Thermal Analysis of Albendazole investigated by HSM

Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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Thermal Analysis of Albendazole investigated by HSM, DSC and FTIR
J.R. Moyano1, J. Liró1, J.I. Pérez1, M.J. Arias1 and P.J. Sánchez-Soto2
1
Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, c/Profesor García
González 2, 41012 Sevilla, Spain, email: jrmoyano@us.es
2
Instituto de Ciencia de Materiales de Sevilla (ICMS), CSIC-Universidad de Sevilla, Centro de Investigaciones Científicas
Isla de La Cartuja de Sevilla, c/Américo Vespucio 49, 41092 Sevilla, Spain, e-mail: pedroji@icmse.csic.es
The thermal behavior of albendazole, an antihelmintic drug, crystallized in different solvents, was analyzed using
Differential Scanning Calorimetry and complemented by Hot Stage Microscopy Studies. Fourier Transform Infrared
Spectroscopy was applied to monitoring the integrity of albendazole during thermal treatment. Thermal analyses revealed
the existence of a low melting form of albendazole. A hypothetic new crystal form generated by crystallization of the melt
was isolated and studied by FTIR spectroscopy, but in this case corresponds to an oxidized form of the original drug.
Keywords: Albendazole; Recrystallization; HSM; DSC; Thermal stability
1. Introduction
Polymorphism is defined as the existence of at least two different solid phases for a molecule. Solid phases can be
crystalline, amorphous or solvates, e.g. crystalline phases that include solvent molecules in their structures (hydrates
when water is the solvent). The research of the nature of different crystalline forms of compounds constitutes a very
important step in pharmaceutical development, not only for active pharmaceutical ingredients (APIs) but also for
excipients. The physicochemical properties of interesting pharmaceutical compounds are strongly related with the
structure at the solid state, such as apparent solubility, hygroscopicity, dissolution rate or rheological behaviour.
Consequently, the study of polymorphism becomes one of the most-important tasks in a serious preformulation process.
Screening for detection of crystal habits, solid phases, determination of their relative stabilities, differentiation between
stable and metastable forms, thermal decomposition and the identification of transition factors are mandatory during the
development of new APIs or dosage forms [1]. For these objectives, the isolation of solid phases by systematic
recrystallization processes is combined by different characterization techniques. The most usual techniques are
Differential Scanning Calorimetry (DSC) and Hot Stage Microscopy (HSM) that constitutes the first step in
polymorphism studies. In particular, HSM enables direct observation of a sample during heating, cooling or under
isothermal conditions. Such investigations provide reliable firsthand evidence about the processes occurring [2,3].
In a second step, a depth characterization is developed by the use of vibrational spectroscopy, as Fourier Transform
Infrared Spectroscopy (FTIR) or RAMAN, X-ray diffraction (powder, single crystal) and solid-state nuclear magnetic
resonance (NMR) [4,5].
Albendazole (ABZ) is an anthelmintic active in neurocysticercosis by larvae of Taenia solium. It is active against
other species such as Ascaris lumbricoides, Wuchereria bancrofti, Trichuris trichiura, Enterobius vermicularis,
Ancylostoma duodenale, Strongyloides stercoralis, Echinococcus granulosus, Necator American, Hymenolepis nana,
Taenia sp. and Giardia lambia [6-8]. ABZ is closely related with mebendazole, another antihelminitc drug, which have
been described up to three polymorphic forms [9,10].
Our investigations are oriented through the thermal history of albendazole (ABZ). Thermal behaviour of samples was
investigated by using DSC. Polarized light optical microscopy and Hot Stage Microscopy (HSM) were applied to study
the crystal patterns of ABZ in stock form and crystallized from different solvents. Different ABZ crystals were prepared
through recrystallization form oversaturated solutions in different common organic solvents. FTIR was used as
complementary technique to confirm the nature of compounds produced during thermal treatment.
2. Materials
ABZ (lot number 035K1421) was purchased from Sigma-Aldrich Co. (St. Louis MI, USA). All of the solvents
employed (Chloroform, Methanol, Acetone, Acetonitrile, Ethyl acetate, Cyclohexane, Diethyl ether, Dichloromethane
and Methyl amine) were of HPLC purity grade.
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Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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3. Methods
3.1 Recrystallization process
Recrystallization from stock ABZ was carried out by its dissolution in different solvents aided by agitation and heating.
The obtained solutions were filtered under vacuum in order to get a particle-free limpid oversaturated solution. The
resultant clean solutions were transferred into a crystallizer, covered and stocked in a quiet place to cool slowly and to
allow crystallization. Alternatively, filtered solutions were crash cooled (by using a blend of ice/salt or liquid nitrogen)
to induce fast precipitation. Solid samples were collected by filtration, dried under vacuum and stored at ambient
temperature prior to analysis.
A summary of solvents employed and treatments to induce precipitation/crystallization is reported in Table 1.
Table 1 Experimental conditions for ABZ recrystallization; a: slow cooling to ambient T; b: crash-cooling in liquid nitrogen and
filtration under vacuum of resultant precipitate; c: crash cooling in ice/salt bath and filtration under vacuum; d: slow solvent
evaporation at ambient temperature and filtration. *: Crystals obtained after fusion of ABZ.
Solvent
Treatment type
Chloroform
a
Chloroform
a
Methanol
a
Acetonitrile
a
Methanol
a
Ethyl acetate
a
Acetonitrile
a
Chloroform
b
Methanol
b
Methanol
c
Chloroform+acetonitrile (1:1)
a
Chloroform+acetonitrile (1:1)
c
Ethyl acetate+chloroform (1:1)
a
Cyclohexane
a
Chloroform+cyclohexane+acetone
d
Diethylether+acetone
d
Methylamine
d
Notes
Nucleation induced by seed plain crystals*
Nucleation induced by seed plain crystals*
Nucleation induced by seed plain crystals*
3.2 DSC
DSC analyses were carried out through a Mettler FP 80 series equipment or a Mettler DSC 821e. Samples of about 10
mg were accurately weighed in open aluminum crucibles. Heating runs were carried out under static air or inert (N2)
atmosphere at 5 or 10 ºC·min-1 rates.
3.3 HSM
Hot stage microscopy studies were performed by using a Mettler FP82HT hot plate linked to a FP85 control unit. The
optical equipment was an Olympus BH2 microscopy and an Olympus Camedia 5600 as recording unit. Polarized light
filter was employed to distinguish between amorphous and crystalline phases. The applied heating rates were the same
as DSC experiments. Isothermal conditions were used in some interesting transitions.
3.4 FTIR
FTIR analyses of samples, before and after thermal analysis, were performed through a Jasco FT/IR 410 apparatus
controlled by Spectra Manager for Windows™, by using KBr press tablet method. Wavenumbers ranged between 600
and 4000 cm-1, with 20 scans for sample and a resolution of 4 cm-1.
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4. Results and discussion
DSC runs of selected samples are collected in Figs. 1 and 2. Stock ABZ is characterized by the presence of two peaks:
first, an acute endothermic effect (onset T 186 ºC; peak T: 202 ºC), which overlaps with another second sharp peak
(peak T: 205 ºC). These data differs from the declared melting point of the substance (215 ºC).
DSC thermogram of ABZ (30-225 ºC, N2 atmosphere, heating rate 10 ºC·min-1).
Fig. 1
Figure 2 reports the DSC analyses of recrystallized samples compared with original ABZ. One of the most
remarkable differences in the DSC runs of crystals obtained by recrystallization in methanol (see Figs. 2b-c) appears as
a clear endo-exo effect at 143 ºC, followed with the endothermic effects described for the stock ABZ. The peaks above
200 ºC appear overlapped thus difficulting their interpretation. An analogous behaviour was observed for other
recrystallized samples. This endo-exo effect corresponds to a solid-solid transition polymorphic transformation of ABZ
obtained by crystallization to stock ABZ [11]. These two polymorphic forms were successfully isolated by other
authors, being characterized to have an enantiotropic relationship [12].
a
b
c
Fig. 2 DSC thermograms of: a) stock ABZ; b) recrystallized from methanol (slow cooling); c) recrystallized from methanol (crash
cooling with liquid N2) (30-225 ºC, N2 atmosphere, heating rate 10 ºC·min-1).
While the nature of endo-exo effect was described in detail in literature, the endothermic events over 200 °C are not
sufficiently explained. In order to elucidate the nature of these phenomena, direct observation by HSM was performed.
Photomicrographs of stock ABZ together with recrystallized samples are reported in Fig. 3. ABZ (Fig. 3a) can be
described as a micronized microcrystalline powder, confirmed by the observation of birefringence under polarized light.
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Fig. 3 Microphotographs (100x magnification) at ambient T of: a) Stock ABZ; recrystallized ABZ from b) methanol (slow
cooled); c) methanol (crash cooled); d) methyl amine; e) acetonitrile:chloroform; f) ethyl acetate:chloroform; g) methanol (seed with
plain crystal); h) cyclohexane.
Crystal growth from different experimental conditions become to the formation of a wide variety of crystal shapes.
Slow cooling from methanol (Fig. 3b) generates the biggest crystals. Crystals from methylamine (Fig. 3d) are irregular,
being similar in shape with those obtained from acetonitrile:chloroform (Fig. 3e). Figure 3g show the ABZ crystal from
methanol seeded with plain crystals (seeds were obtained with thermal treatment of ABZ over 200 ºC) originating
acicular-shaped crystals, very different compared with other methanol samples. Finally, only very small crystals were
obtained from cyclohexane (Fig 3h).
Figure 4 summarizes the thermal behaviour of stock ABZ sample observed under microscopy. ABZ first melting was
registered at 186-190 ºC, followed by a fast recrystallization of the melt. The formed microcrystals melt again at 216 ºC,
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Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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which is in agreement with DSC data. Cooling of this melt to 180 ºC and subsequent heating to 190 ºC did not result in
fusion, indicating the presence of a new solid phase.
Fig. 4
Microphotographs (100x magnification) of stock ABZ at different T: a) 186 ºC (melting onset); b) 190 ºC; c) 190 ºC
(isotherm, recrystallization); d) 190 ºC (isotherm, recrystallized); e) 216 ºC (new melting); f) 190 ºC (isotherm after cooling from 220
to 180 ºC).
Between all the ABZ solid phases elaborated, only HSM analysis of recrystallized ABZ sample from methanol was
selected to be included in this work, because is representative of other recrystallized samples with a similar recorded
behaviour. Photographs at 140 ºC (Fig. 5c), which correspond to the endo-exo effect, did not show any evident change
in crystal shape. Therefore, transformation is present because sample behaviour is similar as compared with stock ABZ,
indicating that at 140 ºC ABZ reverts to its original form. Accordingly, melting-crystallization and final melting (Figs.
5d-h) are similar to stock ABZ.
On the other hand, the change in appearance after first melting could be due either to a polymorphic transformation
or a decomposition process. In fact, following the first hypothesis, some crystals generated by heating over 200 ºC were
employed as seeds, trying to reproduce a similar solid phase by nucleation from some saturated solution of ABZ.
In view of the results obtained by thermal analysis, it was not possible to determine the nature of the material after
melting. Therefore, we used an alternative technique such as FTIR in order to confirm the hypothesis of thermal
decomposition, as literature data suggest with respect to the related compound mebendazole [13] (see Fig. 6).
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Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)
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Fig. 5 Microphotographs of ABZ recrystallized from methanol: a) at ambient T; b) ambient T (polarized light); c) 140 ºC; d) 186
ºC (melting); e) 189 ºC (melting); f) 192.5 ºC (melting); g) 201 ºC; 7) 217 ºC (second melting); h) 220 ºC.
FTIR spectra of selected samples are reported in Fig. 7. The FTIR spectra of the original product and recrystallized
from methanol the same profile (data not shown). However, samples heated up to 220 °C show a new absorption band
at 1029.8 cm-1, which is related to the formation of sulfoxide groups, thereby being demonstrated that melting is
followed by an oxidative process. Consequently, the presence of plain crystal after recrystallization does not match with
a new ABZ form, but a new compound that differs of the starting substance (see Fig. 8). As described in the literature
[7-8], ABZ sulfoxide is the active form of ABZ. In fact, ABZ actually constitutes a prodrug that only becomes active
after biotransformation in plasma in its oxidized form.
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Fig. 6
Fig. 7
Fig. 8
Thermal degradation of mebendazole.
FTIR spectra of: (a) stock ABZ and (b) sample heated to 220 °C.
Original ABZ structure (a) and proposed (b) degradation product (ABZ sulfoxide).
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