Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Chemometric quality inspection control of pyrantel pamoate, febantel

and praziquantel in veterinary tablets by mid infrared spectroscopy
Mrio S. Piantavini a, Flvia L.D. Pontes a, Caroline P. Uber a, Dile P. Stremel b, Marcelo M. Sena c,
Roberto Pontarolo a,
a
Laboratrio de Controle de Qualidade, Departamento de Farmcia, Universidade Federal do Paran, 80210-170 Curitiba, PR, Brazil
b
Universidade Federal do Paran, Palotina, PR, Brazil
c
Departamento de Qumica, ICEx, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 A MIRS method for simultaneous

determination of 3 API in veterinary
tablets.
 siPLS improved the model by
empirically selecting the discriminant
spectral regions.
 Model development without prior
access to excipient composition of the
formulations.
 Analyses of 3 real (commercial)
samples in a situation of multi-
product calibration.
 The proposed MIRS method is more
than ten times faster than the HPLC
one.

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

Article history: This paper describes the development and validation of a new multivariate calibration method based on
Received 1 November 2013 diffuse reectance mid infrared spectroscopy for direct and simultaneous determination of three veter-
Received in revised form 8 January 2014 inary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best
Accepted 14 January 2014
synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions,
Available online 24 January 2014
37153150, 28652583, and 22981733 cm 1, preprocessed by rst derivative and SavitzkyGolay
smoothing followed by mean centering. This model was built with ve latent variables and provided root
Keywords:
mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the
Multi-analyte determination
Quality inspection control
three analytes. The method was validated according the appropriate regulations through the estimate
MIR of gures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction
siPLS deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in
Multivariate analytical validation the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these
Veterinary pharmaceutical formulations commercial products, which was not known a priori, was modeled by an experimental design that
scanned the likely content range of the possible constituents. The results were veried with high perfor-
mance liquid chromatography with diode array detection (HPLCDAD) and high performance liquid chro-
matographytandem mass spectrometry (HPLCMS/MS) and were in agreement with the predicted
values at 95% condence level. The developed method presented the advantages of being simple, rapid,
solvent free, and about ten times faster than the HPLC ones.
2014 Elsevier B.V. All rights reserved.

Corresponding author. Address: Departamento de Farmcia, Universidade Federal do Paran, Av. Pref. Lothrio Meissner, 632, 80210-170 Curitiba, PR, Brazil. Tel.: +55 41
33604094; fax: +55 41 33604106.
E-mail address: pontarolo@ufpr.br (R. Pontarolo).

http://dx.doi.org/10.1016/j.saa.2014.01.078
1386-1425/ 2014 Elsevier B.V. All rights reserved.
 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403
Introduction
Helminthiases are parasitic diseases commonly found in pets,
being considered a public health threat, since they can be trans-
missible to humans. For the treatment and control of helminthia-
ses, combinations of more than one drug are required to increase
the spectrum of action as well as the effectiveness of the treatment
[1,2]. Among the main antiparasitic drugs for pets, febantel, prazi-
quantel and pyrantel pamoate are the most commonly used. Feb-
antel (FB) is a pro-drug, which is converted in two actives
metabolites, fenbendazole and oxfendazole. Its mechanism of ac-
tion involves the inhibition of the microtubule synthesis of the par-
asite and is active against most of the nematode and cestode
worms [3]. Praziquantel (PZ) is highly active against a wide range
of cestodes and all species of schistosome pathogenic to man [4],
and its mechanism of action works by a spastic paralysis of the
worm musculature [5]. Pyrantel pamoate (PP) is effective against
ascaris, enterobius and ancylostoma species and acts by inhibiting
neuromuscular transmissions of the parasite [6,7]. The structures
of these three chemicals are shown in Fig. 1.
Fixed dose combinations containing these three drugs are
available in the market, and the quality control of these medi-
cines becomes important. Many authors have described the
determination of PP, PZ and FB in veterinary pharmaceutical
formulations and/or biological samples. Their individual and
simultaneous determinations have been possible by spectropho-
tometry [8,9], non-aqueous titrimetry [10], voltammetry
[7,8,10,11], HPLC [3,6,10,1217], and LCMS/MS [1822]. How-
ever, none of the proposed methods allow direct, very rapid and
simultaneous determination of these three drugs. In addition,
most of these methods are chromatographic and often require a
series of dilutions, sample ltrations, ultrapure water, complex
sample pretreatments, including removal of interferences and
extraction of the analytes, which sometimes employs enormous
quantities of organic solvents for each sample.
In the last years, the use of reectance infrared spectroscopy
has become a reliable alternative for the quality control of active
pharmaceutical ingredients (API), providing methods of relative
low cost that demand less human intervention and are more
appropriate for the modern quality control. Both near infrared
(NIR) [2326] and mid infrared (MIR) [2731] spectroscopy have
been recently used for developing these methods. The presence
of signicant spectral overlapping among the analyte and excipi-
ents makes the direct determination almost impossible and re-
quires the combined use of multivariate calibration methods,
such as partial least squares (PLS) [32]. The situation is even more
complex in the case of multi-analyte determinations. The simulta-
neous determination of three or more analytes in one pharmaceu-
tical formulation by using NIR or MIR spectroscopy is very rare. To
the best of our knowledge, only one paper has determined more
than two API in a pharmaceutical preparation by using NIR and
PLS [33].
Fig. 1. Chemical structures of (a) PP, (b) PZ and (c) FB.
397
In spite of the growing number of papers published in the last
years that developed methods for API determination in human for-
mulations by NIR/MIR, quantication in veterinary formulations is
also very rare or inexistent. In the Brazilian market, a particularity
of the veterinary formulations is that, unlike human formulations,
sometimes the name of the excipients is not mentioned in the
package leaet. As this work was carried out in a university labo-
ratory, without direct access to the information from the manufac-
turers about the composition of excipients, which is not public, a
previous experience of one of the authors with veterinary formula-
tions was used to choose and test the more appropriate compo-
nents. The selected composition of excipients was varied in a
certain range, in order to incorporate the likely variability present
in several formulations, and the developed model was applied to
three products of different manufacturers available in the Brazilian
market. In this sense, the development of this MIR model can be
considered an example of a multi-product calibration [34,35].
Since practically all the NIR/MIR models for quality control of API
have been specic for one formulation of one manufacturer, an
obvious advantage of multi-product calibration models is the pos-
sibility of their use by public supervision.
In addition to vary the excipients composition, the experimen-
tal design for developing this model employed a central composite
design (CCD) with three factors, corresponding to the three ana-
lytes [36]. This approach reduces the number of experiments, im-
proves statistical interpretation and can indicate whether
parameters interact. Another important issue tackled in this work
is the multivariate validation, a prerequisite for the ofcial recog-
nition of new quantitative infrared methods. Nevertheless, almost
all of the traditional guidelines used in pharmaceutical analysis
[3739] are still conceived in a univariate way and need to be har-
monized in order to comprehend the specic aspects of multivari-
ate methods, such as the lack of requirement for total selectivity,
the inexistence of calibration curves and the concept of net analyte
signal (NAS), the part of the analytical signal uniquely related to
the analyte and orthogonal to the interferences. A deeper discus-
sion about multivariate validation can be found in the appropriate
Refs. [25,4042].
Thus, the aim of this work was to develop and validate a simple
and rapid method for simultaneous determination of pyrantel
pamoate, praziquantel and febantel in powder samples of veteri-
nary formulations from different manufacturers by using diffuse
reectance MIR spectroscopy and synergy interval partial least
squares (siPLS). For the multivariate analytical validation, the fol-
lowing gures of merit were estimated: trueness, precision, linear-
ity, range, selectivity (SEL), sensitivity (SEN), analytical sensitivity
(c), limits of detection (LOD) and quantication (LOQ), bias, and
residual prediction deviation (RPD). The siPLS is a variant of PLS
that split the data set into a number of intervals (variable-wise)
and calculate all possible PLS model combinations of two, three
or more intervals, in order to obtain a judicious selection of the
spectral regions of better predictive ability [30,31].
398 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403
Materials and methods
Reagents and samples
The PP, PZ and FB standards were obtained from SigmaAldrich
(St. Louis, MO, USA) and stored protected from light. The choice
excipients, colloidal silicon dioxide, sodium lauryl sulphate, micro-
crystalline cellulose, butylated hydroxytoluene, talc and corn
starch, were purchased from certied suppliers and used without
further purication. Acetonitrile and methanol were of HPLC grade.
Ultrapure water was obtained using a Milli-Q purication system
from Millipore (Bedford, MA, USA). Powder samples were prepared
by weighing with an analytical balance (0.0001 g), according to an
experimental design. All the three analyzed commercial veterinary
formulations have the same composition of API: 144 mg of PP,
50 mg of PZ and 150 mg of FB per tablet. Their compositions of
excipients are not publicly available.
Apparatus and software
The spectral dataset was acquired from a Bruker Alpha FT-IR
spectrometer, equipped with a diffuse reectance accessory, and
under controlled temperature (20.0 0.2 C) and humidity
4555%). The spectra were obtained in the absorbance mode by
using OPUS (OPtical User Software) for windows, version 6.0, from
Brucker Optik (Bremen, Germany). Data were handled using OPUS,
MATLAB software, version 7.13 (The Math-Works, Natick, USA),
and PLS Toolbox, version 6.5 (Eigenvector Technologies, Manson,
USA).
Experimental design
Fifty six powder samples were prepared according to a CCD
(arotability = 1.6818; aortogonality = 1.2872) with three factors, PP, PZ
and FB, and 8 levels plus a central point, as depicted in Fig. 2.
The total mass of each sample was xed in 100.0 mg. The range
of each factor was varied from about 80.0% to 120.0% of its nominal
content in the analyzed formulations (Section 2.1). This range was
chosen in order to cover from 90.0% to 110.0% of the API contents,
which are the acceptable limits commonly established by the phar-
macopoeias. The central point of CCD corresponds to 28.8 mg of PP,
10.0 mg of PZ and 30.0 mg of FB per 100 mg of sample. Each sample
was completed to 100 mg with a mixture of excipients, which was
prepared based on another experimental design. This design con-
sisted of three factors that were varied in certain ranges: the two
major excipients, microcrystalline cellulose and corn starch, and
a mixture of the minor excipients, colloidal silicon dioxide, sodium
lauryl sulphate, butylated hydroxytoluene and talc. Nevertheless,
this design was not shown here for reasons of commercial interest.
The excipient mixtures obtained from the second design were
Fig. 2. Experimental design (CCD) used for varying PP, PZ and FB contents.
Calibration (circles) and validation (down triangles) samples.
randomly mixed with the samples from the rst design, in order
to obtain a robust model.
Procedure
The powder samples were prepared, manually homogenized
and directly measured. The spectra were recorded from 4000
to 400 cm 1 as the average of 64 scans and with a resolution
of 4 cm 1. Six replicates of samples from the central point were
also obtained for evaluating repeatability. These results were
compared with similar replicates obtained on another day by
a different analyst for estimating intermediate precision. Twenty
spectra of the empty cell were also recorded for estimating the
instrumental noise.
Analysis of real samples
Tablets of three different manufacturers available in Brazil were
purchased in Curitiba, PR. The contents of 20 tablets of each formu-
lation were crushed and mixed into a homogeneous powder. The
MIR spectra of these samples were acquired in the same conditions
described in Section 2.4, in six replicates. These same commercial
samples were also analyzed by HPLCDAD and HPLCMS/MS, as
described in Section 2.6.
Chromatographic analysis
The validation by HPLCDAD was adapted from a published
method14 and was carried out with an Agilent 1100 HPLC System
(Wilmington, NC, USA) that consisted of a G1311A quaternary
pump, a G1379A degasser, a G1329A autosampler, a G1316A
column oven and a G1315B DAD detector. The analytes were
separated on an XBridge C18 250  4,6 mm (5 lm particle size)
column coupled with an XBridge C18 20  4.6 mm (5 lm particle
size) guard column. The mobile phase was water (pH 3.5, adjusted
with 85% phosphoric acid) and acetonitrile in gradient mode. A
ow rate of 1.2 mL min 1 and detection at 215 and 312 nm were
used. The injection volume was 10 lL, and the column temperature

was maintained at 40 C.
The analysis by HPLCMS/MS was performed as described by
Pontes et al. [22] and was carried out with an Agilent 1200 HPLC
system (Santa Clara, CA, USA) coupled with a triple quadrupole
API 3200 from Applied Biosystems MDS Sciex Instruments (Foster
City, CA, USA). The analytical separations were achieved on an
XBridge C8 50  2.1 mm (5 lm particle size) column coupled with
an XBridge C8 10  2.1 mm (5 lm particle size) guard column

maintained at 20 C. The mobile phase consisted of water/acetoni-
trile (15:85 v/v) containing 0.1% formic acid and 3 mmol L 1 of
ammonium formate. The isocratic ow rate was 200 lL min 1
and the injection volume was 20 lL. The electrospray ionization
source was operated in the positive mode under the following
working conditions: ion spray voltage of 5000 V; source tempera-

ture of 450 C; nebulizer and dryer gas (nitrogen) of 45 and 40 psi,
respectively; collision activated dissociation gas (CAD) of 4 psi and
curtain gas (CUR) of 10 psi. Quantication was performed in multi-
ple reaction monitoring (MRM) mode, maintaining a dwell time of
150 ms.
In both methods, a mass equivalent to one tablet was dissolved
in acetonitrile/methanol (50:50, v/v), sonicated and ltered
through a quantitative lter paper (J Prolab, 0.28 lm pore size).
Then, the ltered solution was diluted in a volumetric ask con-
taining the mobile phase, according to the appropriate concentra-
tion. All samples were prepared under low light exposure and
ltered through a 0.22 lm PVDF syringe lter prior to injection.
All the injections were repeated three times and each sample
was analyzed in triplicate.
 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403
Results and discussion
Selection of excipients and experimental design
The oral absorption and therapeutic efcacy of any drug de-
pends on its aqueous solubility and gastrointestinal permeability.
The Biopharmaceutics Classication System (BCS) [43] is a scien-
tic framework used to predict bioavailability of drugs based on
the properties of solubility and permeability [44]. Furthermore,
BCS has been used as criterion for excipient selection of pharma-
ceutical formulation considering pharmacotechnical aspects
which contribute to the drug dissolution and consequent absorp-
tion [45]. According to BCS, drug substances are divided in four
classes: high solubility and high permeability (I); low solubility
and high permeability (II); high solubility and low permeability
(III); and low solubility and low permeability (IV). In agreement
with this classication, PP and PZ are classied as Class II drugs
[45] and therefore their bioavailability are limited by the dissolu-
tion rate. Classication of FB was not available, and thus, only PP
and PZ data were considered to select the excipients. Considering
this context and a specic knowledge of the most common excip-
ients used in veterinary formulations, six excipients were chosen.
The two major excipients were corn starch, used as diluent and
lubricant, and microcrystalline cellulose, used as diluent, lubricant
and disintegrant. The four minor excipients were talc, used to pre-
vent sticking during manufacturing, sodium lauryl sulphate, used
as lubricant tensoactive and agglutinant, colloidal silicon dioxide,
used as absorbent, and butylated hydroxytoluene, used as antiox-
idant. Mixtures of excipients were prepared according to an
experimental design with three factors, as already mentioned in
Section 2.3. The content ranges of these factors were varied
20% around the expected values (not shown). Then, the excipient
mixtures were randomly combined with the samples from the
CDD (Fig. 2). This procedure aims to obtain a representative and
robust model that is able to predict samples with composition
of excipients varying in the studied range. In addition, the random
combination between the experimental designs of the analytes
and excipients aims to avoid chance correlations, also contribut-
ing for a robust model [26].
MIR PP, PZ and FB spectra
MIR spectra of pure PP, PZ and FB powder samples are shown in
Fig. 3. The interpretation of these spectra helps in highlighting the
Fig. 3. Diffuse reectance MIR spectra of pure (a) PP, (b) PZ and (c) FB powder
samples.
399
regions that may most contribute for predictive models. For each
analyte the main bands were identied [46]. In the spectrum of
PP (Fig. 3a), it is possible to observe a broad band with peaks at
3067, 2969 and 2892 cm 1, which are related to CAH, phenolic
OAH and carboxylic OAH stretching vibrations, respectively; a
strong peak at 1659 cm 1, related to the aromatic carboxylic
C@O stretching; a peak at 1612 cm 1, related to the cyclic imine
C@N bond; a peak at 1150 cm 1, related to the CAN stretching of
tertiary amine; and a peak at 713 cm 1, related to the out-of-plane
CAH bending of tiophene ring [47]. In the spectrum of PZ (Fig. 3b),
it can be observed a small band with two peaks at 3291 and
3234 cm 1, related to the rst overtones of the C@O stretching
vibrations; two strong peaks at 2898 and 2848 cm 1, related to
the stretching vibrations of ACAH bound to tertiary amines in
the lactam ring; a strong band between about 1700 and
1600 cm 1 and centered at 1666 cm 1, related to the stretching
vibrations of the two amide carbonyl groups; and a strong band
centered around 1450 cm 1, characteristic of di-substituted
amides. In the spectrum of FB (Fig. 3c), it can be observed a broad
band with a peak at 3209 cm 1, corresponding to the carbamate
NAH stretchings; two peaks at 2977 and 2825 cm 1, correspond-
ing to the CAH stretching from the OACH3 groups; strong peaks
at 1769 and 1687 cm 1, related to carbamate C@O stretchings;
and a peak centered at 1514 nm, related to carbamate NAH
bending.
siPLS model
The spectra of all 56 prepared samples (Fig. 4) were divided into
36 for the calibration set and 20 for the validation set, according to
an experimental design (Fig. 2). The validation samples were se-
lected in order to test the predictive ability of the model by ensur-
ing a homogeneous and representative distribution of the three
analytes within the analytical ranges. Preliminary tested models
indicated that no signicant differences were observed between
the results of PLS1 and PLS2. Thus, for simplicity reasons PLS2
was chosen. In PLS2, in contrast to PLS1, all the analytes are pre-
dicted simultaneously from the same set of loadings. The use of
preprocessing techniques for PLS models developed from reec-
tance infrared spectra of powder samples is almost mandatory,
due to the presence of non-linear baseline deviations, caused by
the multiplicative light scattering. In this work, the most used
techniques multiplicative scatter correction (MSC), standard nor-
mal variate (SNV), and rst derivative with smoothing [48] were
tested. The lowest root mean square error of cross validation
(RMSECV) was obtained with rst derivative followed by
Fig. 4. MIR spectra of all the 56 prepared samples.
400 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403
Savitsky-Golay smoothing (15 points in lter and rst order poly-
nomial t) and mean centering. The best PLS model was selected
by random subsets (with 6 splits) cross validation and with 5 latent
variables (LV). Considering this number of LV, the minimum rec-
ommended number of calibration and validation samples for infra-
red multivariate quantitative models was assured [49].
Since full spectrum PLS models use to include uninformative
wavenumbers that may negatively affect them by increasing the
errors [31], variable selection by siPLS2 was employed for improv-
ing the predictive ability of the model. The spectra were divided in
8, 12, 20, 30 and 50 intervals combined in up to 5 subintervals. The
best results, as shown in Fig. 5, were obtained with spectra divided
in 12 intervals and combined in 5 subintervals. This siPLS2 model
also used 5 LV and accounted for 97.87% of the total variance in the
X block and 97.99% in the Y block. The average (three analytes)
RMSECV of 1.21 mg/100 mg for the PLS2 model was signicantly
decreased to 0.74 mg/100 mg in this siPLS2 model. The RMSECV
for each analyte is shown in Table 1. The selected intervals (in light
gray in Fig. 5) correspond to three spectral regions, between 3715
3150 cm 1, 28652583 cm 1, and 22981733 cm 1. These selected
regions include specic absorptions of each analyte, such as a small
and broad band (2130 and 1790 cm 1) centered at 1923 cm 1 of
PP; small peaks of C@O stretching rst overtones in the region of
33003220 cm 1, and a peak at 2848 cm 1, related to a stretching
vibration of ACAH bound to a tertiary amine of PZ; and a strong
band centered at 3209 cm 1, characteristic of carbamates, and a
carbonyl stretching at 1769 cm 1 of FB (but not the carbonyl vibra-
tions from the other analytes). It should be noted that the entire
spectral region with wavenumbers below 1700 cm 1 led to a de-
crease in the prediction ability of the model (Fig. 5).
Finally, an attempt to improve the model by outlier detection
was carried out based on the identication of samples with ex-
treme leverages, large residuals in the spectral data or large resid-
uals in the analytical concentration values, all with 95% condence
intervals. Nevertheless, no outlier was detected.
Validation of the multivariate model
Once the model was developed, the analytical validation is
essential for quality control purposes. All the multivariate gures
of merit estimated for this validation are listed in Table 1. The aver-
age trueness was evaluated through the parameters RMSECV,
Fig. 5. Results of siPLS2. The mean spectrum of 36 calibration samples and the average (three analytes) RSMECV values for each interval. The numbers at the squares bottom
indicate the number of LV for each interval and the dashed line indicates the RMSECV for the full spectrum model. The selected spectral regions are in light gray.
RMSEC (root mean square of calibration) and, mainly, RMSEP (root
mean square of validation). The estimated RMSEP for the three
analytes were between 0.69 and 0.34 mg/100 mg. The trueness
can also be evaluated through the relative errors of prediction for
each sample. The observed relative prediction errors for the valida-
tion samples were between +3.9% and 2.6% for PP, +5.3% and
3.9% for PZ, and +5.1% and 4.4% for FB. These results were con-
sidered acceptable, since between 60% and 70% of the validation
samples for each analyte presented relative errors below 2%, the
acceptable limits of trueness commonly adopted for univariate
methods in the pharmaceutical industry [50]. The precision was
assessed at two levels, based on the relative standard deviation
(RSD) for six replicates. The RSD for repeatability (intra-run preci-
sion) were between 1.5% and 1.8%, while for intermediate precision
(inter-run) they were between 2.2% and 3.3%. All of these values
are in accordance with the Brazilian regulations [37], which pre-
scribes a maximum RSD of 5%. The results of trueness and precision
allow assuring that the method was considered accurate. The line-
arity of multivariate methods can be evaluated through the plot of
the reference versus predicted values, as shown in (Fig. 6ac) for
PP, PZ and FB. The correlation coefcients (r) were 0.99 for PP
and FB, and 0.96 for PZ. The linearity of this method was assured
by verifying the random behaviors of the residuals distributions
for the three analytes, as can be seen in Fig. 6df. Considering
the linearity and accuracy evaluations, the working ranges of
this method were established from 19.00 to 39.0 mg/100 mg for
PP, 6.70 to 13.40 mg/100 mg for PZ, and 20.00 to 40.0 mg/100 mg
for FB.
The gures of merit selectivity (SEL), sensitivity (SEN), analyti-
cal sensitivity (c), limits of detection (LOD) and quantitation
(LOQ) were estimated based on the concept of NAS, as described
in the appropriate Refs. [25,41,42]. The interpretation of the SEL
concept for multivariate methods is different from univariate ones
and has no practical interest for quality control purposes. The SEL
denition is only useful within a certain group of samples of sim-
ilar qualitative composition and for this method its estimate indi-
cate that about 21%, 30% and 20% of the analytical signal were used
for predicting PP, PZ and FB, respectively. Since the pure SEN is not
appropriate for comparison with other methods, their values were
divided by the estimative of the instrumental noise (e = 7.3.10 4)
and the more useful c was estimated. The inverse of c indicated
that the method was able to discriminate minimum content
 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403 401

Table 1
Parameters estimated for validating the developed siPLS2-MIR method.

Figures of merit Parameter Value

PP PZ FB
Trueness RMSECVa 0.96 0.54 0.72
RMSECa 0.63 0.38 0.56
RMSEPa 0.67 0.34 0.69
Precision RSDrepeateability 1.82% 1.80% 1.53%
RSDintermediate.precision 2.49% 3.31% 2.20%
Linearity Slopeb 0.98 0.95 0.98
Intercepb 0.67 0.49 0.76
r 0.99 0.96 0.99
Rangea 19.0039.00 6.7013.40 20.0040.00
Selectivity 20.8% 20.1% 30.1%
Sensitivityc 0.03 0.03 0.05
Analytical sensitivity (c)d 41.1 40.2 68.2
1a
c 0.02 0.03 0.02
LODa 0.08 0.08 0.05
LOQa 0.24 0.25 0.15
Bias 0.189 0.612 0.075 0.723 0.046 0.215
RPDe Calibration 4.9 3.4 6.4
Validation 3.0 3.1 3.2
a
mg per 100 mg.
b
Values for the lines tted to the calibration samples.
c
Values expressed as the ratio between units of absorbance and (mg/100 mg).
d
(mg/100 mg) 1.
e
Dimensionless units.

Fig. 6. Plots of reference versus predicted values for the calibration (circles) and validation (down triangles) samples of (a) PP, (b) PZ and (c) FB. PLS residuals for the
predictions of (d) PP, (e) PZ and (f) FB.

differences between 0.02 and 0.03 mg/100 mg of the analytes, con-
sidering the random instrumental noise as the only source of er-
rors. Though not necessary for this kind of method, LOD and LOQ
were also estimated based on the e.
The bias should be estimated only for the validation samples
and the values in Table 1, along with their standard deviation, were
used in t tests with 20 degrees of freedom and at 95% condence
level. The estimated t values were all below the critical t value
(2.086), demonstrating the absence of systematic errors in the
model. The RPD51 is the ratio of natural variation in the samples
to the size of probable errors occurring during the prediction,
and it is more useful for comparing models on different data sets
or in absolute terms. It was calculated for the calibration and val-
idation sets and the minimum RPD was estimated as 3.0 for the PP
prediction in the validation set, which is above 2.4, the lower limit
desirable for good calibration equations [51].
Analysis of commercial tablets
The developed method was applied to the simultaneous quan-
tication of PP, PZ and FB in three different commercial formula-
tions (tablets), and the results were veried by independent
HPLCDAD and HPLCMS/MS methods. The samples were
analyzed in six replicates by the developed mid infrared method
and in triplicates by both the chromatographic methods. The pre-
dicted mean values and their standard deviations are shown in Ta-
ble 2. According to non-paired t tests with 7 degrees of freedom,
there were no signicant differences between the predictions of
the three methods for all the analytes in the formulations #1 and
#3, and for PP and FB in the formulation #2, at 99% condence level
(all the estimated t values below the critical t = 3.499). The results
for predicting PZ in the formulation #2 were not in agreement be-
tween the MIR and the chromatographic methods. Clearly, the pre-
diction of the MIR method was discrepant. A possible explanation
for this result is the exclusive presence of a palatabilizing agent in
the formulation #2, which is indicated in its package, but not iden-
tied. Since this substance was not included in the model and
could absorb overlapping selective PZ peaks, it would interfere spe-
cically in the quantication of PZ. For overcoming this problem, it
would be necessary to identify this substance and include it in the
experimental design. As we are not able to identify this palatabiliz-
ing substance, an alternative adopted was to try to predict PZ in the
formulation #2 by testing sub-models built with each one of the
three continuous spectral regions selected by siPLS (Fig. 5). This
was successfully achieved by selecting only the region between
3715 and 3150 cm 1, where PZ presents specic vibrations related
402 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403
Table 2
Mean values and standard deviations for the simultaneous determination of PP, PZ and FB in three commercial formulations by the HPLCDAD (n = 3), HPLC/MSMS (n = 3) and
the proposed MIR spectroscopy (n = 6) methods.
Label claim (mg/tablet) HPLCDAD (mg/tablet)
PP PZ FB PP PZ FB
144a 50 150 157.4 5.0 49.7 0.3 155.3 2.1 155.3 4.4
144b 50 150 153.8 6.2 49.8 0.2 158.3 3.0 154.7 7.7
144c 50 150 144.9 0.6 52.8 1.3 148.8 2.0 145.9 0.5
a
Commercial tablet brand #01.
b
Commercial tablet brand #02.
c
Commercial tablet brand #03.
to the rst overtones of the C@O stretching vibrations (Fig. 3b).
This sub-model used the same previous preprocessing, 4 LV and ac-
counted for 91.78% of the variance in X and 91.84% in Y. It pre-
dicted 51.0 2.9 mg/tablet of PZ in formulation #2, a value with
no signicant differences from the results obtained by HPLC
DAD, 49.8 0.2 mg/tablet, and HPLCMSMS, 45.1 0.5 mg/tablet
(Table 2), as veried by a t test at 99% condence level. The sub-
models built with the other two spectral regions provided poor
predictions for PZ in this formulation, indicating that they may
be prone to interference of the palatabilizing substance.
Conclusions
A diffuse reectance MIR spectroscopy method for the simulta-
neous determination of three API, pyrantel pamoate, praziquantel
and febantel, in veterinary formulations was successfully devel-
oped and validated through the estimative of multivariate gures
of merit. The use of siPLS2 allowed improving the model predic-
tions through the empirical selection of the most discriminant
spectral ranges. For real samples, the developed method provided
results statistically similar to those obtained by two different HPLC
methods in almost of all the cases and presented the advantages of
being more rapid (about ten times faster), less expensive, no need
for reagents or solvents and no generation of chemical waste. This
model was considered suitable for routine quality control determi-
nations in laboratories of industries and handling pharmacies. An
aspect that should be highlighted is that this model was developed
without direct access to the excipient composition of the commer-
cial formulations. The employed experimental design searched to
incorporate the most likely substances present in the excipients
in the adequate content ranges, in a situation of multi-product cal-
ibration. Since practically all the multivariate infrared (NIR and
MIR) models for direct API determinations have been developed
for a specic product, aiming at the industrial quality control, the
strategy of multi-product calibration by incorporating the compo-
sition of several formulations is suitable for the future develop-
ment of multivariate methods for public quality inspection
control by the ofcial organisms.
Acknowledgements
M.S.P thanks CAPES-REUNI and CNPq for fellowships.
References
[1] Q.A. McKellar, F. Jackson, Trends Parasitology 20 (2004) 456461.
[2] P. Taweethavonsawat, S. Chungpivat, P. Satranarakun, R.J. Traub, R. Schaper,
Parasitol. Res. 106 (2010) 533537.
[3] S.C. Su, H.H. Chou, P.C. Chang, C.H. Liu, S.S. Chou, J. Food Drug Anal. 12 (2004)
244253.
[4] A. Davis, D.H. Wegner, Bull. World Health Organ. 57 (1979) 767771.
[5] S.F. Andrade, Manual de Teraputica Veterinria, 2 ed., Roca, So Paulo, 2002.
[6] A.A. Fasanmade, A.O. Akanni, A.A. Olaniyi, A.A. Fasanmade, F. Tayo, Biopharm.
Drug Dispos. 15 (1994) 527534.
[7] R. Jain, N. Jadon, K. Radhapyari, Talanta 70 (2006) 383386.
HPLC/MSMS (mg/tablet) Multivariate model (mg/tablet)
PP PZ FB PP PZ FB
44.4 1.8 151.4 3.1 158.9 1.1 48.5 1.8 155.7 1.9
45.1 0.5 154.8 2.6 160.2 1.3 70.4 1.2 158.6 1.3
54.2 1.4 142.5 2.9 143.7 2.0 53.5 1.3 146.9 2.3
[8] G.A. Forcier, R.F. Mushinsky, R.L. Wagner Jr., J. Pharm. Sci. 60 (1971) 111113.
[9] M.S. Piantavini, F.L.D. Pontes, L.B. Cerqueira, P.G. Peralta-Zamora, R. Pontarolo,
Journal of Analytical Chemistry (2014). in press.
[10] European Pharmacopoeia, sixth ed., Strasbourg, France, 2007.
[11] M.M. Ghoneim, M.M. Mabrouk, A. Tawk, J. Pharm. Biomed. Anal. 30 (2002)
13111318.
[12] W.J. Allender, J. Chromatogr. Sci. 26 (1988) 470472.
[13] A.P. Argekar, S.V. Raj, S.U. Kapadia, Talanta 44 (1997) 19591965.
[14] W. Bialecka, A. Kulik, Acta Pol. Pharm. 67 (2010) 463468.
[15] J. Li, Y. Wang, A. Fenwick, T.A. Clayton, Y.Y. Lau, C. Legido-Quigley, J.C. Lindon, J.
Utzinger, E. Holmes, J. Pharm. Biomed. Anal. 45 (2007) 263267.
[16] J.Z. Sabbatini, J. AOAC Int. 92 (2009) 2633.
[17] V.A. Thorpe, J. Chromatogr. Sci. 26 (1988) 545550.
[18] S. Babic, D. Mutavdzic-Pavlovic, D. Asperger, M. Perisa, M. Zrncic, A.J. Horvat,
M. Kastelan-Macan, Anal. Bioanal. Chem. 398 (2010) 11851194.
[19] G. Balizs, J. Chromatogr. B 727 (1999) 167177.
[20] P.S. Bonato, A.R. de Oliveira, F.J. de Santana, B.J. Fernandes, V.L. Lanchote, A.E.
Gonzalez, H.H. Garcia, O.M. Takayanagui, J. Pharm. Biomed. Anal. 44 (2007)
558563.
[21] R.M. Lima, M.A. Ferreira, T.M. Ponte, M.P. Marques, O.M. Takayanagui, H.H.
Garcia, E.B. Coelho, P.S. Bonato, V.L. Lanchote, J. Chromatogr. B 877 (2009)
30833088.
[22] F.L.D. Pontes, R. Pontarolo, F.R. Campos, J.C. Gasparetto, M.A. Cardoso, M.S.
Piantavini, A.C.L.B. Trindade, Asian J. Pharm. Clin. Res. 6 (2013) 191200.
[23] M.H. Ferreira, J.F.F. Gomes, M.M. Sena, J. Braz. Chem. Soc. 20 (2009) 1680
1686.
[24] W.F.C. Rocha, A.L. Rosa, J.A. Martins, R.J. Poppi, Determination and validation of
nimesulide in pharmaceutical formulation by near infrared spectroscopy, J.
Braz. Chem. Soc. 21 (2010) 19291936.
[25] M.A.M. Silva, M.H. Ferreira, J.W.B. Braga, M.M. Sena, Talanta 89 (2012) 342
351.
[26] D. Xiang, J. Berry, S. Buntz, P. Gargiulo, J. Cheney, Y. Joshi, B. Wabuyele, H. Wu,
M. Hamed, A.S. Hussain, M.A. Khan, J. Pharm. Sci. 98 (2009) 11551166.
[27] A.A. Bunaciu, H.Y. Aboul-Enein, S. Fleschin, Appl. Spectrosc. Rev. 45 (2010)
206219.
[28] A.A. Kandhro, A.H. Laghari, S.A. Mahesar, R. Saleem, A. Nelofar, S.T. Khan, S.T.H.
Sherazi, Spectrochim. Acta Part A 115 (2013) 5156.
[29] S. Mazurek, R. Szostak, Vib. Spectrosc. 57 (2011) 157162.
[30] A.L.H. Mller, R.S. Picoloto, M.F. Ferro, F.E.B. da Silva, E.I. Mller, .M. Flores,
Drug Test. Anal. 4 (2012) 500506.
[31] F.E.B. Silva, M.F. Ferro, G. Parisotto, E.I. Muller, E.M.M. Flores, J. Pharm.
Biomed. Anal. 49 (2009) 800805.
[32] D.M. Haaland, E.V. Thomas, Anal. Chem. 60 (1988) 11931202.
[33] M. Blanco, M. Alcal, Eur. J. Pharm. Sci. 27 (2006) 280286.
[34] E. Micklander, K. Kjeldahl, M. Egbo, L. Norgaard, J. Near Infrared Spectrosc. 14
(2006) 395402.
[35] Y. Zheng, X. Lai, S.W. Bruun, H. Ipsen, J.N. Larsen, H. Lowenstein, I. Sondergaard,
S. Jacobsen, J. Pharm. Biomed. Anal. 46 (2008) 592596.
[36] G.E.P. Box, J.S. Hunter, Ann. Math. Stat. 28 (1957) 195241.
[37] Agncia Nacional de Vigilncia Sanitria, (ANVISA), Guia para Validao de
Mtodos Analticos e Bioanalticos, Resoluo RE no 899, Brazil, 2003.
[38] International Conference on Harmonisation, Tripartite Guideline Q2A Text
on Validation of Analytical Procedures, Fed. Regist., 60 FR 11260, USA, 1995.
[39] International Conference on Harmonisation, Tripartite Guideline Q2B
Validation of Analytical Procedures: Methodology, Fed. Regist., 62 FR 27464,
USA, 1997.
[40] M.H. Ferreira, J.W.B. Braga, M.M. Sena, Microchem. J. 109 (2013) 158164.
[41] A.C. Olivieri, N.M. Faber, J. Ferr, R. Boqu, J.H. Kalivas, H. Mark, Pure Appl.
Chem. 78 (2006) 633661.
[42] P. Valderrama, J.W.B. Braga, R.J. Poppi, Quim. Nova 32 (2009) 12781287.
[43] TRSL, Therapeutic Systems Research Laboratories. BCS (Biopharmaceutics
Classication System), <http://www.tsrlinc.com/resources/services/>,
(accessed October 2013).
[44] G.L. Amidon, H. Lennernas, V.P. Shah, J.R. Crison, Pharm. Res. 12 (1995) 413
420.
[45] M. Martinez, L. Augsburger, T. Johnston, W.W. Jones, Adv. Drug Deliv. Rev. 54
(2002) 805824.
[46] R.M. Silverstein, F.X. Webster, D.J. Kiemle, Spectrometric Identication of
Organic Compounds, seventh ed., John Wiley & Sons, New York, 2005.
 M.S. Piantavini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 396403 403

[47] F. Hegelund, R.W. Larsen, M.H. Palmer, J. Mol. Spectrosc. 247 (2008)
100114.
[48] A. Rinnan, F.v.d. Berg, S.B. Engelsen, TrAC Trends Anal. Chem. 28 (2009)
12011222.
[49] Standard Practices for Infrared Multivariate Quantitative Analysis E1655-05,
ASTM International, West Conshohocken, USA, 2012.
[50] L. Huber, Validation and Qualication in Analytical Laboratories, second ed.,
LabCompliance, New York, USA, 2007.
[51] P. Williams, Implementation of Near-infrared Technology, in: P. Williams, K.
Norris (Eds.), Near-infrared Technology in the Agricultural and Food
Industries, second ed., American Association of Cereal Chemists Inc, St. Paul,
USA, 2001.