Beruflich Dokumente
Kultur Dokumente
, 2014.
ARTICLES
Antiparasitic drugs are commonly used in veterinary Toxascaris leonine [3]. The structures of the two chemicals
practice for the prevention and treatment of parasitic in are shown below. The use of combinations of anthelm
fections. Among the main groups of parasiticides are feb intics have been effectively used in veterinary treatment to
antel and pyrantel pamoate [1]. Febantel is a prodrug extend the spectrum of antiparasitic activity [4]. The liter
that is directly converted into the active metabolites fen ature has described several analytical methods for the de
bendazole or oxfendazole following administration [2]. termination of FB and PP in pharmaceuticals and other
FB acts by inhibiting the microtubule synthesis of the par
asite and is active against Ancylostoma caninum, Toxocara samples using spectrophotometry [5], potentiometry [6],
canis, Toxocara cati and Taenia spp [3]. Pyrantel pamoate voltammetry [7], HPLC [8–18] and LC–MS–MS [19–
is a cholinergic agonist and acts by inhibiting the neuro 22]. However, to the best of our knowledge, no analytical
muscular transmissions of the parasite [2]. It is effective method has been described that uses UV spectrophotom
against Ancylostoma caninum, Ancylostoma tubaeforme, etry for the simultaneous determination of FB and PP in
Uncinaria stenocephala, Toxocara canis, Toxocara cati and 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 949
Pyrantel
bration method used for building regression models is
partial leastsquares (PLS) [24]. The combination of
PLS and UV spectrophotometry has been used for the 7
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 5 7 9 11 13
ticles quantify two drugs in two different pharmaceuti Febantel
cal formulations (capsules and oral suspensions).
Therefore, there is a significant and a valuable applica Fig. 1. The composition (µg/mL) of calibration (䉬) and
tion of the methodology. prediction (䉫) set for spectrophotometric method.
Owing to the widespread use of FB and PP by the
pharmaceutical industry and magistral pharmacies, calibration models, 51 synthetic mixtures were pre
the aim of this work was to develop and validate a sim pared, according to a full experimental design, con
ple, reliable and cheap analytical method for quantify taining 6–12 µg/mL of FB and 5.76–11.52 µg/mL of
ing these drugs in antiparasitic products. PP, dissolved in diluent solution (Fig. 1). The model
ing amplitude was chosen based on the nominal con
EXPERIMENTAL centration in commercial medications and a maximal
variation of ±35%. Multivariate models were devel
Standards, samples, chemicals and reagents. Stan oped with 38 synthetic mixtures, reserving the other
dards of PP and FB were obtained from SigmaAld 13 as external validation set. The PLS models were ob
rich (St. Louis, MO, USA). Stock solutions of PP and tained using internal validation (cross validation
FB at concentrations of 1 mg/mL were prepared sep method – leave one out), preprocess for Yblock and
arately in a diluent solution of methanol and acetoni Xblock and different numbers of latent variables. Pre
trile (50 : 50, v/v) and stored protected from light at diction errors were expressed as RMSEP (root mean
4°C. From these solutions, an intermediate solution square error of prediction), defined as follows:
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 RMSEP =
∑ (y r − yˆ p )2
,
calibration and validation sets. Oral suspension and n
capsules were purchased from a magistral pharmacy where n is the number of samples, yr is the standard
located in Curitiba, Paraná state, Brazil. Acetonitrile (real) value and yˆ p is the value predicted by the model.
and methanol were HPLC grade. Pharmaceutical sample preparation. There are two
Equipment and software. Electronic absorption different pharmaceutical formulations available on the
measurements were carried out on a Shimadzu 1800 market that contain PP and FB in association: cap
double beam UVvis and a UVvis Agilent 8453E sules, 144 mg of PP and 150 mg of FB, and oral sus
spectrophotometer using 1.00 cm quartz cells. The pensions, 14.4 mg/mL of PP and 15 mg/mL of FB.
spectral data was recorded between 200 and 400 nm The excipient composition is not provided by industry,
using a spectral resolution of 0.5 nm. HPLC analysis so the mixtures were made based on the properties of
was performed using an Agilent 1100 HPLC System drugs solubility and permeability by the Biopharmaceu
(Wilmington, NC, USA). The analytes were separated tics Classification System [26]. The contents of 20 cap
on an XBridge C18 150 × 4.6 mm (5 µm) column cou sules were crushed and mixed into a homogeneous pow
pled with an XBridge C18 20 × 4.6 mm (5 µm) guard der. A mass equivalent to 100 mg of febantel and 96 mg
column. The injection volume was 10 µL, and the col of pyrantel pamoate was accurately weighed and trans
umn temperature was maintained at 40°C. Data ac ferred to volumetric flasks to obtain a final concentra
quisition was performed using ChemStation A.10.02 tion of 1 mg/mL of FB and 0.96 mg/mL of PP. The
software. The data treatment was carried out using oral suspension was diluted with a diluent solution to
Statistica 8 (StatSoft). PLS models were developed us obtain the same final concentration and the same pro
ing PLSToolbox 3.0 (Eigenvector Research, Inc.) op cedure as described for the capsules was subsequently
erating in Matlab 7.0.1 (Math Work Inc.). performed on the oral suspension solution.
Procedure. Calibration set, validation set and syn Analysis of the pharmaceutical samples. For the
thetic mixtures. For the development of multivariate electronic spectroscopic analysis, an aliquot of the fil
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 was found 5 × 10–3 µg/mL for FB and 5 × 10–3 µg/mL
mediate precision assessments gave RSD values lower for PP.
than 1.45% for FB and PP, indicating that the method Concerning to the robustness, the proportion of
is precise (Table 1). Recoveries were determined and solvents in the diluent solutions, the temperature and
gave values close to 100%. The RSD values were all the reading time showed no influence on the results of
lower than 1.94%, indicating good reliability and ac the proposed method. The ANOVA statistical method
curacy (Table 2) and in accordance with guidelines. was used to detect any significant differences. The re
The pvalues for the repeatability, intermediate preci sults showed that there were no significant differences
sion and accuracy indicate that the values are statisti among the three parameters studied.
cally identical.
The multivariate model was applied to quantify the
The determination of some figures of merit (FOM) FB–PP combination in commercial drugs (capsules
such as sensitivity and selectivity were estimated and and oral suspensions). The results (Table 3) from this
can be used to compare analytical methods and to model indicate a good agreement between the multi
compare calibrations constructed from spectroscopic variate and chromatographic methods, with low rela
data collected using two analytical instruments [29]. tive errors. According to the ttest the pvalue for the
When expressing FOM for multivariate calibration comparison HPLC and UV used to study PP in cap
methods, the part of the signal that relates uniquely to sules and for FB in oral solution were up to 0.05.
the analyte of interest is more important than the total Therefore the values were statistically equal to the true
signal. This unique signal is termed net analyte signal value. The pvalues for the comparison HPLC and UV
and is defined as the part of the signal that is orthogo for FB in capsules and PP in oral solution were 0.035
nal to the signal of the interferences present in the and 0.028, respectively. This means that the values are
sample [30, 31]. The results for selectivity were 32.80% statistically different, but analytically they are very
(FB) and 38.95% (PP). The sensitivities were 1.21 similar.
(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 PLS, a powerful and widely used tool in multivari
cific technique employed. For inverse of sensitivity ate calibration methods, was successfully employed
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 15. Sabbatini, J.Z., J. AOAC Int., 2009, vol. 92, p. 26.
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(capsules and oral suspensions). The calibration mod Chou, S.S., J. Food Drug Anal., 2004, vol. 12, p. 244.
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and meets the requirements imposed by ANVISA and 18. USP, The United States Pharmacopoeia, Rockville,
ICH. Therefore, this method could be easily used as 2008.
an analysis technique in the quality control laborato 19. Babic, S., Mutavdzic Pavlovic, D., Asperger, D., Perisa, M.,
ries of industries and manipulation pharmacies. Zrncic, M., Horvat, A. J., and KastelanMacan, M.,
Anal. Bioanal. Chem., 2010, vol. 398, p. 1185.
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