European Journal of Pharmaceutical Sciences 165 (2021) 105939

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Design of experiments applied to stress testing of pharmaceutical products:

A case study of Albendazole
Jorge Armando Ardila a, Benedito Roberto de Alvarenga Junior a, Luis Cuadrado Durango a,
Frederico Luis Felipe Soares b, Bruno Perlatti c, Josiane de Oliveira Cardoso a,
Regina Vincenzi Oliveira a, Moacir Rossi Forim a, Renato Lajarim Carneiro a, *
a
Department of Chemistry, Federal University of São Carlos, 13565-905, São Carlos, São Paulo, Brazil
b
Department of Chemistry, Federal University of Paraná, 81530-900, Curitiba, Paraná, Brazil
c
Department of Chemistry and Biochemistry, University of California, 90095, Los Angeles, California, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Forced degradation tests are studies used to assess the stability of active pharmaceutical ingredients (APIs) and
Design of Experiment their formulations. These tests are performed submitting the API under extreme conditions in order to know the
forced degradation main degradation products in a short period of time. The results of these studies are used to assess the degra­
albendazole
dation susceptibility of APIs and to validate chromatographic analytical methods. However, most of degradation
degradation products
stress testing, Pummerer reaction
studies are performed using one-factor-at-the-time (OFAT) which does not consider the interactions between
degradation variables. This work proposes the use of Design of Experiment (DoE) approach in forced degradation
of albendazole (ABZ). It was used a central composite design (CCD) to evaluate the forced degradation in a
multivariate way. Experiments were performed taking into account the variables pH, temperature, oxidizing
agent (H2O2) and UV radiation. It was verified the influence of the variables and their interactions on the ABZ
degradation. The ABZ oxidation showed to be the main degradation route for ABZ, which is strongly influenced
by the temperature. The hydrolysis was relevant at alkaline medium and high temperature. LC-IT-MSn was used
to identify the degradation products. It was found three known degradation products (albendazole-2-amino,
albendazole sulfoxide and albendazole sulfone) and a new derivate of albendazole molecule (albendazole sulf­
oxide with a chlorine). This last one was isolated and characterized by UPLC-QToF-MS and NMR analyses.

1. Introduction Albendazole (ABZ), chemical name methyl 5-(propylthio)-2-benzi­

Forced degradation tests, or stress degradation studies, are common
and important tests performed by pharmaceutical companies to assess
the stability of active pharmaceutical ingredients (API), to know
degradation products and reveal the degradation pathway of every
compound yielded during the API degradation (Klick et al., 2005; Singh
et al., 2013a). These tests involve the exposition of an API under mod­
erate or extreme conditions, such as, temperature, oxidizing agents (e.g.
H2O2 solutions), UV radiation (usually from 290 to 400 nm), and basic
and acidic medium (usually HCl or NaOH 0.1 mol L− 1) (Baertschi et al.,
2011; Blessy et al., 2014; ICH, 2003). The results of these studies are
useful to determine the storage conditions, the shelf life of the API, the
formulation and also to validate analytical methods (Chen et al., 2014;
Singh et al., 2013b).
midazolecarbamate, is a drug marketed worldwide under various names
(Albenza, Alworm, Andazol, Eskazole, Noworm, Zentel, Alben-G, ABZ,
Cidazole, Wormnil and others). It is an anthelmintic compound
belonging to the class of benzimidazole methylcarbamates, used in both
veterinary and human treatment against different types of parasitic
worms. ABZ is one of the most important anthelmintic drugs, along with
praziquantel and ivermectin. Its major metabolite is the ABZ sulfoxide
(ABZ-SO), which is further metabolized to an apparently inactive form,
ABZ sulfone (ABZ-SO2) (Belaz et al., 2008; Zhang et al., 2011). The ABZ
stability is known and its metabolites can also be easily produced in
forced degradation reactions, since they are oxidation products of ABZ.
Forced degradation studies of ABZ has been performed for validation of
HPLC methods and some of its degradation were identified (Gomes and
Nagaraju, 2001; Jajikore et al., 2015; KALAS et al., 2016; Nikalje and

* Corresponding author.
E-mail address: renato.lajarim@ufscar.br (R.L. Carneiro).

https://doi.org/10.1016/j.ejps.2021.105939
Received 9 March 2021; Received in revised form 28 May 2021; Accepted 28 June 2021
Available online 17 July 2021
0928-0987/© 2021 Elsevier B.V. All rights reserved.
J.A. Ardila et al.
Gadikar, 2019; Patel Asmita et al., 2015).
In general, forced degradation studies are performed considering
only one variable at a time, this approach is commonly called one-factor-
at-the-time (OFAT, (Mäkelä, 2017; Organization, 2009). In fact, there
are no studies in literature related to forced degradation of albendazole
involving other approach (Ahmed et al., 2018; Attia et al., 2017; Ragno
et al., 2006). In this context, Design of Experiments (DoE) rises like an
useful tool that allow to study all variables simultaneously with a
pre-determined number of experiments (Leardi, 2009), and avoids waste
of time and chemical consumption by unnecessariness experiments
(Ferreira et al., 2018). Besides, when one variable is taken at the time in
the forced degradation studies, the variables are completely indepen­
dent and the interactions between degradation variables are not taken
into account (Hibbert, 2012; Sakkas et al., 2010). DoE approach allows
to assess the synergism between variables and statically quantify the
influence of each variable and its interactions on the system (Candioti
et al., 2014). Furthermore, as variables are evaluated simultaneously,
new degradation conditions are studied, which increases the possibility
to find new degradation compounds, as verified in this work.
DoE has been used in the drug-excipient compatibility studies as a
more realistic approach, where the physical and chemical stability of the
drug are evaluated in different mixtures using thermal analyses (Fer­
reira-Nunes et al., 2018; Lima et al., 2018; Pires et al., 2017). To the best
of our knowledge, there have been only few works in the literature
proposing the use of DoE for evaluation of variables in the forced
degradation studies. Among such works is Sonawane and Gide, which
used experimental design to bring about forced degradation of lulico­
nazole and evaluate the influence of degradation variables(Sonawane
and Gide, 2016). Kurmi et. al. applied DoE to develop the
stability-indicating method and also found the conditions of furosemide
in the degradation range of 20–30%(Kurmi et al., 2014).
Besides DoE, forced degradation studies can generate a large amount
of data and chemometric tools are important to extract useful chemical
information. Chemometric tools such as Principal Component Analysis
(PCA), Partial Least Squares (PLS), Artificial Neural Network (ANN),
Multivariate Curve Resolution (MCR), and their applications in the
forced degradation context are shown in the Roberto de Alvarenga Ju­
nior and Lajarim Carneiro’s review (Roberto de Alvarenga Junior and
Lajarim Carneiro, 2019).
Albendazole was selected in this work because it is widely used as an
anthelmintic with wide spectrum of activity, but no forced degradation
study using multivariate approach was reported in the literature so far.
We proposed the forced degradation study of ABZ using central com­
posite design (CCD) in order to evaluate the combination of stress pa­
rameters in the degradation profile. Additionally, liquid
chromatography coupled to an ion trap multiple-stage mass spectrom­
eter (LC-IT-MSn) was used for the identification of degradation products.
Nuclear magnetic resonance (NMR) and ultra-performance liquid
chromatography - quadrupole/time of flight mass spectrometry (UPLC-
QToF-MS) were employed to characterize a new product obtained in a
multivariate experiment at 60◦ C, where H2O2 and HCl were simulta­
neously present.
2. Material and methods
2.1. Materials
All reagents used in the experiments were of analytical grade (PA).
Albendazole (Sigma-Aldrich, ≥ 98 %) solutions for degradation were
freshly prepared. For these solutions, it was used ultrapure water (re­
sistivity > 18.2 MΩ cm at 25◦ C) from a Milli-Q system (Millipore®),
hydrochloric acid (HCl, Sigma-Aldrich, 37 %), sodium hydroxide
(NaOH, Synth, 97.0 %) and hydrogen peroxide (H2O2, Synth, 30 %).
Other reactants used in the work were Dimethyl sulfoxide (DMSO,
Sigma-Aldrich, ≥ 99 %), Dimethyl sulfoxide-d6 (Sigma-Aldrich, DMSO-
d6, 99.96 atom % D) and ethyl acetate (CH3COOC2H5, Sigma-Aldrich
2
European Journal of Pharmaceutical Sciences 165 (2021) 105939
99.8 %). For the chromatography methods it was used acetonitrile
(CH3CN, J. T. Baker, 99.9 %, HPLC grade) and formic acid (CH2O2,
Sigma-Aldrich 98 – 100 %, analytical grade).
2.2. Forced degradation studies using design of experiments
The degradation tests of albendazole were carried out using a
modified central composite design (CCD). The variables temperature,
pH, concentration of oxidizing agent (H2O2) and UV radiation (294 nm)
were evaluated while the degradation time was fixed at 60 min. The time
of degradation and the other experimental ranges were based on rec­
ommendations of ICH and mainly from previous studies in which
Albendazole did not present complete degradation. The continuous
variables were studied at five levels (-1, -0.5, 0, 0.5 and 1) and the
discrete one (UV radiation) was evaluated at two levels (presence “+1”
or absence “-1”).
The experiments performed with oxidizing agent were performed
only in absence of UV radiation, once this combination could lead to
non-selective oxidation reactions due to uncontrolled formation of hy­
droxyl radical. Table 1 shows the coded values of variables in the
experimental design. The degradation experiments were carried out
randomly in glass tubes of 10 mL in an oven with digital control of
temperature. The UV lamp was adapted in the oven to perform experi­
ments with simultaneous temperature and UV radiation. UV lamp was
turned off and tubes were wrapped with aluminum foil when UV radi­
ation was not desirable.
Table 2 presents the experimental setup for the 26 experiments. The
central points are the experiments 15, 16, 17 (without UV radiation),
and 26 (using UV radiation). The ABZ stock solution 1.0 mmol L− 1 was
freshly prepared in HCl 0.1 mol L− 1. An aliquot of 1.0 mL was diluted 10
times, being the pH adjusted with NaOH 0.1 mol L− 1 or HCl 0.1 mol L− 1
followed by addition of an aliquot of H2O2 30% to obtain the desired
concentration according to each experiment of Table 2. The final con­
centration of ABZ in the degradation experiments was 1 × 10− 4 mol L− 1
and degradation reaction time was 60 min, defined by previous assays
which demonstrate no excessive degradation of the API. After the re­
action time, tubes were placed in an ice bath and the solutions were
diluted using the mobile phase. The chromatographic analyses were
performed sequentially to the experiments.
2.3. HPLC-UV analyses
The chromatographic condition for the HLPC-UV determination of
ABZ and its degradation products was adapted from the literature (Wu
et al., 2005). HPLC analyses were carried out using an Agilent Tech­
nology chromatograph 1200 Series equipped with degasser, quaternary
pump, autosampler, column oven (35.0◦ C), UV/Vis detector settled at
294 nm and controlled by EZChrom SI software. A Phenomenex Gemini
C18 (150 mm × 4.6 mm i.d., 5 µm particle size) chromatographic column
was used. The mobile phase was 0.5 % (v/v) of acetic acid (solvent A)
and methanol (solvent B) and the following gradient program was
employed: 0 to 10 min, 30% B; 10 to 18 min, 30 to 50% B; 18 to 25 min,
50 to 30% B and from 25 to 30 min, 30% B. The flow rate was 1.0 mL
min− 1 and 15 µL of injection volume.
Table 1
Codification of variables for the central composite design (CCD).
Level Variable
pH H2O2 (%v/v) Temperature (◦ C) UV radiation
-1 1.0 0.00 30.0 no
-0.5 4.0 0.25 37.5 -
0 7.0 0.50 45.0 -
0.5 10.0 0.75 52.5 -
1 13.0 1.00 60.0 yes
J.A. Ardila et al. European Journal of Pharmaceutical Sciences 165 (2021) 105939

Table 2 media to 9 (in order to neutralize the degradation products). The organic
Conditions of each experiment performed for degradation of ABZ. phase was dried at room temperature and the product was solubilized
Exp. pH (x1) H2O2 (x2) Temperature (x3) UV (x4) Degradation (%) with DMSO for posterior chromatographic isolation.
The compound was isolated by fraction collection using an Agilent
1 -1 -1 -1 -1 0.22
2 1 -1 -1 -1 0.12 Technology chromatograph 1200 Series coupled to an Agilent Tech­
3 -1 1 -1 -1 46.42 nologies 1260 Infinity analytical-scale fraction collector, and controlled
4 1 1 -1 -1 11.56 by EZChrom SI software. A Xtimate C18 (150 mm × 4.6 mm i.d., 5 µm
5 -1 -1 1 -1 0.25 particle size) chromatographic column was used. The injection volume
6 1 -1 1 -1 5.36
7 -1 1 1 -1 80.00
was 50 µL and the mobile phase, column temperature, and flow rate
8 1 1 1 -1 52.19 were the same of chromatograph method of the Section 2.4 LC-IT-MSn
9 -0.5 0 0 -1 6.97 analyses. The compound was dried at room temperature and solubilized
10 0.5 0 0 -1 6.98 in DMSO for LC-MS analyses and DMSO-d6 for NMR analyses.
11 0 -0.5 0 -1 2.18
The QToF analysis was performed by a Waters UPLC-MS Acquity H-
12 0 0.5 0 -1 6.37
13 0 0 -0.5 -1 2.19 Class UPLC coupled to a Xevo-G2 Q-TOF MS/MS and electrospray mode
14 0 0 0.5 -1 8.00 for ionization. The separation was carried out using a Waters column
15 0 0 0 -1 8.08 UPLC-BEH C18 (1.7μm, 2.1 × 50mm). The gradient was the same of
16 0 0 0 -1 8.64 Section 2.4, flow rate, injection volume and column temperature were,
17 0 0 0 -1 10.50
18 -1 -1 -1 1 0.40
respectively, 100 µL min− 1, 1.0 µL and 35 ◦ C. For the mass analysis, an
19 1 -1 -1 1 0.43 ESI interface was selected and used in the positive ion mode with
20 -1 -1 1 1 0.36 capillary potential 1.2 kV, desolvation gas flow 750 L h− 1, cone voltage
21 1 -1 1 1 6.93 40 V, low and high collision energy 0 V and 5 V, respectively. It was used
22 -0.5 -1 0 1 0.15
MSE mode to collect data in range from 50 to 1000 Da and processed by
23 0.5 -1 0 1 0.15
24 0 -1 -0.5 1 0.18 Unifi 1.7 software (Waters, Milford, MA, USA).
25 0 -1 0.5 1 0.20 The NMR characterization was performed in a Bruker 400 MHz,
26 0 -1 0 1 0.17 model Avance III with auto sample, 5 mm BFO (smart probe with
ATMA®), unit generating of field gradient and unit of temperature
control. Approximately 4 mg of the isolated compound was solubilized
2.4. LC-IT-MSn analyses
in 700 µL DMSO-d6 and 1H NMR, COSY, and 13C NMR spectra were
acquired.
For the LC-IT-MSn analyses, the chromatographic conditions
described for the HPLC-UV detection was adapted in order to obtain
3. Results
higher ionization efficiency. LC-IT-MSn experiments were carried out
using a Shimadzu LC system (Kyoto, Japan) equipped with two LC-20AD
3.1. Design of experiment
pumps, a SIL-20A autosampler, a DGU-20A5 degasser and a CBM-20A
interface, coupled to a Brucker Esquire 6000 3D Ion Trap (3D IT) mass
Central composite design was divided in two blocks: the first one in
spectrometer (Bruker Daltonics, Bremen, Germany). Chromatographic
the absence of UV radiation and using H2O2 as variable (experiments
separation was performed using the same Phenomenex Gemini C18
from 1 to 17) and; the second one with UV radiation in the absence of
column at room temperature. A gradient elution was performed using
H2O2 (experiments from 18 to 26). Temperature and pH were evaluated
formic acid 0.3 % (v/v, pH 3.0, solvent A) and acetonitrile (solvent B) by
in both blocks. Figure 1 presents the chromatograms of the experiments
the following gradient program: 0 to 11 min, 15 to 85% B; 11.1 to 12.1
that presented more intense degradation.
min, 85 to 15% B; 12.1 to 15 min, 15% B. The mobile phase flow rate
Multiple regression and Analysis of Variance (ANOVA) were per­
was 100 µL min− 1 and sample injection volume was 20 µL.
formed on the two blocks of Table 2 in order to find the most important
An electrospray ionization (ESI) interface was selected and used in
stress variables and their interactions.
the positive ion mode. The 3D IT was operated under the following
conditions: nebulizer gas 30 psi, dry gas flow 7.0 L min− 1, dry gas
temperature 325◦ C and capillary potential 4.5 kV. For data acquisition,
the 3D IT was operated in the full-scan MS mode using an accumulation
time of 100 ms, a target of 30000, and an acquisition range from m/z 70
to m/z 400 in conjunction with data-dependent MS/MS acquisitions on
the most intense ions selected from the full-scan MS spectrum. Data
acquisition and processing were performed using the Data Analysis
software (Bruker Daltonics, Bremen, Germany).

2.5. Isolation and characterization of an unreported degradation product

An unreported degradation product found in this work, posteriorly

called ABZ-SOCl, was isolated and characterized by QToF and NMR.
In order to isolate this molecule for NMR characterization, it was
prepared 10 mls of ABZ solution 1.0 × 10− 2 mol L− 1 with 0.1 mol L− 1
HCl and H2O2 2 % v/v. The solution was transferred to glass tubes
wrapped with aluminum foil to avoid the presence of any UV radiation
and heated in oven at 70 ◦ C during 7 hours (a kinetic study showed the
decreasing of this compound after 7 hours, as presented in Figure S1 –
Supplementary Material). After the degradation, a preconcentration step Figure 1. UV-chromatograms (294 nm) of experiments: 5 (pH 1.0 at 60◦ C); 6
was performed using liquid-liquid partition with ethyl acetate (4:1 (pH 13.0 at 60◦ C); 7 (pH 1.0, H2O2 1.0 % at 60◦ C) and; 8 (pH 13.0, H2O2 1.0 %
degraded solution:ethyl acetate) after adjusting the pH of the reactional at 60◦ C). A1 to A4 are the degradation products.

3
J.A. Ardila et al. European Journal of Pharmaceutical Sciences 165 (2021) 105939

3.1.1. Experiments without UV radiation

For the experiments without UV radiation, it was found as significant
parameters: H2O2 (x2), Temperature (x3) and the interactions pH ×
H2O2 (x1x2) and pH × Temperature (x1x3). The Equation 1 presents a R2
0.751 (75.1 % of explained variance) at 90 % of confidence level:
% deg.ABZ = 15.06 + 21.91x2 + 9.69x3 − − 8.46 x1 x2 + 8.62 x1 x3 (1)
2
R and % of explained variance values represent the adjustment of an
equation to empirical results. Due to the high complexity of degradation
reactions and the simultaneous variation of three variables, it is not
expected to obtain a mathematical model which describes perfectly the
reaction. However, the mathematical model help us to understand the
behaviour of our degradation as function of the studied variables. In that
way, the R2 and % of explained variance value were consider satisfac­
tory. The 24.9 % of explained variance which was not explained came
from the lack of adjust among the empirical values and the predicted
values by the Equation 1.
Figure 2 shows the response surface for the Equation 1. It is worth to
say that, in the case of this study, ANOVA is used to identify the most Figure 3. Results of ABZ degradation with UV radiation. Points represent each
significant variables and a significative mathematical model is not experiment of experimental design.
mandatory.
Equation 1 shows that the increasing of H2O2 concentration and the
representation of ABZ degradation according to pH and temperature.
increasing of temperature promote a higher ABZ degradation, as ex­
Only experiment 21 showed a high degradation due to combination
pected. However, H2O2 presents more influence in the degradation
between variables pH 13, temperature at 60 ◦ C and UV radiation.
when changed from lower levels to higher levels. In fact, Table 2 shows
Again, such results corroborates that the multivariate approach is
experiments 7 and 8 presenting highest ABZ degradation, 80.00 % and
more adequate to perform degradation studies, since the isolated pH or
52.19 %, respectively, both performed at high levels for temperature
temperature did not show significant degradation.
and H2O2.
The lack of knowledge about the interaction of stress parameters can
The negative value for pH × H2O2 interaction indicates that higher
lead to problems during the manufacturing, where temperature is pre­
degradation is obtained in basic condition with low H2O2 concentration
sent in unit operations such as drying and compression (due to the
or acidic condition at high H2O2 concentration. The interaction pH ×
friction among particles and the heating of the tablet compression
Temperature indicates that basic medium and high temperatures
machine).
(higher levels) or acidic medium and low temperatures (lower levels)
promotes more degradation.
These results clearly show the advantage of performing the stress 3.2. Identification of degradation compounds
testing in a multivariate way, being possible to assess the interactions
among factors, which can be present during the product manufacturing In order to identify the degradation compounds observed at different
or during the product shelf life. This approach is also important to retention times, it was selected the degradation conditions from exper­
evaluate the critical process parameters (CPPs) and critical material iments 6, 7 and 8, since they contained all observed degradation prod­
attributes (CMAs) in the Quality by Design (QbD) approach (Roberto de ucts. In order to produce high amounts of degradation products, these
Alvarenga Junior and Lajarim Carneiro, 2019). experiments were performed during 120 minutes. Four degradation
products were found in the UV chromatographic method and detected in
3.1.2. Experiments with UV radiation the LC-IT-MSn method. These four products were named as A1, A2, A3,
For the experiments in absence of H2O2, it was evaluated UV radia­ and A4 and are presented in the ion chromatograms at Figure 4. As
tion, temperature and pH. It was not possible to obtain a significant mentioned in the topic 2.4, the chromatographic mobile phase was
model by ANOVA due to the lack of fit. However, it is possible to observe different for UV and LC-IT-MSn methods in order to improve the ioni­
in Table 2 that a significant degradation occurs only in the experiment zation when using the mass detector. The RTs for the UV method was
21 with 6.93 % of degradation, i. e. ABZ did not degrade easily at basic around 2.5 times the RTs for LC-IT-MSn method.
or acidic conditions at low temperature, but at basic pH (higher level) The experiment 6 (pH 13 and temperature 60 ◦ C) presented only the
and high temperature, the degradation occurs. Figure 3 presents a better compound A1 (RT 4.2 min, Fig. S2a – Supplementary Material). The
extracted ion chromatogram (EIC) of A1 is presented in Figure 4a and its

Figure 2. Surface response from Eq. 1 for ABZ degradation at pH levels -1, 0 and +1.

4
J.A. Ardila et al.
Figure 4. a) ABZ degraded in pH 13.0 and 60.0◦ C and selected ion chromatogram of A1 (C10H14N3S), (m/z 208.1). b) total ion concentration chromatogram of ABZ
degraded in pH 1.0, 60.0 ◦ C and 1.0 % H2O2 (30 % v/v) solution.
identification was performed by structure elucidation of the molecular
ion and subsequent fragmentation using MS2 experiment. The A1 pro­
tonated ion at m/z 208.1 and its MS2 fragmentation yielded fragments at
m/z 165.0 m/z (C7H7N3S) and m/z 133.1 (C7H7N3) suggesting a loss of
(C3H7) and (C3H7S), respectively (Fig. S2b – Supplementary Material). It
indicates that A1 comes from the hydrolysis of the amide group of ABZ,
yielding an amine known as ABZ-NH2.
The total ion chromatogram LC/MS and selected ion chromatogram
of compounds A2 and A3 in the experiment 8 (pH 13, temperature at 60
◦
C and 1% of H2O2) are presented at Fig S3 – Supplementary Material.
The retention time of ABZ was 8.4 minutes and its degradation products
A2 and A3 eluted at 5.4 and 6.7 minutes, respectively.
The MS2 and MS3 isolation and fragmentation of the ion m/z 266.1 at
8.4 minutes results in three product ions at m/z 234.1, m/z 192.0 and at
m/z 159.0, which could be easily assigned to the starting material
(Fig. S4 – Supplementary Material). Compound A2 showed a protonated
ion [M-H]+ at m/z 282.1, suggesting an oxidation step by the increase of
16 Da in the molecular mass of ABZ. The most common degradation
product of ABZ is the sulfoxide form (ABZ-SO, C12H15N3O3S) also known
as ricobendazole. The ABZ-SO originates from the oxidation of the sul­
fide group of ABZ yielding a sulfoxide group (Wu et al., 2005). The
multiple-stage fragmentation of the precursor ion m/z 282.1 produced
two product ions at m/z 240.1 and m/z 208.0 (Fig. S5 – Supplementary
Material). The MS2 spectrum showed a major product ion at m/z 240.0
(C9H10N3O3S) suggesting a loss of C3H6 [M+H-C3H6]+. The MS3
5
European Journal of Pharmaceutical Sciences 165 (2021) 105939
fragmentation of m/z 240.0 produced m/z 208.0, which suggests a loss
of CH3OH [M+H-CH3OH]+. The degradation product A2 was assigned
to albendazole sulfoxide, which was later confirmed in a comparison
with an ABZ-SO standard.
Compound A3 showed a protonated molecule at m/z 298.1 indi­
cating the introduction of two oxygen atoms in the ABZ molecule due to
an increase of 32 Da. The most probable product is the albendazole
sulfone (ABZ-SO2, C12H16N3O4S), where the sulfoxide group from the
ABZ-SO undergoes into another oxidation producing the sulfone. The
MS2 fragmentation of the precursor ion m/z 298.1 shows the product ion
at m/z 266.1 (C11H12N3O3S) [M+H-CH3OH]+ as the most abundant
fragmentation product ion. Further MS3 experiments show two product
ions at m/z 224.0 and m/z 159.0, suggesting a loss of [M+H-C3H6]+ and
[M+H-C3H7SO2]+, respectively. Therefore, the degradation product A3
was assigned to albendazole sulfone, ABZ-SO2. The mass spectra related
to the characterization of compound ABZ-SO2 is presented in Fig. S6 –
Supplementary Material.
Besides compounds A2 and A3 also detected in basic medium, the
acid conditions presented another degradation product (A4) eluted at
6.4 min (Figure 4b). For LC-IT-MSn analyses, the compound A4 showed a
protonated ion [M+H]+ at m/z 316.1 suggesting the introduction of a
chloride atom due to an increase of 35 Da in the ABZ-SO molecule. The
addition of a chloride in the structure of albendazole is not expected in
degradation reactions, so A4 was isolated and analyzed by NMR and
UPLC-QToF-MS/MS. By analyzing the isotope distribution pattern, the
J.A. Ardila et al.
A4 mass spectra clearly show the presence of a chlorine atom as see in
Figure 5, which shows the multiple-stage fragmentation for the pre­
cursor ion m/z 316.0515. The MS2 fragmentation shows the production
of m/z 240.0444 as the main product ion representing a loss of C3H5Cl
[M+H-C3H5Cl]+. The m/z 208.0177 is the ion product from a loss of
CH3OH [M+H-CH3OH]+. Both m/z 240.0444 and m/z 208.0177 suggest
that the chloride atom is in the aliphatic chain of ABZ. The exact mass
was compared with theorical exact mass and obtained an error below 5
ppm (-2.22 ppm).
The addition of a Cl atom (from the HCl) in the ABZ-SO molecule,
producing ABZ-SOCl (the A4 degradation compound), can be explained
by the Pummerer reaction. In this reaction the activated oxygen of the
sulfoxide is eliminated to give a thionium ion, which is attacked by a
nucleophile, in our case, the Cl− (Smith et al., 2010). In the presence of
an oxidizing agent, like H2O2 in the present reaction, the sulfur is again
rapidly oxidized to sulfoxide. Figure 6 shows a proposition of mecha­
nism for ABZ-SOCl formation, based on the Pummerer reaction.
The ABZ-SOCl is an unpublished compound obtained by
Figure 5. Q-TOF-MS analyses of ABZ-SOCl (A4) isolated. (a) Protonated molecular ion of ABZ-SOCl (C12H15N3O3SCl), [M+H]+ 316.0515. Insert isotopic contri­
bution of chlorine; (b) MS2 product ion spectrum for m/z 316.0515 and proposed fragmentation sites for the generation of the respective product ions.
6
European Journal of Pharmaceutical Sciences 165 (2021) 105939
combination of high temperature, oxidizing agent and HCl. Actually,
this compound is not considered a true degradation product of ABZ, but
an “artefact” of the forced degradation condition, since it could be pro­
duced only in the presence of Cl− ions and after ABZ oxidation to
sulfoxide.
1
3.3. H NMR and COSY analyses
The exact position of chlorine was verified and confirmed by 1H NMR
(Figure 7), COSY (Figure 8), and 13C NMR spectra (Fig S7 – Supple­
mentary Material).
The 1H RMN spectrum presented chemical shifts in δ 7.35 ppm (dd, J
= 8.3, 1.7 Hz, 1H); 7.56 ppm (d, J = 8.3 Hz, 1H) and 7.71 ppm (d, J = 1.6
Hz, 1H) related to aromatic ring characteristic of benzimidazole ring.
The chemical shift in δ 5.01 (dd, J = 9.2, 4.4 Hz, 1H) represents the effect
of the chlorine nucleus and δ 3.76 (s, 3H) indicates a methyl linked to
oxygen atom by single bond (OCH3). A signal in δ 3.3 was identified as
impurity by H2O or HDO from DMSO solvent. The signals in δ 2.15 (ddd,
J.A. Ardila et al.
Figure 6. Proposition of mechanism for ABZ-SOCl synthesis based on the Pummerer reaction.
Figure 7. 1H NMR spectrum of the ABZ-SOCl (400 MHz, DMSO-d6).
J = 14.4, 7.3, 4.5 Hz, 1H) and 1.72 (m, 1H) are related to a methylene
(CH2) with different chemical environment for each proton due to
presence of chlorine in the structure. The last one, a chemical shift in δ
1.07 (t, J = 7.3 Hz, 3H) indicates proton from methyl.
The COSY spectrum of ABZ-SOCl shows that protons of methyl (12)
are correlated to protons of methylene (11a and 11b) and it can be
observed in A and B points. The proton of α-carbon of sulfoxide (10) is
coupled to protons (11a and 11b) of methylene, which is represented by
signals in C and D in Figure 8. COSY spectrum presented no signal regard
to coupling between proton of carbon with chlorine and the protons of
methylene. Therefore, the COSY spectrum contributes to clarify the
position of chlorine is really in carbon adjacent to sulfoxide group of the
compound ABZ-SOCl.
4. Conclusion
Design of experiments showed to be an excellent tool to evaluate
stress test parameters in forced degradation studies. The DoE allows a
holistic evaluation of stress parameters and the obtention of a potential
degradation profile, since it consider the simultaneous presence of more
than one source of stress.
This approach allowed to observe that oxidation is the main degra­
dation route for ABZ, which is strongly influenced by temperature. Using
temperature at lower levels, significant oxidation only happens in the
7
European Journal of Pharmaceutical Sciences 165 (2021) 105939
presence of HCl, producing a new degradation product (actually, an
artefact). The hydrolysis reaction, in the absence of H2O2, is significant
only at high pH and temperature. UV radiation presented low influence
on ABZ degradation.
The degradation conditions yielded three known degradation prod­
ucts (ABZ-NH2, ABZ-SO, ABZ-SO2) and an unreported compound (ABZ-
SOCl). LC-IT-MSn experiments allowed to identify them and ABZ-SOCl
was characterized by UPLC-QToF-MS and NMR.
CRediT authorship contribution statement
Jorge Armando Ardila: Investigation, Formal analysis, Writing –
original draft. Benedito Roberto de Alvarenga Junior: Investigation,
Formal analysis, Writing – original draft, Writing – review & editing.
Luis Cuadrado Durango: Investigation, Formal analysis. Frederico
Luis Felipe Soares: Investigation, Formal analysis. Bruno Perlatti:
Investigation, Formal analysis. Josiane de Oliveira Cardoso: Investi­
gation, Formal analysis. Regina Vincenzi Oliveira: Resources, Super­
vision. Moacir Rossi Forim: Resources, Supervision. Renato Lajarim
Carneiro: Project administration, Resources, Conceptualization, Meth­
odology, Supervision, Writing – original draft, Writing – review &
editing.
J.A. Ardila et al.
Figure 8. COSY spectrum of the ABZ-SOCl.
Declaration of Competing Interest
None.
Acknowledgments
This paper was partially supported by the National Council for Sci­
entific and Technological Development (CNPq) and São Paulo Research
Foundation (FAPESP), processes 2010/16520-5 and 2017/13095-0.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2021.105939.
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