Niclosamide quantification in methyl-β-cyclodextrin after derivatization to aminoniclosamide

Article abstract of DOI:10.1007/s10847-011-9945-2

A fluorescence derivatization method for the quantification of the anthelmintic drug niclosamide in organized media (methyl-β-cyclodextrin) is described. The derivatization reaction is based on a 3 h reduction reaction of the nitro group in niclosamide to an amino group by hydrogenation catalyzed by Pd/C in methanol:ethyl acetate (1:3). The aminoniclosamide exhibits a maximum of fluorescence at 431 nm (excitation at 280 nm) that is markedly enhanced when methyl-β-cyclodextrin is present. The methodology was assessed by the analysis of pure and dosage forms. The method showed a linear range between 0 and 2200 μg L^-1 with a limit of detection of 45.7 nL and a precision (relative standard deviation) of 4.1% in niclosamide. The effect of various interferents, such as niclosamide, fructose and 2-cyano-6- metoxybenzothiazole, in several tolerance ratios, was investigated respected to aminoniclosamide.

Full text of DOI:10.1007/s10847-011-9945-2

J Incl Phenom Macrocycl Chem (2012) 72:89–94
DOI 10.1007/s10847-011-9945-2
ORIGINAL ARTICLE
Niclosamide quantification in methyl-b-cyclodextrin
after derivatization to aminoniclosamide
Manuel Algarra
Francisco G. S a´ nchez
•
Bruno B. Campos
•
•
Jos e´ E. Rodriguez-Borges
•
Juan M. L o´ pez-Romero
•
Joaquim C. G. Esteves da Silva
Received: 14 January 2011 / Accepted: 1 March 2011 / Published online: 23 March 2011
Ó Springer Science+Business Media B.V. 2011
Abstract A fluorescence derivatization method for the
quantification of the anthelmintic drug niclosamide in
organized media (methyl-b-cyclodextrin) is described. The
derivatization reaction is based on a 3 h reduction reaction
of the nitro group in niclosamide to an amino group by
hydrogenation catalyzed by Pd/C in methanol:ethyl acetate
Keywords Niclosamide ꢀ Aminoniclosamide ꢀ Inclusion
complex ꢀ Cyclodextrins
Introduction
0
0
(
1:3). The aminoniclosamide exhibits a maximum of
Niclosamide (2 ,5-Dichloro-4 -nitrosalicylanilide, Fig. 1)
belongs to the family of medicines called anthelmintics
drugs which are effective against most tapeworms,
including the beef tapeworm, the dwarf tapeworm and the
dog tapeworm [1]. Commercially known as bayluscide or
niclocide in USA and Canada or clonitralid and Bayer 73 in
Germany. Its mode of action against tapeworm species is to
uncouple oxidative phosphorylation and it blocks the glu-
cose uptake and inhibits respiration in cestodes and the
anaerobic ATP production [2], widely used as a molluscide
in tropical regions for control of freshwater snails [3] and
also used as an intestinal taeniacide in humans [4].
fluorescence at 431 nm (excitation at 280 nm) that is
markedly enhanced when methyl-b-cyclodextrin is present.
The methodology was assessed by the analysis of pure and
dosage forms. The method showed a linear range between
-
1
0
and 2200 lg L with a limit of detection of 45.7 nL
and a precision (relative standard deviation) of 4.1% in
niclosamide. The effect of various interferents, such as
niclosamide, fructose and 2-cyano-6-metoxybenzothiazole,
in several tolerance ratios, was investigated respected to
aminoniclosamide.
In the literature there are references to several analytical
techniques used in the quantification of niclosamide at
trace levels. Methods for analysis of niclosamide in water
include spectroscopy [5–8], liquid chromatography [9–12]
and voltammetric determination [13, 14]. Residues of
niclosamide have been analyzed by gas chromatography
but this procedure requires indirect analysis of the hydro-
lysis product 2-chloro-4-nitroaniline [10, 15]. A derivati-
zation method based on the reduction of the nitro group in
niclosamide to the amino group using zinc powder in e-
tanolic acid media, followed by a colour development
reaction with p-benzoquinone has been proposed [6].
Niclosamide is not fluorescent but it can be analyzed by
reduction to aminoniclosamide which exhibits a green
fluorescence at 439 nm which can be enhanced using its
ability to form inclusion complexes with cyclodextrins.
Because of the organizing ability of cyclodextrin media,
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-011-9945-2) contains supplementary
material, which is available to authorized users.
M. Algarra (&) ꢀ B. B. Campos
Centro de Geologia do Porto, Faculdade de Ci eˆ ncias,
Universidade do Porto, Rua do Campo Alegre 687,
4169-007 Porto, Portugal
e-mail: magonzal@fc.up.pt
F. G. S a´ nchez ꢀ J. M. L o´ pez-Romero
Departamento de Qu ´ı mica Anal ´ı tica and Qu ´ı mica Org a´ nica,
Facultad de Ciencias, Universidad de M a´ laga, Campus de
Teatinos s/n 29071, M a´ laga, Spain
J. E. Rodriguez-Borges ꢀ J. C. G. Esteves da Silva
Centro de Investiga c¸ a˜ o em Qu ´ı mica (CIQ-UP), Faculdade de
Ci eˆ ncias da Universidade do Porto, Rua do Campo Alegre 687,
4
169-007 Porto, Portugal
123

9
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J Incl Phenom Macrocycl Chem (2012) 72:89–94
O
N
Derivatization reaction
NH2
O-
O
O
H2:Pd/C
Cl
Cl
N
H
N
H
Niclosamide, is one of the halogenated salicylanilides,
which are highly hydrophobic having a very low aqueous
solubility [25] and showing aromatic behaviour, do not
fluoresce as a result of the strong electron-withdrawing
nature of the nitro substituent, for that was planned its
determination in aqueous media by reduction.
Methanol : Ethyl Actetate
(1 : 3)
Cl
Cl
OH
OH
Niclosamide
Aminoniclosamide
Fig. 1 Formation of aminoniclosamide
Aminoniclosamide (Fig. 1) were obtained by the selec-
tive catalytic reduction of niclosamide, under hydrogen
atmosphere (4 bars) in methanol:ethyl acetate (1:3) solu-
tion, using Pd/C (10%) for 3 h at room temperature. Then,
the yellowish solution obtained was filtered through a celite
layer and the solvent was removed on rotary evaporator
yielding the corresponding pure aminoniclosamide (99.9%)
as yellowish solid. The synthesised aminoniclosamide were
luminescent phenomena are enhanced, the molecules in the
internal cavity are isolated from the surrounding environ-
ment and their excited states shielded from extinction
processes. Cyclodextrins (CD) show selectivity depending
on the relative size of the cavity and guest molecules
[
16–18]. The determination of a variety of organic com-
pounds has been developed by exploiting the a-CD
19, 20], b-CD and methyl-b-CD [21–23]. Only a research
1
13
checked by H-NMR and C-NMR spectrometry, included
distortionless enhancement by polarization transfer (DEPT)
and heteronuclear single quantum correlation (HSQC)
analysis (NMR Spectra are presented in the Supporting
information). Mass spectrometry analysis of the product in
[
work has been found to use cyclodextrins and dendrimers
in combination to solve the problem of solubilisation of
niclosamide in aqueous media, but no analytical determi-
nation was done [11].
-
1
a methanol solution resulted in a m/z peak at 296 g mol
which demonstrates the formation of aminoniclosamide.
In the present work a new procedure for the derivati-
zation reduction reaction, based on a catalytic step for the
quantitative transformation of niclosamide to aminonicl-
osamide, followed by the formation of an inclusion com-
plex of aminoniclosamide with a cyclodextrin (CD) is
described. Six CD derivatives were studied in order to
obtain the one that provokes the highest fluorescence
enhancement, which was methyl-b-cyclodextrin. The
inclusion complex was analysed by fluorescence emission
measurements.
0
0
Aminoniclosamide (2 ,5-Dichloro-4 -
aminosalicylanilide) NMR characterization
1
0
H-NMR (DMSO-d , 50 °C): d:6.96 (m, 1H, H-5 ), 7.04 (d,
6
0
0
1
1
7
1
1
H, J = 7.4 Hz, H-3 ), 7.36-7.39 (m, 1H, H-6 ), 7.38 (d,
H, J = 8.8 Hz, exc. D O, NH H ), 7.44 (m, 1H, H-4),
2
a b
.80 (d, 1H, J = 8.8 Hz, exc. D O, NH H ), 7.79-7.82 (m,
a b
2
H, H-3), 8.02 (dd, 1H, J = 7.9 Hz, J = 1.6 Hz, H-6),
1
2
0.52 (br.s, 1H, exc. D O, CONH), 11.55 (br.s, 1H, exc.
2
1
3
0
D O, OH) C-NMR (DMSO-d , 50 8C) d:118.65 (C5 ),
1
2
6
Material and methods
0
0
18.87 (C1), 120.37 (C3 ), 123.33 (C3 ? C1 ), 124.41
0
0
(
C6 ? C5), 130.00 (C2 ), 130.58 (C6), 135.00 (C4),
0
Materials
1
38.65 (C4 ), 159.84 (C2), 168.01 (C=O). Supporting
information contains the NMR spectra of aminoniclosa-
mide and NMR spectra and data of niclosamide.
Niclosamide (99%), a-cyclodextrin (a-CD), b-cyclodextrin
(b-CD), methyl-b-cyclodextrin (with a degree of substitution
of 1.6–2.0 per glucose units, Mb-CD), hidroxypropyl-b-
cyclodextrin (Hp-b-CD), sulphated-b-cyclodextrin (Sb-CD),
c-cyclodextrin (c-CD) and 2-cyano-6-metoxybenzothiazole
Preparation and fluorescence measurements
of the complex of aminoniclosamide with cyclodextrins
(
2Cy6M) were purchased from Sigma-Aldrich Qu ´ı mica SA
Spain). Fructose was obtained from Panreac Qu ´ı mica (Bar-
(
To aliquots of aminoniclosamide in deionised water were
-
2
celona, Spain).Allreagentswereofhighpuritygradeandused
as received.
added increasing volumes of 10 M of the different CDs
solutions and deionised water up to a final volume of 5 mL.
These samples were sonicated for 10 min and their fluo-
rescence spectra were recorded at 431 nm with excitation
at 280 nm at room temperature. Calibration set of six
samples was prepared by transferring appropriate aliquots
of stock solution of pure aminoniclosamide, being the final
-
1
Stock solutions of aminoniclosamide (50 mg L ) were
prepared in deionised water and diluted as required. Ami-
noniclosamide is light sensitive thus stock and standard [24]
solutions were stored in light tight containers. Cyclodextrins
-
2
solutions (10 M) were prepared in deionised water with
-
1
resistivity higher than 4 MX/cm.
concentration between 0 and 2000 lg L .
1
23

J Incl Phenom Macrocycl Chem (2012) 72:89–94
Apparatus
91
M
-
2
analyse the interaction of aminoniclosamide with 10
of a-CD, b-CD, Mb-CD, Hb-CD, Sb-CD and c-CD solu-
tions. Figure 2 shows the fluorescence spectra of aqueous
solution of aminoniclosamide in the presence of CDs.
The analysis of Fig. 2 shows a general fluorescence
intensity enhancement when CD is presence compared
when only in water but it is highest for Mb-CD. The
enhancement of fluorescence is observed because cyclo-
dextrins offer a protective, more constrained microenvi-
ronment to an electronically excited fluorophore and so the
resulting fluorescence is enhanced. The maximum fluo-
rescence obtained for the inclusion complex formed
between the fluorophore and a CD is a consequence of the
aqueous medium surrounding it.
Excitation between 199.4 and 672.8 nm and emission
between 349.7 and 719.7 nm were obtained with a SPEX
3
D luminescence spectrophotometer equipped with a
Xenon pulse discharge lamp (75 W) as light source, and a
CCD detector. After optimization, 5 nm slits and 0.1 s
1
13
integration time were used. H-NMR and C-NMR spectra
were recorded on a Bruker Avance III spectrometer at 400
and 75.47 MHz, respectively, using TMS as an internal
standard (chemical shifts in d values and J in hertz). Mass
spectra were recorded on a Finnigan LCQ DECA XP MAX
(
Finnigan, San Jose, CA) quadrupole IT equipped with API
source, using ESI interface. The vaporizer and the capillary
voltages were 5 and 4 V, respectively. The capillary tem-
perature was set at 300 °C.
So, the relative higher enhancement with Mb-CD
probably results from a better fit of the aminoniclosamide
molecule inside the cavity, helped for the methyl groups
surrounding the host molecule. The fluorescence emission
of aminoniclosamide in the presence of Mb-CD is about
twice that in its absence. Only the Sb-CD provokes a
bathochromic shift which is probably due to the stabiliza-
tion effect of hydrogen bonding between the amino (–NH2)
group and water.
Data analysis
The interaction between aminoniclosamide and CD can be
analysed using a Langmuir-type binding isotherm [26].
This model is described by the following equation:
ꢀ
ꢁ
ꢀ
ꢁ
C
I
1
1
¼
þ
C
ð1Þ
BImax
Imax
Quantification of aminoniclosamide in the presence
where C is the CD concentration, I is the fluorescence
intensity of the complex and B is the binding constant.
The quenching of inclusion complex fluorescence by
interfering species can be described by a Stern–Volmer
equation:
of Methyl-b-CD
The influence of Mb-CD concentration on the aminonicl-
-
1
osamide (0.1 mg L ) fluorescence was studied and the
fluorescence intensity was enhanced when the Mb-CD
concentration increases (between 0 and 6 mM) at room
Io
¼
1 þ Ksv½Interferentꢁ
ð2Þ
I
where I is the fluorescence intensity without interferent,
o
3,5
Mβ-CD
I is the fluorescence intensity observed in the presence
interferent and KSV is the Stern–Volmer constant [27]. The
detection limit of detection (LOD) and limit of quantifi-
cation (LOQ) were calculated as 3.33 times (99.7% con-
fidence degree) and 10 times the standard deviation of
seven blanks divided by the slope of the calibration graph.
The precision of the estimations was determined by ana-
3
β-CD
Hβ-CD
2
1
0
,5
2
S-CD
α-CD
-
1
γ-CD
lysing 10 samples containing 0.1 mg L of aminonicl-
osamide and 6 mM CD.
0
,5
1
Results and discussion
,5
0
Fluorescence study
The increase of the relative fluorescence intensity of ami-
noniclosamide in the presence of different CD in water was
used as a qualitative measure of the complexing stability of
the inclusion complex. Experiments were conducted to
3
50
400
450
500
Wavelength / nm
Fig. 2 Fluorescence spectra of aminoniclosamide: CDs
123

9
2
J Incl Phenom Macrocycl Chem (2012) 72:89–94
6
5
4
3
2
1
0
4,00E-09
αCD
βCD
3
3
,50E-09
,00E-09
γCD
M-βCD
H-βCD
S-βCD
6
4
2
1
2,50E-09
0
,5
,2
0
2
1
,00E-09
,50E-09
0
1,00E-09
5,00E-10
0,00E+00
0
0,001 0,002 0,003 0,004 0,005 0,006 0,007 0,008 0,009
[CD] / M
3
50
400
450
500
Wavelength / nm
Fig. 4 Langmuir-binding isotherms of aminoniclosamide with the
different CDs
Fig. 3 Influence of [Mb-CD] in the fluorescence intensity of
aminoniclosamide
has the highest binding constant is Sb-CD, with a
-
1
B = 3119 ± 192 M , and the CD that provokes the
highest fluorescence intensity increase is b-CD with
IM = 57.3 ± 0.2 9 10 .
temperature (Fig. 3). Because an increasing trend was
always observed, the Mb-CD concentration of 6 mM was
chosen and maintained for maximum intensity of
fluorescence.
5
However, Sb-CD provokes a relatively small fluores-
5
cence intensity increase 17.2 ± 0.4 9 10 and b-CD has a
-
1
Using the optimum values of the experimental factors
the calibration graph obtained by the linear least-squares
treatment (with a correlation coefficient of 0.999) was
relatively small binding constant (B = 507 ± 5 M ). The
CD that shows a relatively large binding constant and
provokes relatively large fluorescence intensity increase is
-
1
(
concentration expressed as mM): I = 917766 [Aminon-
F
Mb-CD with B = 1879 ± 29 M
and IM = 52.2 ±
6
iclosamide] ? 2 9 10 . The calibration linear dynamic
5
0.8 9 10 . Consequently the CD chosen to be used in the
analytical method was Mb-CD.
-
1
range was established between 0 and 2,200 lg L
respected to niclosamide. The LOD and LOQ were 45.7
and 137 nM, respectively for aminoniclosamide and cor-
related to niclosamide. The precision of the estimations
was 4.1% (expressed as relative standard deviation).
Interference studies
In order to assess the selectivity of the proposed fluores-
cence method, an interference investigation was performed
-
1
Analysis of the CD binding capacity
using a solution containing 0.1 mg L of aminoniclosa-
mide to check the effects of concomitant molecules such as
niclosamide, the raw material, and two other possible
interferences, the excipient fructose and 2-cyano-6-
metoxybenzothiazole.
For quantitative purposes the analysis of the binding
capacity of the CD towards aminoniclosamide was studied
using a Langmuir-type binding isotherm (Eq. 1) [26].
Figure 4 shows the plot of the experimental data and
Table 1 resumes the data obtained by linear least-squares
of the plots. The analysis of this Table shows that experi-
mental data fits Eq. 2 quite well and a linear trend is
observed throughout the entire tested concentration range.
The binding constants (B) to all the studied CDs are
summarized in Table 1.
Table 2 shows the results obtained using the method of
tolerance ratios. For fructose the interfering effect quite
small is and for 2-cyano-6-metoxybenzothiazole a 10%
reduction in the recovery is observed for the 1:10 ration.
For niclosamide the effect is quite significative which
shows that the reduction process should be quantitative and
the full conversion of niclosamide to aminoniclosamide is
required.
As far as the analysis of aminoniclosamide is of concern
we are interested in the formation of a stable inclusion
complex, with a relatively high binding constant, but also
that provokes a relatively high fluorescence intensity
increase. The analysis of Table 1 shows that the CD that
The interference of niclosamide is a consequence of the
decrease of the fluorescence signal of the inclusion com-
plex of Mb-CD with aminoniclosamide in aqueous solu-
tion. In order to quantify the quenching provoked by
1
23

J Incl Phenom Macrocycl Chem (2012) 72:89–94
93
Table 1 Fitting results of data to a Langmuir-type binding isotherm
Cyclodextrin
I
max (a.u.)
B
r
N
Concentracion range (mM)
Alfa
4571963.8 ± 99800
5739582.5 ± 21360
2290262.3 ± 56730
5226607.7 ± 81660
5133133.5 ± 3822
600.8 ± 51.3
0.984
0.984
0.999
0.996
0.981
5
5
5
6
5
1–8
Beta
507.1 ± 4.8
2253.6 ± 392.6
1878.6 ± 29.0
872.9 ± 4.7
1–8
Gamma
Methyl-Beta
1–8
0.2–6
0.5–4
Hydroxypropyl-
Beta
Sulphated-Beta
1726768.6 ± 37416
3119.1 ± 192.0
0.981
5
0.2–4
M
I maximum intensity, B binding constant, r correlation coefficient, N number of points
Ci eˆ ncia 2007 program, Ministerio de Educaci o´ n y Ciencia Project #
CTQ2007 62386-PPQ and FSE-FEDER (Project PTDC/QUI/71001/
2006) is acknowledged.
Table 2 Tolerable limits of interfering species
Interference
Aminoniclosamide:Interference Recovery
%)
(
Niclosamide
1:1
96
86
80
98
98
96
96
95
90
1
1
:5
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