Co-determination of two antiparasitics drugs by derivative spectrophotometry and its photodegradation studies

Article abstract of DOI:10.4067/S0717-97072013000300006

This work proposes a method for the co-determination of praziquantel and fenbendazole in veterinary pharmaceutical formulations (VPF) by second-derivative spectrophotometry at 226.4 nm (Zero-crossing method) and 324.6 nm (Graphic method), respectively. The excipients commonly used in VPF do not interfere. To obtain the optimal determination conditions, studies of solvent and light effects, were performed. The results of the light effects show that the expose of both drugs to indirect light and darkness does not produce any chemical change, since their zero-order spectra remained unchanged during the experiment. However, the exposition to the direct light only affects the fenbendazole. The zero order spectrum of FBZ in EtOH-HCl, present two broad bands (200 to 240 nm and 260 to 330 nm), these signals disappear and appear others totally different, at 230 nm and other with two peaks (285 and 295 nm). The FT-IR spectrum shows characteristics signals of the FBZ structure as the amide-NH group (3336.3 cm-1), this signal disappear indicating the bond break of the amide group caused by the exposition to sunlight for a time. These spectral changes evidenced a structural transformation as a result of photochemical decomposition in acid medium.

Full text of DOI:10.4067/S0717-97072013000300006

J. Chil. Chem. Soc., 58, Nº 3 (2013)
CO-DETERMINATION OF TWO ANTIPARASITICS DRUGS BY DERIVATIVE SPECTROPHOTOMETRY
AND ITS PHOTODEGRADATION STUDIES
CÉSAR SOTO*1, ROMINA OTIPKA2, DAVID CONTRERAS1, JORGE YÁÑEZ 1, M. INÉS TORAL 2
1Department of Analytical and Inorganic Chemistry, Faculty of Chemical Sciences, University of Concepción, PO Box: 160-C, Concepción, Chile.
*1Address correspondence to this author at the Department of Inorganic and Analytical Chemistry, Faculty of Chemical Sciences,
University of Concepción, PO Box: 160-C, Concepción, Chile.
2Department of Chemistry, Faculty of Sciences, University of Chile, PO Box: 653, Santiago, Chile.
(Received: March 3, 2013 - Accepted: June 12, 2013)
ABSTRACT
This work proposes a method for the co-determination of praziquantel and fenbendazole in veterinary pharmaceutical formulations (VPF) by second-derivative
spectrophotometry at 226.4 nm (Zero-crossing method) and 324.6 nm (Graphic method), respectively. The excipients commonly used in VPF do not interfere.
To obtain the optimal determination conditions, studies of solvent and light effects, were performed. The results of the light effects show that the expose of both
drugs to indirect light and darkness does not produce any chemical change, since their zero-order spectra remained unchanged during the experiment. However, the
exposition to the direct light only affects the fenbendazole. The zero order spectrum of FBZ in EtOH-HCl, present two broad bands (200 to 240 nm and 260 to 330
nm), these signals disappear and appear others totally different, at 230 nm and other with two peaks (285 and 295 nm). The FT-IR spectrum shows characteristics
signals of the FBZ structure as the amide-NH group (3336.3 cm-1), this signal disappear indicating the bond break of the amide group caused by the exposition to
sunlight for a time. These spectral changes evidenced a structural transformation as a result of photochemical decomposition in acid medium.
Keywords: Fenbendazole; Praziquantel; Derivative Spectrophotometry; Veterinary Formulations; Photochemical Decomposition.
(CASDPV)13. In feeds and pharmaceutical formulations by techniques such
INTRODUCTION
as, CASDPV13, colorimetric method14, second derivative spectrophotometry
and multivariated calibration methods15, amperometric detection with
The antiparasitic are drugs that have been used for about 50 years in crop
flow injection16, HPLC capillary electrophoresis17, HPLC18,19, gas liquid
protection, human and animal health. The treatment of antiparasitic diseases is a
topic of great importance in these days for the pharmaceutical industry, because
chromatography (GLC)20, densitometric determination21, voltammetric
determination with poly(3-methylthiophene)-modified glassy carbon
electrode22, voltammetric determination16, 23, voltametric stripping methods24
and polarographic assay25.
it becomes increasingly necessary to improve the pharmaceutical formulations
due to the mix presentation of these diseases in many animals. The compounds
of antiparasitic can include benzimidazoles and pyrazinoisoquinoline
derivatives. The antihelminthics are widely used in veterinary medicine to
treat gastrointestinal infections1 and in food-producing animals, and therefore,
could be hazardous to humans. Part of the clinical efficacy of the antiparasitic
depends on its penetration and of concentrations achieved within the cells
of the parasite. The application of the treatment in young cattle, produce an
antiparasitic immunity. Since the antihelminthic spectra of most drugs used
for treatment is limited, combinations of more than one active ingredient are
required to control helminthic infections2 .In this context the mix of two drugs
such as Fenbendazole, methyl N-(6-phenylsulfanyl-1H-benzoimidazol-2-yl)
carbamate, (FBZ) (Fig.1a), this drug is thought to bind to tubulin and thereby
preventing its polymerization to form microtubules, but it also inhibits fumarat
reductase and glucose transport. As a result, parasites may die of starvation3
and Praziquantel, (RS)-2-(Cyclohexyl-carbonyl)-1,2,3,6,7,11,b-hexahydro-
4H-pyrazino (2,1-alpha) isoquinoline-4-one, (PZQ) (Fig.1b), is a synthetic
antihelminthic agent, used against many cestodes, and for the treatment of
schistosomiasis, as well other fluke infections pathogenic to humans4, 5 , are
commonly used in domestic animals for the prevention and control of a wide
variety of parasitic diseased, in a pharmaceutical combination with a 1:10 mass
ratio.
There are studies where is proved that these drugs are more effective
together than separately, one example of that is an evaluation of therapeutic
effects against coenurosis in sheep6 this study indicates the efficacy of co-
administration of fenbendazole together with praziquantel against coenurosis,
highlighting the benefits to the use of the combination of both drugs. The
evaluation of the veterinary preparations require of the determination of their
active ingredients in different samples such as drug formulations and biological
matrices. Numerous methods for the quantification of either one or a mixture
of FBZ and PZQ with others compounds has been reported in the literature,
in biological matrices using techniques such as, liquid membrane and solid
phase extraction with liquid chromatography electrospray mass spectrometry
(LC ES MS)7 liquid membrane extraction and liquid chromatography
(LC)8 , high performance liquid chromatography (HPLC)9, LC10,11 polymer
solid phase extraction coupled to square wave voltammetry at carbon fibre
microelectrodes12, cathodic adsorptive stripping differential pulse voltammetry
Figure 1. Molecular structure for (a) FBZ and (b) PZQ.
In veterinary pharmaceutical formulations, only the British Pharmacopoeia
presents an official method for FBZ assay26. In 33th United States Pharmacopoeia
version, has been reported methods for the individual determinations of PZQ
and FBZ, in pharmaceutical formulations for human treatments27, 28. Although
the determination of these drugs has been reported by several methods in
conjunction with other veterinary drugs, there are not reported methods
that consider the simultaneous determination of these drugs alone. The aim
e-mail: cesarsoto@udec.cl
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
of this study was to develop and to validate analytical methods for the co-
determination of FBZ and PZQ by derivative spectrophotometry in veterinary
pharmaceutical formulations. This technique has been used successfully in the
for these drugs (Fig. 2). It was also made a study of solvent in the pharmaceutical
formulations, using EtOH-HCl 0.01 and 0.1 mol/L. The result shows that in
EtOH-HCl 0.01 mol/L the tablets were slightly soluble and in EtOH-HCl 0.1
mol/L were completely soluble. The solvent select for these simultaneous
determinations was EtOH-HCl 0.1 mol/L, because analytical signals are higher
and defined for both drugs and the pharmaceutical formulation.
determination of the others drugs in different samples29, 30
.
EXPERIMENTAL
Instruments
Table 1. Qualitative studies of the solubility of PZQ and FBZ 1.0 x 10-3
Absorption spectra were recorded on
a
Shimadzu UV-1603
mol/L.
spectrophotometer. All samples were measured using a 1 cm path-length quartz
cuvette. For all the tested solutions, the second derivative spectra were recorded
on the range between 190-400 nm against solvent, using 0.2 nm sampling
intervals and scanning of 480 nm/min. The spectral data were processed by
software Shimadzu kit version 3.7 (P/N 206-60570-04). All solid samples were
weighted with a 0.01 mg of uncertainty using a Sartorius R 200D balance.
Solvent
Acetonitrile
PZQ
Not soluble
Slightly soluble
Slightly soluble
Soluble
FBZ
Not soluble
Slightly soluble
Slightly soluble
Soluble
Ethanol
Methanol
Reagents
Analytical standards of PZQ and FBZ were purchased from Sigma-
Aldrich®. Stock solutions of PZQ and FBZ were prepared by dissolving
the accurately weighed reference compounds in HCl 0.10 mol/L in ethanol
solution (EtOH-HCl) to give a final concentration of 1.0 x 10-3 mol/L. These
solutions were then serially diluted with the same solvent to achieve other
ranges of concentrations for standard working solutions. The pharmaceutical
product, Fentel® (Laboratory KUALCOS SRL, BA, Argentina) containing
both compounds was also dissolved using the same solvent. Furthermore, in
order to carry out a study of solvent, solutions of PZQ and FBZ were prepared
dissolving the amount previously described for each drug in different solvents.
HCl 0.01 mol/L in ethanol
HCl 0.01 mol/L in methanol
HCl 0.1 mol/L in ethanol
HCl 0.1 mol/L in methanol
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Procedures
Calibration graphs of FBZ and PZQ in mixtures
Aliquots of the stock solution of PZQ and FBZ were simultaneously
diluted in EtOH-HCl 0.1 mol/L to obtain a concentration range between 1.0
x 10-6 – 6.0 x 10-6 mol/L for PZQ and 1.0 x 10-5 – 6.0 x 10-5 for FBZ. The
calibration procedures were determined for each drug in the presence of 5.0
x 10-5 mol/L of FBZ and 5.0 x 10-6 mol/L of PZQ, respectively. In all cases,
the corresponding absolute values of the second derivative spectra at 226.4
nm for PZQ and 324.6 nm for FBZ, were obtained and plotted versus the
corresponding concentrations. The exposure to direct light (sunlight) of all
working solutions, for a long time must be avoided.
Simultaneous determination of PZQ and FBZ in synthetic samples
The stock solutions of each drug were appropriately diluted to obtain
solutions that contain molar ratios between 1:4 and 1:14 (PZQ: FBZ). For each
solution, three repetitions were made. Then, the second derivative spectra were
evaluated and the concentrations of each compound were calculated, allowing
the determination of the corresponding values of recovery and relative standard
deviation (RSD).
Figure 2. Zero-order spectra of PZQ and FBZ in EtOH-HCl 1.0 mol/L.
PZQ 5.0 x 10-5 mol/L and FBZ 5.0 x 10-5 mol/L.
Photo-stability studies of PZQ and FBZ
Individual solutions of PZQ and FBZ containing 4.0 x 10-5 mol/L of each
drug in EtOH-HCl 0.1 mol/L solution were used in photo-stability studies. The
solutions were exposed to direct light, indirect light and darkness. The zero-
order spectra of each drug were evaluated every 30 min over 5 hours.
Photo-stability studies
In order to establish the photo stability of PZQ and FBZ in EtOH-HCl
0.10 mol/L, a 4.0 x 10-5 mol/L solution for each individual drug was used. All
solutions were exposed to: direct light (sunlight), indirect light (laboratory
light) and darkness for 5 hours by measuring the absorbance every 30 minutes.
When exposed to indirect light and darkness none of the drugs change, since
their zero-order spectra remained unchanged during the experiment. But when
exposed to direct light only FBZ spectral behavior was altered (Fig. 3).
Simultaneous determination of PZQ and FBZ in pharmaceutical
formulation (Tablet)
Ten Fentel® tablets were weighed and finely powdered. The average
weight of tablets was determined with the help of the weight of 10 tablets.
The nominal contain is 500 mg of FBZ and 50 mg of PZQ in the tablet. A
quantity of 15 mg of powder was weighed and diluted in 50 mL of EtOH-HCl
0.1 mol/L. The solution was in a sonic bath for 30 min.
The zero order spectra of FBZ in EtOH-HCl 0.1 mol/L, has features such
as signals two broad bands between 200 and 240 nm and between 260 and
330 nm, as this drug is degraded photochemically, these signals disappeared
and two bands appear, one centered at 230 nm and the other with two peaks
centered at 285 and 295 nm. These spectral changes evidenced a structural
transformation as a result of photochemical decomposition in acid medium.
Because of this, FBZ and FBZ degraded were analyzed by FT-IR (Fig. 4).
RESULTS AND DISCUSSION
Solvent selection and spectral behavior
The selection of solvent was based in find a solvent that satisfy the
solubility of both drugs for the laboratory works. The spectral behavior should
also be adapted to carry out the simultaneous determination for derivative
spectrophotometry, developing a method, according to the analytical aims, of
precision and accuracy. The solvents evaluated in this study for both drugs are
shown in the Table 1. Using EtOH-HCl 0.1 mol/L the solubility and spectral
behavior of both drugs shows the possibility of the simultaneous determination
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
The IR spectrum shows signals of structural characteristics FBZ,
highlighting the amide-NH group to 3336.3 cm-1 31, because this signal is not
present in the FT-IR spectrum of FBZ degraded, indicating the existence of
bond breaking of the amide group as a result of “stress” caused by sunlight for
a time. Thus it is postulated that this break is of type photochemistry, since,
when applied indirect light and darkness, no change in the spectral signals.
Table 2 summarizes the assignment of FT-IR signals to FBZ and degraded
FBZ. It is known that elucidate the mechanism of a photochemical reaction, has
a high difficulty due to the high reactivity of the radicals formed, since these
can react with any other species present in the reaction medium. Based on this,
and knowledge of UV-Vis spectra start and end of the study, it was postulated
a radical type reaction. Fig.5 presents possible products. It should be noted that
the method of analysis developed, is not invalidated, with the results previously
obtained only if the solutions are not exposed to direct sunlight, since, when
working under indirect light or darkness, the drug did not show any alteration
in their spectral behavior. In fact, when the DU is plotted against time, there
is constancy in the concentration of both drugs, confirming that the drug is
degraded only if exposed to direct light (Fig. 6).
Table 2. Assignment of the functional groups in the spectra FT-IR for FBZ
and degraded FBZ.
1/l (cm-1)
FBZ
1/l (cm-1)
Degraded FBZ
Functional groups
Tension C-H
-N-H Amide
-C-N
3050.9-2954.5
3336.3
1442.5
1708.6
1222.7
736.7
3062.5-2985.3
-
1461.8
1743.4
1223
-C=O
-C-O
Fenil
752.1
Fenil-(o)
Bencenthiol
-O-H Carboxílic
1099.2
690.4
1083.8
694.3
-
3063.5-2831
Figure 3. Effect of exposure time to direct light (sunligth) on zero-
order spectra of PZQ and FBZ in EtOH-HCl 0.1 mol/L, respectively. Drugs
concentration 4.0 x 10-5 mol/L. (a) PZQ (A) 0 h, (B) 1 h, (C) 3 h and (D) 5 h.
(b) FBZ (E) 0 h, (F) 1 h, (G) 3 h and (H) 5 h.
Figure 5. Schema of reaction postulated for the photochemical degradation
of FBZ.
Spectral features
PZQ and FBZ were directly evaluated against the solvent selected, PZQ
showed one maximum absorption peak centered at 205.8 nm; under identical
conditions FBZ presented three maximum absorption peaks centered at 304.4,
229.6 and 208.8 nm (Fig. 2). Derivative spectrophotometry technique was used
for the simultaneous determination of both drugs, since the spectral bands of
the drugs under study are highly overlapping.
Figure 4. FT-IR spectra of: (a) FBZ recently prepared. (b) FBZ after 5 h
of exposure to direct light (sunlight).
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
Figure 6. Photo-stability studies: (a) Indirect light (Laboratory light). (b)
Darkness.
Selection of spectral variables
Figure 7. Derivative spectra of PZQ and FBZ in HCl 0.1 mol/L in ethanol
solution. (a) First order. (b) Second order. For both derivatives, FBZ: (A) 3.0
x 10-5 (B) 4.0 x 10-5 (C) 5.0 x 10-5 mol/L, PZQ: (D) 3.0 x 10-5 (E) 4.0 x 10-5 (F)
5.0 x 10-5 mol/L.
Derivative order
Different derivative orders of the spectra were obtained digitally from zero-
order spectra, this is based on a mathematic computational model proposed by
Savitzky and Golay32, sacrificing the spectral resolution in order to increase
strongly the signal/noise ratio, favoring the accuracy and the precision of the
results and of the method, respectively. To select the optimum derivative order,
the first to the fourth derivative spectra of the solutions containing separately
the respective drugs were recorded. In Fig. 7 the first and second derivative
are shown; third and fourth derivative order were discarded because the
sensibilities and signal/noise ratio decreases. The first derivative spectra do not
present zero crossing or zone characteristics for the simultaneous determination
of both drugs, only the FBZ determination by graphic methods between 305.0
and 340.0 nm is possible. The first derivative is more sensitive that the second
order, but this, present wavelength values for the simultaneous determination;
for PZQ shows a zero-crossing at 226.4 nm and FBZ at 324.6 nm can be
determined by graphic method. Because the resolution was satisfactory the
second order derivative was selected.
Analytical wavelength selection
The analytical wavelength selection was carried out taking into account
the second derivative signal of both analytes individually. FBZ determination
was carried out by zero-crossing at 218.9, 266.2 and graphic methods between
320.0 and 330.0 nm (Fig. 7 b). 324.6 nm present analytical signals higher
and well defined, furthermore presents the higher proportionality between
DU and analyte concentration. Based on this, 324.6 nm was selected to the
FBZ determination. FBZ second derivative signal show only one zero-
crossing at 226.4 nm to the PZQ determination with high sensibility (Fig. 7
b). Based on this, 226.4 nm was selected to the PZQ determination. In order
to select suitable analytical wavelengths is important to study the effect of
the concentration of each drug on the other, in pharmaceutical formulation
Fentel®, with a molar ratio PZQ: FBZ of 1:10. Different mixtures contained
both drugs, with concentrations of FBZ between 1.0 x 10-5 to 5.0 x 10-5 mol/L
and constant concentration PZQ 5.0 x 10-6 mol/L, including the mass ratio
contained in pharmaceutical formulations. After were evaluated used the
optimum parameters, second-order derivative, smoothing factor 8,000 and
scale factor 100,000. The Fig. 8 includes spectra of a mixture of drugs with
the pharmaceutical formulation mass ratio PZQ: FBZ and solution prepared
from the tablet dissolved under the selected conditions containing: 5.0 x 10-5
mol/L of FBZ and 5.0 x 10-6 mol/L of PZQ. The independence of the analytical
wavelength values selected of the drugs concentration, is experimentally
demonstrated in Figure 8, both spectrum are similar. In these real samples
also is possible the simultaneous determination of the drugs because the other
compounds presents in the tablet do not affect the determination.
Selection of the smoothing and scale factor
By using the derivative order selected (second), the smoothing factor could
be optimized when the values defined by defect are varied, according to ∆λ
value used to obtain the spectra. For a ∆λ of 150 nm the smoothing factor can
be studied by taking the following values: 2,000; 4,000; 8,000 and 16,000. The
smoothing factor increment produces a decrease in the heights of derivative
signal and a higher signal/noise ratio. The only smoothing factor value that
produce a high signal/noise ratio was 8,000, for this reason was selected.
The scale factor must be studied to determine whether the system presents a
distortion effect. To facilitate, the reading of the analytical signal, eliminate
the distortion effects and not to affect the sensibility, a value of 100,000 was
selected as scale factor.
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J. Chil. Chem. Soc., 58, Nº 3 (2013)
Table 4. Recovery rates of PZQ and FBZ in different proportions.
PZQ:FBZ
1:4
PZQ ( % recovery)
94.6 ± 6
FBZ (% recovery)
110.1 ± 3
1:6
96.0 ± 5
116.9 ± 1
1:8
98.4 ± 3
101.8 ± 1
1:10
1:12
1:14
99.4 ± 2
98.1 ± 1
115.1 ± 14
107.0 ± 6
107.4 ± 1
104.7 ± 4
Application of the proposed method
In the commercial pharmaceutical formulation Fentel® the developed
methods was applied. This formulation has a tablet average weight of 860 mg,
containing nominally 500 mg of FBZ and 50 mg of PZQ. The results were
satisfactory (Table 5).
Table 5.Simultaneous determination of PZQ and FBZ in veterinary
pharmaceutical formulations.
Fentel®
(nominal content mg)
Amount found per tablet
mg
Figure 8. Spectra of the pharmaceutical formulation (A) and the synthetic
Drug
mixture 5.0 x 10-5 mol/L of FBZ and 5.0 x 10-6 mol/L for PZQ (B).
FBZ
PZQ
500
50
490 ± 6
49 ± 1
Analytical features
Using the optimal parameters selected, a linear relation was observed
between corresponding DU and the respective analytes concentrations.
Calibration graphs were obtained by plotting the second derivative values for
FBZ, DU (λ= 324.6 nm) and PZQ, DU (λ= 226.4 nm), using a smoothing factor
of 8,000, a scale factor of 100,000 and a ∆λ of 150 nm, versus the respective
drug concentrations. The criteria of the ICH Harmonized Tripartite Guideline
were used for to obtain the analytical features. The following equations were
used: 3σ/S and 10σ/S, respectively, where S is the slope of the calibration curve
and σ the standard deviation corresponding to the response of 11 blanks to
calculate the DL and QL.
The repeatability, expressed as relative standard deviation (RSD), was
obtained by using 9 standard samples containing a mixture of the drugs in
proportion 1:4, 1:6 and 1:9 (PZQ:FBZ). The determination range was defined
between the QL and the loss of linearity. In this context, it is possible to obtain
and an acceptable linearity degree with accuracy and precision in the results
when the proposed method is applied. The results are show in Table 3.
CONCLUSIONS
The spectral behavior for both drugs is different and according to the
results obtained the simultaneous determination of the binary mixtures of FBZ
and PZQ by second derivative spectrophotometry is effective. The proposed
methods are accurate, precise, simple, less time-consuming, inexpensive and
more sensitive. The recoveries of FBZ and PZQ from the synthetic samples,
including the proportion contained in veterinary commercial pharmaceutical
formulation, were near 100%. The amounts found for both drugs are very
similar to the nominal content in commercial tablets. Excipients contained in
pharmaceutical matrix not affect significantly on the determination of the two
analytes. Moreover the higher mass ratio present in tablets (PZQ: FBZ (1:10))
was overcome successfully.
ACKNOWLEDGEMENTS
Table 3. Analytical features.
The authors declare no conflict of interests and are grateful to the
Dirección de Investigación de la Universidad de Concepción (DIUC) Project
Nº 210.021.029-10, and the Fondo Nacional de Investigación en Ciencia y
Tecnología (FONDECYT), Project Nº 1100103, for the financial support.
Analytical features
PZQ
FBZ
Detection limit (mol/L)
6.31 x 10-8
1.96 x 10-8
Quantification limit
2.10 x 10-7
6.54 x 10-8
(mol/L)
Determination range
(mol/L)
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