ISSN 10619348, Journal of Analytical Chemistry, 2014, Vol. 69, No. 10, pp. 948–952. © Pleiades Publishing, Ltd.

, 2014.

ARTICLES

Simultaneous Spectrophotometric Determination of Pyrantel

Pamoate and Febantel in Pharmaceutical Preparations Using Partial
LeastSquares Regression1
M. S. Piantavinia, F. L. D. Pontesa, L. B. Cerqueiraa, P. G. PeraltaZamorab, and R. Pontaroloa, *
a
Laboratório de Controle de Qualidade, Departamento de Farmácia
Universidade Federal do Paraná, Av. Pref.Lothário Meissner, 632, 80210170, CuritibaPR, Brasil
*email: pontarolo@ufpr.br
b
Laboratório de Química Ambiental e de Materiais, Departamento de Química
Received January 21, 2013; in final form, July 3, 2013

Abstract—The multivariate spectroscopic determination of the combination of pyrantel pamoate (PP)

and febantel (FB) in pharmaceutical preparations was carried out by partial leastsquares regression.
The UV absorbance spectra of standard mixtures of PP and FB were measured at concentrations rang
ing from 5.76–11.52 and 6–12 μg/mL, respectively, in a diluent solution of methanol and acetonitrile.
The best model was selected with variable selection (280–320, 380–400 nm), 2 latent variables and
mean centered. External validation was performed using 13 different mixtures, which provided predic
tion errors lower than 1%. The method was validated in accordance with international and Brazilian guide
lines. This method was successfully employed to quantify the drug content in two different pharmaceutical
formulations (solid – capsules, liquid – oral suspensions) with good prediction capacity. Furthermore, its
advantages include the use of simple and accessible reagents and equipment, and a low operating cost.
Keywords: pyrantel pamoate, febantel, multivariate calibration, chemometric, UV spectrometry, PLS, validation
DOI: 10.1134/S1061934814100104

Antiparasitic drugs are commonly used in veterinary
practice for the prevention and treatment of parasitic in
fections. Among the main groups of parasiticides are feb
antel and pyrantel pamoate [1]. Febantel is a prodrug
that is directly converted into the active metabolites fen
bendazole or oxfendazole following administration [2].
FB acts by inhibiting the microtubule synthesis of the par
asite and is active against Ancylostoma caninum, Toxocara
canis, Toxocara cati and Taenia spp [3]. Pyrantel pamoate
is a cholinergic agonist and acts by inhibiting the neuro
muscular transmissions of the parasite [2]. It is effective
against Ancylostoma caninum, Ancylostoma tubaeforme,
Uncinaria stenocephala, Toxocara canis, Toxocara cati and
Toxascaris leonine [3]. The structures of the two chemicals
are shown below. The use of combinations of anthelm
intics have been effectively used in veterinary treatment to
extend the spectrum of antiparasitic activity [4]. The liter
ature has described several analytical methods for the de
termination of FB and PP in pharmaceuticals and other
samples using spectrophotometry [5], potentiometry [6],
voltammetry [7], HPLC [8–18] and LC–MS–MS [19–
22]. However, to the best of our knowledge, no analytical
method has been described that uses UV spectrophotom
etry for the simultaneous determination of FB and PP in
pharmaceutical preparations.

H3C O
CH3 OH OH O
N HOOC COOH HN S
S ⋅
N
O N
H3C
O NH NH
PP CH3
O O
FB
The chemical structure of pyrantel pamoate and febantel.

1
The article is published in the original.

948
 SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION
The simultaneous determination of FB and PP by
UV spectrophotometry represents a challenge in the
field of analytical chemistry because of the spectral in
terference of the analytes. However, the use of multi
variate calibration makes it possible to quantify these
analytes [23] and the most popular multivariate cali
bration method used for building regression models is
partial leastsquares (PLS) [24]. The combination of
PLS and UV spectrophotometry has been used for the
simultaneous determination of several common active
compounds that are associated in pharmaceutical for
mulations, such as albendazole and praziquantel [25],
but, to the best of our knowledge, none or very few ar
ticles quantify two drugs in two different pharmaceuti
cal formulations (capsules and oral suspensions).
Therefore, there is a significant and a valuable applica
tion of the methodology.
Owing to the widespread use of FB and PP by the
pharmaceutical industry and magistral pharmacies,
the aim of this work was to develop and validate a sim
ple, reliable and cheap analytical method for quantify
ing these drugs in antiparasitic products.
EXPERIMENTAL
Standards, samples, chemicals and reagents. Stan
dards of PP and FB were obtained from SigmaAld
rich (St. Louis, MO, USA). Stock solutions of PP and
FB at concentrations of 1 mg/mL were prepared sep
arately in a diluent solution of methanol and acetoni
trile (50 : 50, v/v) and stored protected from light at
4°C. From these solutions, an intermediate solution
of each standard was prepared at a concentration of
150 µg/mL. These intermediate standards were used
to prepare mixtures of the standard solutions for the
calibration and validation sets. Oral suspension and
capsules were purchased from a magistral pharmacy
located in Curitiba, Paraná state, Brazil. Acetonitrile
and methanol were HPLC grade.
Equipment and software. Electronic absorption
measurements were carried out on a Shimadzu 1800
double beam UVvis and a UVvis Agilent 8453E
spectrophotometer using 1.00 cm quartz cells. The
spectral data was recorded between 200 and 400 nm
using a spectral resolution of 0.5 nm. HPLC analysis
was performed using an Agilent 1100 HPLC System
(Wilmington, NC, USA). The analytes were separated
on an XBridge C18 150 × 4.6 mm (5 µm) column cou
pled with an XBridge C18 20 × 4.6 mm (5 µm) guard
column. The injection volume was 10 µL, and the col
umn temperature was maintained at 40°C. Data ac
quisition was performed using ChemStation A.10.02
software. The data treatment was carried out using
Statistica 8 (StatSoft). PLS models were developed us
ing PLSToolbox 3.0 (Eigenvector Research, Inc.) op
erating in Matlab 7.0.1 (Math Work Inc.).
Procedure. Calibration set, validation set and syn
thetic mixtures. For the development of multivariate
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 69
949
11
9
Pyrantel
7
5 7 9 11 13
Febantel
Fig. 1. The composition (µg/mL) of calibration (䉬) and
prediction (䉫) set for spectrophotometric method.
calibration models, 51 synthetic mixtures were pre
pared, according to a full experimental design, con
taining 6–12 µg/mL of FB and 5.76–11.52 µg/mL of
PP, dissolved in diluent solution (Fig. 1). The model
ing amplitude was chosen based on the nominal con
centration in commercial medications and a maximal
variation of ±35%. Multivariate models were devel
oped with 38 synthetic mixtures, reserving the other
13 as external validation set. The PLS models were ob
tained using internal validation (cross validation
method – leave one out), preprocess for Yblock and
Xblock and different numbers of latent variables. Pre
diction errors were expressed as RMSEP (root mean
square error of prediction), defined as follows:
RMSEP =
∑ (y r − yˆ p )2
,
n
where n is the number of samples, yr is the standard
(real) value and yˆ p is the value predicted by the model.
Pharmaceutical sample preparation. There are two
different pharmaceutical formulations available on the
market that contain PP and FB in association: cap
sules, 144 mg of PP and 150 mg of FB, and oral sus
pensions, 14.4 mg/mL of PP and 15 mg/mL of FB.
The excipient composition is not provided by industry,
so the mixtures were made based on the properties of
drugs solubility and permeability by the Biopharmaceu
tics Classification System [26]. The contents of 20 cap
sules were crushed and mixed into a homogeneous pow
der. A mass equivalent to 100 mg of febantel and 96 mg
of pyrantel pamoate was accurately weighed and trans
ferred to volumetric flasks to obtain a final concentra
tion of 1 mg/mL of FB and 0.96 mg/mL of PP. The
oral suspension was diluted with a diluent solution to
obtain the same final concentration and the same pro
cedure as described for the capsules was subsequently
performed on the oral suspension solution.
Analysis of the pharmaceutical samples. For the
electronic spectroscopic analysis, an aliquot of the fil
No. 10 2014
950 PIANTAVINI et al.
2.5 3 PP intermediate standards solutions were added to ob
2.0
2 115 and 120% of the test concentration (9 µg/mL of
Absorbance
1.5
1.0 1 tween 200 and 400 nm using a spectral resolution of
0.5
0 studying the effects of varying the ratio of solvents in
200 250 300 350 400
Wavelength, nm
Fig. 2. UV absorption spectra of FB (1, 9.0 µg/mL), PP (2,
8.64 µg/mL) and their mixture (3) in methanol–acetoni
trile (50 : 50, v/v).
tered capsule solution was diluted with a diluent solu
tion, resulting in a calculated concentration of
9 µg/mL of FB and 8.64 µg/mL of PP. For the chro
matographic analysis, an aliquot of the filtered capsule
solution was transferred to a volumetric flask to obtain
a final concentration of 400 µg/mL of FB and
384 µg/mL of PP. The chromatographic measure
ments were performed under gradient conditions us
ing an optimized procedure described by Bialecka
et al. [10]. Each measurement was performed in tripli
cate, and the same process was used for the oral sus
pension.
Validation of the method. Analytical validation was
performed according to the guidelines set forth by
ANVISA [27] and the International Conference on
Harmonisation [28]. The predictive capacity of the
multivariate model was appraised with regards to the
external validation set and the crossvalidation rou
tine. The linearity was estimated using a standard
curve with mixtures of FB and PP, yielding standards
with concentrations that ranged between 6–12 and
5.76–11.52 µg/mL of FB and PP, respectively. The
measurements were performed in triplicate and the
correlation coefficient and the regression equation
were assessed. Repeatability was determined using
samples at concentrations of 9 µg/mL FB (n = 6 × 3)
and 8.64 µg/mL PP (n = 6 × 3). Intermediate preci
sion was also verified using samples of FB (9 µg/mL,
n = 6 × 3) and PP (8.64 µg/mL, n = 6 × 3) on different
days with different equipment and different analysts.
The precision was determined using the variation co
efficient.
The accuracy of the method was determined using
a standard addition technique. For the capsules, the
FB and PP intermediate standard solutions were add
ed to a placebo (Aerosil® silica 0.8%; sodium lauryl
sulfate 2%; sodium starch glycolate 8%; microcrystal
line cellulose 22.3% and lactose 66.9%) to obtain three
concentration levels corresponding to 80, 100 and
120% of the test concentration (9 µg/mL of FB and
8.64 µg/mL of PP). For the oral suspension, FB and
JOURNAL OF ANALYTICAL CHEMISTRY
tain three concentration levels, corresponding to 110,
FB and 8.64 µg/mL of PP). Each solution was pre
pared in triplicate. All spectral data were recorded be
0.5 nm. Data were verified using a ttest.
The robustness of the procedure was analyzed by
the diluent solutions, the temperature and the reading
time. To assess the influence of variations in the com
position of the diluent, three different proportions of
methanol–acetonitrile were prepared (45 : 50, 50 : 50
and 55 : 45, v/v). To evaluate the effect of varying the
temperature, samples were maintained at 20, 25 and
30°C and readings were taken at 0, 24 and 96 h. To as
certain the effect of changing the reading time, mea
surements were taken at room temperature at 0, 24 and
96 h after the initial sample preparation. The analyses
were performed in triplicate using a diluted solution of
FB (9 µg/mL) and PP (8.64 µg/mL). All spectral
data were recorded between 200 and 400 nm using a
spectral resolution of 0.5 nm. Data were verified using
a ttest for paired samples and oneway analysis of
variance (ANOVA).
RESULTS AND DISCUSSION
PP and FB UV absorption spectra. The individual
UV spectra of PP, FB and the spectrum of a synthetic
mixture of the analytes in a diluent solution have a
maximum absorption of 236.5 nm for PP and near to
213 and 282 nm for FB (Fig. 2). The region in between
325–400 nm has a signal majorly given by PP and
250–320 nm better discriminates FB. There is a clear
overlap of the spectroscopic signal of both species, a
fact that usually hinder their simultaneous quantifica
tion by direct UV absorbance measurements. To solve
this problem, probably, the best method is PLS regres
sion.
Multivariate models. The best model was developed
with variable selection (280–320, 380–400 nm), 2 LV
and mean centered. It was selected for subsequent
studies, mainly due to the low values of the predic
tion errors (FB 0.57%, PP 0.82%), RMSEC (FB
0.044 µg/mL, PP 0.060 µg/mL) and RMSEP (FB
0.058 µg/mL, PP 0.060 µg/mL) observed under these
conditions. Two latent variables can explain the major
ity of the variance in the analytical data (99.96% of the
Xblock and 99.94% of the Yblock). Outliers can ad
versely affect the predictive capability of the model
and, therefore, were investigated by analysis of the stu
dentized residuals versus leverage graphs. In this eval
uation outliers were not observed.
Validation of the multivariate model. The linearity
was estimated by correlation coefficient (r) for FB
(0.9970), PP (0.9994) and the regression equation was
assessed for FB (y = 0.9937x + 0.0565) and PP
Vol. 69 No. 10 2014
 SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION 951

Table 1. Results of the precision (repeatability and intermediate precision) study

Febantel Pyrantel
Analyst
creal* cpredicted* RSD, % recovery, % creal* cpredicted* RSD, % recovery, %
Repeatability
9.00 8.97 1.38 99.69 8.64 8.59 1.45 99.46
Intermediate precision
1 9.00 8.97 1.38 99.69 8.64 8.59 1.45 99.46
2 9.00 8.96 0.96 99.60 8.64 8.65 1.21 100.13
* Concentration, µg/mL.

Table 2. Results of the accuracy test performed by the addition of an FB and PP intermediate standard solution to a placebo
of capsules and oral solutions
Febantel Pyrantel
Formulation Level, %
creal* cpredicted* RSD, % recovery, % creal* cpredicted* RSD, % recovery, %
Capsules 120 10.80 10.69 1.45 98.98 10.37 10.21 0.45 98.46
100 9.00 9.05 0.77 100.56 8.64 8.60 1.94 99.54
80 7.20 7.18 0.95 99.72 6.91 6.85 0.81 99.13
Oral suspension 120 10.80 10.91 1.52 101.02 10.36 10.50 0.50 101.35
115 10.35 10.31 0.95 99.61 9.93 10.01 0.19 100.81
110 9.90 10.06 0.37 101.62 9.50 9.58 0.12 100.84
* Concentration, µg/mL.

(y = 0.9989x + 0.0097). The repeatability and inter
mediate precision assessments gave RSD values lower
than 1.45% for FB and PP, indicating that the method
is precise (Table 1). Recoveries were determined and
gave values close to 100%. The RSD values were all
lower than 1.94%, indicating good reliability and ac
curacy (Table 2) and in accordance with guidelines.
The pvalues for the repeatability, intermediate preci
sion and accuracy indicate that the values are statisti
cally identical.
The determination of some figures of merit (FOM)
such as sensitivity and selectivity were estimated and
can be used to compare analytical methods and to
compare calibrations constructed from spectroscopic
data collected using two analytical instruments [29].
When expressing FOM for multivariate calibration
methods, the part of the signal that relates uniquely to
the analyte of interest is more important than the total
signal. This unique signal is termed net analyte signal
and is defined as the part of the signal that is orthogo
nal to the signal of the interferences present in the
sample [30, 31]. The results for selectivity were 32.80%
(FB) and 38.95% (PP). The sensitivities were 1.21
(FB) and 1.58 (PP). With the inverse of sensitivity it is
possible to establish a minimum concentration differ
ence that is discernible by the analytical method in the
absence of experimental error, independent of the spe
cific technique employed. For inverse of sensitivity
was found 5 × 10–3 µg/mL for FB and 5 × 10–3 µg/mL
for PP.
Concerning to the robustness, the proportion of
solvents in the diluent solutions, the temperature and
the reading time showed no influence on the results of
the proposed method. The ANOVA statistical method
was used to detect any significant differences. The re
sults showed that there were no significant differences
among the three parameters studied.
The multivariate model was applied to quantify the
FB–PP combination in commercial drugs (capsules
and oral suspensions). The results (Table 3) from this
model indicate a good agreement between the multi
variate and chromatographic methods, with low rela
tive errors. According to the ttest the pvalue for the
comparison HPLC and UV used to study PP in cap
sules and for FB in oral solution were up to 0.05.
Therefore the values were statistically equal to the true
value. The pvalues for the comparison HPLC and UV
for FB in capsules and PP in oral solution were 0.035
and 0.028, respectively. This means that the values are
statistically different, but analytically they are very
similar.
***
PLS, a powerful and widely used tool in multivari
ate calibration methods, was successfully employed

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 69 No. 10 2014

952 PIANTAVINI et al.

Table 3. The results (µg/mL) of the chromatographic and multivariate spectroscopic determination of FB and PP in cap
sules and oral suspension (mean, n = 3)
Label claim HPLC Multivariate model
Formulation
FB PP FB PP FB PP
Capsules* 150 144 155.63 136.75 152.28 137.60
(0.64)*** (1.38) (0.11) (0.05)
Oral suspension** 15 14.4 15.50 15.79 15.54 15.27
(1.19) (0.92) (0.63) (0.17)
* Each capsule was labeled to contain 150 and 144 mg of febantel and pyrantel pamoate, respectively.
** 1 mL of oral suspension was labeled to contain 15 and 14.4 mg of febantel and pyrantel pamoate, respectively.
*** In parentheses are the values of RSD, %.

for the simultaneous spectrophotometric quantifica
tion of FB and PP in two pharmaceutical preparations
(capsules and oral suspensions). The calibration mod
el (mean centered, two latent variables) was validated
and meets the requirements imposed by ANVISA and
ICH. Therefore, this method could be easily used as
an analysis technique in the quality control laborato
ries of industries and manipulation pharmacies.
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