and
Radu (2015) evaluated detection of sibutramine in herbal slimming
food supplements by using FTIR spectroscopic technique and gas
chromatography technique and showed that sibutramine could
be detected in dietary supplements using the absorbtion bands at
821 cm 1, 1011 cm 1, 1370 cm 1 and 1428 cm 1. Although
Popescu and Radu (2015) accomplished to detect sibutramine in
one of ten test samples and validated the findings with GC tech-
nique; nevertheless, they could ensure detection of sibutramine
by comparing FTIR spectra of samples and the study was limited
by the absence of any useful and robust chemometric technique,
which would also limit the robustness and accuracy of the detec-
tion. This limitation means that their findings need to be inter-
preted cautiously. Deconinck et al. (2014) found that ATR-IR
technique combined with k-Nearest Neighbours (k-NN) chemo-
metrics can be used with minimum false prediction for detection
of sibutramine in dietary supplements (Deconinck et al., 2014).
However, the main weakness of this study was the false predic-
tions obtained by the method; that is to say, three samples were
misclassified by the preferred k-NN chemometric method as false
positive. Accordingly, the authors themselves recognized that
model has to be rendered more robust. Although Deconinck et al.
(2014) successfully utilized the fingerprint spectral range between
2000 and 650 cm 1 for sibutramine detection and classification;
nevertheless, no attempt was made to define absorbtion bands in
this region and to assign the vibrational bands with functional
groups which are specific to the sibutramine hydrochloride mono-
hydrate; thus, their study suffers from chemical insight of the
developed FTIR spectroscopic technique. Another source of uncer-
tainty was associated with samples which were analyzed in their
study. As the authors noted, capsules, gel capsules, powders, pow-
ders for fruit and beverages and instant coffee were employed in
the study. However, it is impossible to understand whether their
method would also be applicable to detect sibutramine in other
dietary supplement products like green tea, green coffee and
mixed herbal tea since the sample diversity and composition were
not defined clearly in their study.
5. Conclusions
Our study provides the first comprehensive assessment of sibu-
tramine hydrochloride monohydrate detection in the dietary sup-
plements; e.g. green tea, green coffee and mixed herbal teas. In
525
this study, unadulterated and adulterated tea and coffee samples
could be perfectly discriminated with respect to their sibutramine
contents. The spectral range 27462656 cm 1 was used to gener-
ate the dendrogram through Euclidian distance and the Wards
algorithm. Therefore, one of the strengths of this study is that
the developed method is based on a significant spectral region
(27462656 cm 1) since the absorbtion band at 2698 cm 1 in this
region resulted from NAH stretching vibrations of hydrochloride
salts of tertiary amines which is specific to the sibutramine
hydrochloride monohydrate (Blachut et al., 2006; Lin-Wien et al.,
1991).
Findings highlight the potential usefulness of FTIR-ATR tech-
nique combined with chemometrics for sibutramine detection in
herbal slimming teas. This research has shed a contemporary light
on the contentious issue of active matter adulteration in slimming
teas. In this study, discrimination and classification of unadulter-
ated and adulterated samples were successfully performed by
FTIR-ATR technique without any sample preparation. Discrimina-
tion of all samples in relation with their sibutramine content was
accomplished at 100% accuracy. One of the strengths of this study
is that method works perfectly without any false prediction when
the sibutramine adulteration is implemented between 0.375 and
12 mg in totally 1.75 g of green tea, green coffee and mixed herbal
tea. The current data highlight the robustness of the method since
0.375 mg sibutramine could be detected in complex herbal matrix
of the mixed tea samples which contained 10 different types of
ingredients from leaf, fruit and wood sources. Finally, it can be con-
cluded that the developed FTIR-ATR technique is cost effective,
rapid, easy to operate, non-destructive and can be mentioned as
green analytical technique since no solvents and reagents have
been used during the study.
Conflict of interest
The authors declare no competing financial interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
02.072.
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