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Krishna R. Gupta et al. / International Journal of Advances in Pharmaceutical Research
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SOLID-STATE CHARACTERIZATION OF THIOCOLCHICOSIDE
Rachana R. Joshi and Krishna R. Gupta*
Department of Pharmaceutical Chemistry, Smt. Kishoritai Bhoyar College of Pharmacy,
New Kamptee-441002, Nagpur (MS,) India.
Received on 17 - 12 - 2012 Revised on 24 - 12 - 2012 Accepted on 21- 01 - 2013
ABSTRACT
The present study deals with the generation and characterization of various solid-state forms of Thiocolchicoside, an
anti-inflammatory analgesic agent. The drug was subjected to polymorphic screen using different solvents to explore
the possibility of existence of different solid Forms. The solvents like distilled water, methanol, propanol and
acetonotrile were selected based on solubility of drug. Detailed methods of preparation of the polymorphs and
pseudopolymorphs are described. All these solid-state forms were characterized by thermoanalytical (DSC, TGA),
Crystallographic (XRD), microscopic (SEM), and spectroscopic (FTIR) techniques.
Key Words: Thiocholchicoside, Solid state characterisation, polymorphism
INTRODUCTION
Thiocolchicoside (TCD) is an anti-inflammatory
analgesic agent with muscle relaxant action.
Thiocolchicoside is a glucoside extracted from the
seeds of Colchicum autumnale. It has muscle
relaxant, anti-inflammatory, analgesic and anesthetic
actions with minimal side effects and is used
topically for the treatment of mascular spasms and
for rheumatologic, orthopaedic and traumatologic
disorders. Thiocolchicoside [1] chemically is (s)-N-
[3-(B-D-glucopyranoxyloxy) – 5, 6, 7, 9- terahydro-
1, 2 dimethoxy- 10- (methyl thio)-9- oxobenzo [a]
heptalen-7yl] acetamide.
Polymorphism is defined as the ability of a
substance to exist as two or more crystalline phases
or forms that have different arrangements and/or
conformations of the molecule in crystal lattice [2].
The possibility of polymorphism or
pseudopolymorphism may exist for any particular
compound, but the conditions required for unknown
polymorphs or pseudopolymorphs are not easily
determined [3]. It has been estimated that large
number of pharmaceuticals exhibit polymorphism.
The frequent occurrence of polymorphism of
pharmaceutical solids is known for a long time past;
as well as the fact that different polymorphic forms of
active ingredients may have different bioavailability,
and physical/chemical stability [4,5]. Because of the
regulatory constraints of the last few decades, which
force pharmaceutical companies to deal with
Research Paper
ISSN: 2230 – 7583
polymorphism of active ingredients [6,7], and even
more for the economic potential of its new solid
forms [8,9], the relevance of the pharmaceutical
polymorph analysis is constantly growing. This is
evidenced by many excellent recent publications,
which discuss the subject in detail [8, 10-12]. Several
techniques for solid-state measurements have been
reported in the study of polymorph: microscopy, IR
spectroscopy [13], differential scanning calorimetry
(DSC) [14-16], powder X-ray diffractometry (XRD)
[17-18] and solid-state NMR [19-22].
In the present study, various solid-state
forms of Thiocolchicoside were generated and
characterized by thermal, crystallographic,
microscopic, infrared technique.
MATERIAL AND METHODS
Material
Thiocolchicoside was kindly provided from Sanofi
Aventis. All solvents were AR/HPLC grade.
Preparation of Solid state forms
The first sequence of study entails crystallisation of
the substance out of a variety of solvents. Solvents
have been selected on the basis of their protondonating,
proton-accepting and dipole-interaction
abilities. The solubility of Thiocolchicoside (TCD)
was observed in different solvents selected on trial
and error basis. TCD was found to be soluble in
distilled water, methanol, 2-propanol and acetonitrile.
Hence, these solvents were selected for the
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preparation of polymorphs or solvatomorphs. To
avoid confusion in the nomenclature of the
polymorphs it was denoted by Roman letters.
A brief outline of the preparative procedure is as
follows:
Form I was obtained by dissolving TCD in distilled
water with the aid of heat and constant stirring at 30
rpm to get saturated solution. The solution was
filtered to remove all nuclei, and then the filtered
solution was cooled at room temperature to get
crystals. The resultant crystals of obtained on cooling
were harvested by filtration and dried at RT. They
were stored separately in ambered colour glass
bottles and kept in desiccator over silica pellets at
RT.
Form II was obtained by dissolving TCD in
methanol with the aid of heat and constant stirring at
30 rpm to get saturated solution. The solution was
filtered to remove all nuclei, and then the filtered
solution was cooled at room temperature to get
crystals. The resultant crystals obtained on cooling
were harvested by filtration and dried at RT. They
were stored separately in ambered colour glass
bottles and kept in desiccator over silica pellets at
RT.
Form III was obtained by dissolving TCD in 2propanol
with the aid of heat and constant stirring at
30 rpm to get saturated solution. The solution was
filtered to remove all nuclei, and then the filtered
solution was cooled at room temperature to get
crystals. The resultant crystals obtained on cooling
were harvested by filtration and dried at RT. They
were stored separately in ambered colour glass
bottles and kept in desiccator over silica pellets at
RT.
Form IVwas obtained by dissolving TCD in
acetonitrile with the aid of heat and constant stirring
at 30 rpm to get saturated solution. The solution was
filtered to remove all nuclei, and then the filtered
solution was cooled at room temperature to get
crystals. The resultant crystals obtained on cooling
were harvested by filtration and dried at RT. They
were stored separately in ambered colour glass
bottles and kept in desiccator over silica pellets at
RT.
Characterization
X-ray powder diffraction (XRD)
The X-ray patterns of solid state forms of
Thiocolchicoside were collected using MiniFlex2 Xray
Difftactometer. Radiations generated from Cu
source and filtered through Kb filters at 15mA and 30
kV were used to study the X-ray diffraction patterns.
The instrument was operated over the 2θ range of 5–
80 ° .
Scanning electron microscopy (SEM)
The samples were viewed under JEOL Model JSM -
6390LV Scanning Electron Microscope (Jeol, Japan)
at magnification within the range of 5× - 300,000×
was used and micrographs were taken at several
magnifications using an accelerating voltage of 5
keV.
Differential scanning calorimetry (DSC)
Dupont USA 990 Differential scanning calorimeter
was used to record the thermogram of the different
solid state forms of Thiocolchicoside. Temperature
axis and cell constant were calibrated using indium.
The samples were exposed to heating rate of
10 0 C/min over a temperature range of 30–300 0 C
under nitrogen purging in pin-holed aluminium pans.
Thermogravimetric analysis (TGA)
Thermogravimetric analysis was carried out using a
Perkin Elmer Diamond TG/DTA at the heating rate
of 10 0 C/min from 30-300 0 C under nitrogen purging.
Fourier transform infrared spectroscopy (FTIR)
Infrared Spectra of all solid forms of
Thiocolchicoside were obtained on a Shimadzu
FTIR- 8400S (Kyoto, Japan) with prior sample
preparation by physically mixing the drug with
potassium bromide to prepare a pellet for further
analysis.
Solubility determination
The solubilities of the prepared solid forms were
determined at room temperature. An excess amount
of the samples were added to 10 mL beaker
containing 5 mL water. The samples were rotated on
magnetic stirrer at 30 rpm for period of time in
excess of that required for equilibrium (24 hr). After
equilibrium, the solutions were filtered rapidly
through whatmann filter paper. The filtered solutions
were analyzed using proposed validated
spectrophotometric procedure described later in
chemical characterization of the drug.
Determination of stability
The Standard TCD and all forms were packed in
aluminium foils and kept in an hot air oven set at
55°C for one month. The samples were characterized
using FTIR.
RESULT AND DISCUSSION
Characterization
SEM (Scanning electron microscopy)
Crystal morphology plays a valuable role in
pharmaceutical processing and product development
of solid dosage forms. Differences in crystal habit
may strongly influence the particle orientation,
modify flow ability, packing, compaction,
compressibility and dissolution characteristics of a
drug powder. SEM is the technique of choice to
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Krishna R. Gupta et al. / International Journal of Advances in Pharmaceutical Research
obtain information at high magnification levels or
when a three-dimensional view of particle surface is
required.
SEM photographs (Fig. 1) showed a distinct
difference in the morphology of different solid-states
forms.The SEM image showed cylindrical tube like
structure for TCD. The SEM image showed thin
cylindrical shaped tube like structure of crystal for
Form I. The SEM image showed thin flat plate like
irregular arrangement of crystal for Form II, III and
IV.
XRD (X-ray powder diffraction)
XRD is one of the most sensitive and a
foolproof method for solid-state characterization as
the results are obtained directly from the molecular
arrangements of the crystalline material (Chao and
Vail, 1987). Fig. 2 shows the XRD patterns of solidstate
forms of TCD. Crystalline forms of TCD
showed sharp diffraction peaks. The XRD of TCD
showed characteristic peaks at 10.46°, 11.38°, 13.34°,
17.32° and 14.48° 2θ those corresponding only to the
TCD. The X-ray powder diffraction pattern showed
distinct differences in peak positions and peak
intensities than other forms prepared by
recrystallization from solvents. The X-ray powder
diffraction (XRPD) pattern of Form I showed
significantly different distinct peak positions (2θ) and
peak intensities (I/I0) as campared to TCD. This
observation indicates that the form I contains solvent
molecule (water) having a different crystal structure
from the non-solvated form TCD and can be
described as polymorphic solvate or hydrate. The
XRD of Form I showed characteristic peak at 12.26°,
12.60°, 18.06° and 20.94° 2θ. The X-ray powder
diffraction pattern of Form II, III and IV was devoid
of diffraction peaks and a halo pattern was observed,
confirming the lack of three-dimensional long range
ordered structure. Hence it was amorphous in nature.
Thermal analysis (Differential scanning calorimetry
and Thermogravimetric analysis)
Fig. 3 and 4 shows DSC and TG
thermograms of five modifications of
Thiocolchicoside. DSC thermogram of TCD showed
an endothermic peak with maxima at 272.6°C
(enthalpy of fusion, Hf-152.1 J/g) due to melting.
TGA analysis revealed weight loss from the sample
over temperature range of 30–280°C because of the
weak hydrogen bond interaction of water molecules
in the crystal structure.
DSC thermogram of Form I showed two endothermic
peaks at 111.5°C and 270.2°C (Enthalpy of fusion,
Hf-86.6.1 J/g). The TGA thermogram show 7.78%
weight loss in temperature range of 55°C-115°C
which corresponds to desolvation of the crystal and
correlates with the first endothermic peak observed in
DSC pattern of Form I. The 9.09% weight loss shown
in temperature range of 130°C-290°C is due to
decomposition of the sample which corresponds to
the melting point of the crystals.
DSC thermogram of Form II showed two
endothermic peaks at 109.8°C and 267.4°C (enthalpy
of fusion, Hf-83.3 J/g) while one exothermic peak at
222.4°C. The first endothermic peak corresponds to
desolvation of the crystals followed by
recrystallization process indicated by exothermic
peak at 222.4°C and the melting point corresponding
to the second endothermic peak at 267.4°C. The TGA
thermogram of Form II showed 17.68% weight loss
in the temperature range of 40°C-240°C which
corresponds to desolvation.
DSC thermogram of Form III showed two
endothermic peaks at 113.44°C and 250.6°C
(enthalpy of fusion, Hf-23.3 J/g). The first peak
corresponds to desolvation of the crystals while the
second peak corresponds to the melting point of the
crystals. TGA thermogram of Form III showed
5.46% weight loss in temperature range of 40°C-
120°C. The weight loss occurred correlates with the
first endothermic peak observed in DSC of Form III.
The 7.99% weight loss shown in temperature range
of 130°C-260°C was due to decomposition of the
sample.
DSC thermogram of Form IV showed two
endothermic peaks at 95.2°C and 266.7°C (Enthalpy
of fusion, Hf-84.87 J/g). The first endothermic peak
corresponds to desolvation of the crystals while the
second endothermic peak corresponds to the melting
point of the crystals. TGA thermogram of Form IV
showed 4.071% weight loss in temperature range of
40°C-100°C. The weight loss occurred in temperature
range correlates with the first endothermic peak
observed in DSC of Form IV. The 3.57% weight loss
shown in temperature range of 180 0 C-290 0 C is due to
decomposition of the sample.
FTIR (Fourier transform infrared spectroscopy)
FTIR spectroscopy has been successfully
used for exploring the differences in molecular
conformations, crystal packing and hydrogen
bonding arrangements for different solid-state forms
of an organic compound . Spectra of TCD (Fig. 5)
showed characteristic carbonyl group (Amide I) band
at 1525.5 cm -1 , C=O str (tropane ring) at 1664.4 cm -1 ,
Amide II band (N-H str) at 3325.9cm -1 , thioether
band at 2360.7cm -1 and hydroxyl group stretching
band at 3400.2cm -1 .
FTIR spectra of Form I (Fig. 5) showed characteristic
carbonyl group band at 1558.3 cm -1 , C=O str (tropane
ring) at 1654.8cm -1 , Amide II band (N-H str) at
3247.9cm -1 , thioether band at 2376.1cm -1 and
hydroxyl group stretching band at 3400.2cm -1 and
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free -OH stretching band at 3545.7cm -1 . The shift in
frequency of C=O str (tropane ring) and amide II
band at lower frequency in Form I as compared with
TCD indicate the involvement in intermolecular
hydrogen bonding with distilled water which was
used as solvent for the preparation.
FTIR spectra of Form II (Fig. 5) showed
characteristic carbonyl group band at 1527.5cm -1 ,
C=O str (tropane ring) at 1647.1cm -1 , Amide II band
(N-H str) at 3325.0cm -1 , thioether band at 2376.1cm -1
and hydroxyl group stretching broad band at
3336.6cm -1 which indicate the intermolecular
hydrogen bonding between TCD and methanol in
Form II. The shift of carbonyl group (tropane ring) at
lower frequency in Form II when compared with
TCD also confirmed the intermolecular hydrogen
bonding with methanol which was used as solvent for
the preparation.
FTIR spectra of Form III (Fig. 5) showed
characteristic carbonyl group band at 1526.9 cm - 1,
C=O str (tropane ring) at 1701.1cm -1 , Amide II band
(N-H str) at 3326.9cm -1 , thioether band at 2360.7cm -1
and hydroxyl group stretching broad band at
3336.6cm -1 which indicate the intermolecular
hydrogen bonding between TCD and 2-propanol in
Form III.
FTIR spectra of Form IV (Fig. 5) showed
characteristic carbonyl group band at 1521.7 cm - 1,
Table 1: Results of Solubility study
Sample Solubility (mg/mL)
Form I 12.2
Form II 24.4
Form III 13.1
Form IV 9.5
(a) (b)
C=O str (tropane ring) at 1627.8cm -1 , Amide II band
(N-H str) at 3326.9cm -1 , sharp band at 2360.7cm -1
indicate –CN stretching (acetonitrile) and also band
for thioether appeared at the same region. Broad band
at 3336.6cm -1 due to hydroxyl group stretching
indicate the intermolecular hydrogen bonding
between TCD and acetonitrile in Form IV.
Solubility determination
The solubility data (Table 1) revealed that the
solubility of Form II, III was more as compared to
Form I. The above results correlates with the
literature 4 that hydrates are less soluble in water than
the corresponding solvates formed from other
solvents. If the solvent is water-miscible, are more
soluble in water than the corresponding non solvated
form.
Form II > Form III > Form I > Form IV.
Stability study
The study of FTIR spectral pattern of various forms
reveals that form I shows similar IR spectral pattern
as TCD non- solvated form on exposure to 55°C on
1 st day, form II on 7 th day and form IV on 3 rd day.
When the spectral patterns were recorded on 21 st day
of exposure to 55°C no prominent change in the
spectral pattern was observed.
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(c) (d)
(e)
Figure 1: SEM Photographs of solid forms of TCD (a) TCD (b) Form I (c) Form II (d) Form III (e)Form IV
(a) (b)
(c) (d)
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(e)
Figure 2: X-ray Powder Diffraction Pattern of Thiocolchicoside and its polymorphs (a) TCD (b) Form I (c)
Form II (d) Form III (e) Form IV
(a) (b)
(c) (d)
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(e)
Figure 3 : DSC Thermograms of Thiocolchicoside and its polymorphs (a) TCD (b) Form I (c) Form II (d)
Form III (e) Form IV
(a) (b)
(c) (d)
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(e)
Figure 4: TGA Thermograms of Thiocolchicoside and its polymorphs (a) TCD (b) Form I (c) Form II (d)
Form III (e) Form IV
(a) (b)
(c) (d)
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(e)
Figure 5: FTIR Spectra of Thiocolchicoside polymorphs and solvates (a) TCD (b) Form I (c) Form II (d)
Form III (e) Form IV
CONCLUSION
Based on crystallization experiments and solid state
characterization it was concluded that
Thiocolchicoside form polymorphs (solvates) with
Distilled water and the amorphous form of
Thiocolchicoside was formed using solvents like
methanol, propanol and acetonotrile. The solvates
exhibited plate-like crystal habit in contrast to
cylindrical crystal habit of the stable form. The Xray
diffractograms and FTIR absorption spectra of
the crystalline and amorphous forms also differed
distinctively. The desolvation peak in DSC curves,
weight loss in TGA confirmed the existence of
solvent in the crystal lattice. The higher aqueous
solubility of solvates and amorphous forms indicate
the usefulness of solid-state manipulation as a tool to
overcome biopharmaceutical hurdles in drug
delivery.
ACKNOWLEDGEMENT
The authors are thankful to the Principal, Smt.
Kishoritai Bhoyar college of Pharmacy for providing
necessary facility for carrying out the research work.
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