INTRODUCTION:

In design of dosage forms crystalline materials are often employed. Some may exist in different crystalline forms. There are varieties of reasons for such changes in space lattice of crystal. It largely depends on how the crystallization of drug is conducted, the nature of solvent(s) used, the processing conditions such as temperature, pressure, cooling rate, agitation, use of the co-solvents, presence of other solutes and ions.^1-3 Though the polymorphs are chemically identical, they exhibit different physicochemical properties such as melting point, solubility, dissolution behavior, X-ray diffraction pattern etc.^4-5 These physicochemical properties further affect the biological properties of drug molecules.⁶

Chemicals that are capable of forming hydrogen bonding can exhibit polymorphism.⁷

Aqueous solubility of drug is important for bioavailability and drug action. Since dissolution is an important prerequisite for drug absorption in most of drugs, the polymorphism influences the drug absorption to a great extent.^8-9

As different polymorph arise through different arrangements of the molecules or ions in the lattice. This will have different interaction energies in the solid state. Under a given set of condition the polymorphs with the lowest free energy will be the most stable one and other polymorphs will tend to transform into it.⁵

The present work involves the study of effect of solvents of different polarity and processing conditions on crystallization (or polymorphism) of drug, optimization of the experimental conditions for obtaining polymorphs, characterization of the crystal habit and the study of solubility and dissolution profile of the crystals.

The drug selected for the study is aceclofenac, a poorly soluble drug, categorized as an analgesic and anti-inflammatory drug. It is chemically [[[2-[(2, 6-Dichlorophenyl) amino] phenyl] acetyl] oxy] acetic acid. It works by blocking the action of a substance in the body called cyclo-oxygenase, which is involved in the production of various chemicals in the body, some of which are known as prostaglandins. Prostaglandins are produced in response to injury or certain diseases and would otherwise go on to cause pain, swelling and inflammation. Arthritic conditions are one example of this. Aceclofenac is used to relieve pain and inflammation in arthritic conditions.^10-13

The objective of present work was to study the effect of solvents of different polarity and processing conditions on the crystallization of Aceclofenac, to optimize the experimental conditions for obtaining polymorphs, to characterize the crystal habit and to study the solubility and dissolution behavior of these crystals.

MATERIALS AND METHODS:

MATERIALS:

Aceclofenac pure drug was procured from Aarti Drugs. (Mumbai, India). Acetone, Methanol, Ethanol, 2-Propanol, Ethyl acetate were procured from S.D. Fine Chemicals Limited, Mumbai. All other chemicals used for the study were of analytical grade.

METHODS:

Preparation of Crystals:

Among the so many solvents tried for crystallization, only five solvents gave encouraging results for aceclofenac. The other solvents such as hexane, water and chloroform did not give crystals, may be due to poor solubility.¹⁴ The saturated solutions of drug was prepared and subjected for different processing conditions like, Deep freezing, Low temperature cooling and Room temperature cooling techniques etc. They were selected as processing conditions to check the effect of cooling temperature on crystallization process.

Deep freezing technique:

In deep freezing method, the containers were always closed with stoppers. Occasional agitation was used to verify the crystals. The nuclei were separated and acted as seeds for further crystal growth. The crystals from the solution were separated, dried at room temperature and stored in dessicator.

Low temperature cooling technique:

The low temperature cooling method was used by keeping the saturated solutions of drug in different solvents in lower compartment of the freeze at around 8 to 15˚C for 8-10 days to complete the nucleation and crystal growth. The crystals from the solution were separated and dried at room temperature for two days and stored in dessicator.

Room temperature cooling technique:

In room temperature cooling (no stress conditions), samples were prepared by keeping the saturated solutions of drug in the solvents at room temperature for 8-10 days to complete nucleation and crystal growth. The crystals from the solution were separated, dried at room temperature for two days and stored in dessicator.

In all the above methods, the surface moisture was removed by storing the sample in dessicator containing self-indicating silica crystals for 1 to 2 weeks. The influence of different polarity solvents and processing conditions on the crystal habit could be highlighted. The crystals were prepared three times in order to get reproducibility. Large scale crystallization (5 to 10 gms) was also attempted so as to get sufficient quantity for XRD spectra.

Characterization of Crystals:

Microscopical Observation:

Crystal shape was observed under the binocular microscope with 40x magnification and photographs were taken to compare the crystals with pure drug.

Melting Point:

The determination of melting point of the crystals was carried out in open capillaries by using digital melting point apparatus (Veego Instruments Pvt. Ltd., India).⁴

Solubility Study:

The solubility study of aceclofenac (pure drug) and crystals was performed in distilled water.¹⁵ About 10 mg of crystals were added to 10 ml of distilled water in specific gravity bottles. This amount was sufficient to obtain saturated solution. These specific gravity bottles were shaken for 8 hours at 25°C by keeping in a cryostatic constant temperature reciprocating shaker bath. The bottles were then opened and solutions were filtered with the help of Whatman filter paper. The absorbance of the solution was measured at 274.5 nm by using UV-Visible Spectrophotometer (UV-1700, Shimadzu, Japan). This method was performed in triplicate.

Dissolution Study:

USP XXI dissolution apparatus Type-I (Dissolution Apparatus – TDT 06P, Electrolab, Mumbai) was employed for the present study. The sample (100 mg) was encapsulated in a hard gelatin capsule and transferred to the dissolution medium (900 ml distilled water). The dissolution medium was stirred at 100 RPM by maintaining the temperature at 37±0.5°C. 5 ml of the aliquot was withdrawn at the interval of 10 minutes for 2 hours with the help of guarded pipette. The absorbance was measured at 274.5nm.¹⁶

Fourier Transformed Infra-Red (FT-IR) Spectroscopy:

In FT-IR analysis, sample powder was dispersed in KBr powder; pellets were prepared from it and analyzed. FT-IR spectra were obtained by powder diffused reflectance on a FT-IR spectrophotometer (FT-IR 1600 Perkin-Elmer).⁴

X-Ray Diffraction Study:

The X-ray diffraction patterns of pure drug and optimized crystals obtained from different solvents and processing conditions were recorded using PAN Analytical diffractometer system (X’pert pro PW 30-40/60) with a copper target. The operating conditions were: voltage − 40 kV; current−30 mA; scanning speed−0.05°/sec; temperature of acquisition - room temperature; detector- scintillation counter detector; sample holder- non-rotating holder. The diffraction pattern was analyzed for the range from 20 to 45-2θ.¹⁷

Differential Scanning Calorimetry (DSC) Study:

Differential scanning calorimetry was performed using DSC-60 (Shimadzu, Tokyo, Japan) calorimeter to study the thermal behaviour of drug and optimized crystals prepared from different solvents and processing conditions. The instrument comprised of calorimeter (DSC 60), flow controller (FCL60), thermal analyzer (TA 60WS) and operating software (TA 60). The samples were heated in hermetically sealed aluminum pans under air atmosphere at a scanning rate of 10°C/min from 25°C to 300°C. Empty aluminum pan was used as a reference.

Scanning Electron Microscopy (SEM) Study:

The surface characteristics of pure drug and prepared crystals were studied by SEM (JEOL, JSM 5610LV, Tokyo, Japan) at 250X, 500X and 1,000X. The samples were mounted on double-sided carbon adhesive tape that had previously been secured on brass stubs and then analyzed. The accelerating voltage was 15 kV.

Model Dependent Method:

Release kinetic was analyzed by various mathematical models, which were applied considering the amounts of drug released from 0 to 120 min.^18-20 Dissolution of all the batches were fitted to zero-order, first order, Higuchi, Hixon-Crowell, Korse-meyer and Peppas and Weibull model to ascertain the kinetic modeling of drug release. Model fitting was done using FORTRAN software. The least value of sum of square of residuals (SSR) and Fisher’s ratio (F) were used to select the most appropriate kinetic model.²¹

RESULTS AND DISCUSSION:

Different techniques for identification of habit and characterization of amorphous or crystalline solids are available. One method may not give correct proof for these modifications. Therefore, all the methods were analyzed to decide whether morphological changes did occur. Among the solvents used for crystallization, only five solvents gave encouraging results for aceclofenac. The other solvents such as hexane, water and chloroform, did not give crystals, may be due to poor solubility of drug.

Microscopical Observation:

Aceclofenac pure drug sample and the crystals obtained from different solvents and processing conditions were observed using magnifying lens and microscopic method (40x). Photographs were taken by CCD camera to draw certain conclusions for morphological characterization. The pure drug sample of aceclofenac (Figure 1) and crystals obtained from different processing conditions like room temperature cooling, low temperature cooling and deep freezing techniques were observed by microscopic method (Figure 2 to 4).

Figure 1: Microscopic observation of Aceclofenac pure drug

(A) Acetone

(B) Ethanol

C) Methanol

(D) 2-Propanol

Fig. 2: Microscopic observation of Aceclofenac crystals obtained by Room temperature cooling method from solvent (A) acetone (B) ethanol (C) methanol (D) 2-propanol (E) ethyl acetate

(B) Ethanol

(C) Methanol

(D) 2-Propanol

(D) 2-propanol

Fig. 3: Microscopic observation of Aceclofenac crystals obtained by Low temperature cooling method from solvent (A) acetone (B) ethanol (C) methanol (D) 2-propanol C

(A) acetone

(B) Ethanol

‘Methanol

(D) 2-Propanol

(E) Ethyl acetate

Fig. 4: Microscopic observation of Aceclofenac crystals obtained by Deep freezing method from solvent (A) acetone (B) ethanol (C) methanol (D) 2-propanol (E) ethyl acetate

Aceclofenac pure drug was found to be in crystalline state having hexagonal shape and small in size. The microscopic observation of Fig. 2 to 4 shows that crystals obtained from different solvents and processing conditions gave variety in size and shape. General findings are given in Table 1 to 3.

Table 1: Microscopic observations (40x) of Aceclofenac crystals obtained by room temperature cooling method.

Sr. No.	Solvent of crystallization	Description of crystals
1	Acetone	Large size, irregular shape, platy crystals
2	Methanol	Platy square shape and blunt at both the ends. Transparent crystals
3	Ethanol	Small size, irregular shape, transparent crystals
4	2-Propanol	Small size, irregular shaped crystals
5	Ethyl acetate	Large size, irregular shape, pitted and transparent crystals

Table 2: Microscopic observations (40x) of Aceclofenac crystals obtained by low temperature cooling method.

Sr. No.	Solvent of crystallization	Description of crystals
1	Acetone	Transparent, small size, transparent crystals
2	Methanol	Thick needles, pointed at one end, transparent with little bit broad shape crystals
3	Ethanol	Irregular shape with small size crystals
4	2-Propanol	Thin needles, shorter in length, opaque crystals
5	Ethyl acetate	Pitted square shaped crystals with little thickness

Table 3: Microscopic observations (40x) of Aceclofenac crystals obtained by deep freezing method.

Sr. No.	Solvent of crystallization	Description of crystals
1	Acetone	Small size, square shaped and thick crystals
2	Methanol	Small size, hexagonal shaped, transparent and thin as compared to crystals obtained from Acetone.
3	Ethanol	Somewhat larger in size, square shaped and thick crystals
4	2-Propanol	Small size, irregular shaped and thick crystals
5	Ethyl acetate	Small size, almost triangular shaped, thin crystals

Melting point:

Melting point of the aceclofenac pure drug sample and crystals were determined using digital melting point apparatus by open capillary method. The melting point of aceclofenac pure drug was found to be 152.2⁰C. The data of melting point of crystals of aceclofenac obtained from different solvents and processing conditions were also collected (Table 4). There was no much difference in melting point of pure drug and crystals obtained from different solvents using different processing conditions. Reproducible results were obtained though the melting points were monitored over a period of 45 days. In other words, the polymorphic transformation during the storage was not observed.

Solubility study:

Solubility data in distilled water for aceclofenac pure drug as well as crystals obtained from different solvents and processing conditions after shaking for 8 hours at 25±1°C was studied (Table 5). Acetone was one of the best solvents for deep freezing and low temperature cooling method. In case of room temperature method ethyl acetate gave greatest solubility and proved to be the best solvent. In almost all the cases, solubility of crystals showed 3 to 7 time higher solubility as compared to aceclofenac pure drug. Aceclofenac pure drug was found to have solubility 7.3 ± 0.1 µg/ml.

Dissolution study:

For the dissolution studies, distilled water was selected as a dissolution medium. Dissolution studies were carried out by Dissolution Apparatus Type-I.¹⁸ Percent drug dissolved of Aceclofenac crystals obtained from different solvents and processing conditions at 2 hrs were observed (Table 6).

In this study, crystals were filled in the hard gelatin capsule and added to the dissolution medium. The dissolution rate-time profiles for aceclofenac crystals obtained from different processing conditions (Figure 5 to 7). After 2 hrs the percent drug dissolved for aceclofenac pure drug was found to be 17.55 ± 0.44 %.

From the solubility and dissolution study of pure drug as well as crystals obtained from different solvents and processing conditions, few types of crystals were selected for further analysis and characterization. The selection of

crystals was done on the basis of difference in their solubility and dissolution study. The selected crystals were having maximum solubility and dissolution as compared to other samples obtained from the same method of crystallization but with other solvents. The selected samples were crystals obtained from ethyl acetate by room temperature cooling (B5), from acetone by low temperature cooling (C1) and from acetone by deep freezing technique (D1).

The list of samples of aceclofenac crystals which were selected for the further study (Table 7).

Fig. 5: Comparative dissolution profile of Aceclofenac crystals obtained from Room temperature cooling method

Fig. 6: Comparative dissolution profile of Aceclofenac crystals obtained from Low temperature cooling method

Fig. 7: Comparative dissolution profile of Aceclofenac crystals obtained from Deep freezing method

FT-IR Spectroscopic Analysis:

The FT-IR spectra were obtained for the selected crystals of aceclofenac from different solvents and processing conditions and are presented in figure 8. The pure drug was used for comparison. The spectral analysis was done in two parts: the first was for the identification of drug using characteristic bands and the second was for identification of polymorphs. From the structure of aceclofenac the characteristic bands were identified and are given in Table 8. From Table 8, it can be inferred that the compound under study was aceclofenac only. The crystals prepared in this work also showed the characteristic bands.^22-23

A perusal to Figure 8 indicated that all the crystals of aceclofenac had no considerable changes in the spectra as compared to that of aceclofenac pure drug, though all these types of crystals were prepared in different experimental conditions by using different solvents. The IR spectra have failed to show any characteristic band in the finger print region.

Table 8: Comparison of characteristic bands between literature and pure drug sample values of Aceclofenac

Characteristic bands	Literature values, cm^-1	Observed in this study, cm^-1
Secondary Amine Stretching	3319	3311
C-H Stretching	2970 & 2935	2983 & 2937
C=O Stretching (Acid)	1716	1718
Primary amine stretching	1589 & 1506	1587&1508
C=C Stretching	1479	1481
C-H₂Deformation	1344	1344
C-O Stretching	1280 & 1255	1249
N-H Wagging	665	667

X-Ray Diffraction:

The XRD of pure drug sample (A) was taken and compared with selected crystals obtained from different solvents and processing conditions (B5, C1 and D1) (Figure 9).

A perusal to Table 9 indicated the XRD of all the selected crystals as well as aceclofenac pure drug. Here all the samples showed similar X-ray pattern and no considerable changes were observed. Moreover the reduction in intensities was again an indication of reduction in crystallinity and improvement of dissolution.²⁴ Further analysis with DSC was required for confirmation.

Differential Scanning Calorimetry Study:

Crystalline materials in powdered state showed characteristic DSC patterns made up of peaks of varying intensities in certain positions. Each powder pattern of the crystal lattice is characteristic for a given polymorph.

The DSC thermograms of aceclofenac pure drug was taken and compared with crystals obtained from ethyl acetate by room temperature cooling (B5), acetone by low temperature cooling (C1) and acetone by deep freezing technique (D1). (Table 10).

The perusal to Figure 10 revealed that there was no significant decrement in melting point as well as fussion temperature in the crystals of ethyl acetate by room temperature cooling (B5), acetone by low temperature cooling (C1) and acetone by deep freezing technique (D1) as compared to aceclofenac pure drug.²⁵ The crystal obtained from acetone by deep freezing method (D1) was having highest solubility among all other crystals as well as aceclofenac pure drug. But, sample (D1) was failed to show any difference in DSC study with compare to aceclofenac pure drug.

Table 9: XRD for characteristic crystalmorphic (crystal habit) changes Aceclofenac crystals.

Sr. No.	Samples of crystals	Peak 2θ value	Intensity	Other	Inference
1	Commercial sample (A)	22.225 24.425 25.875	557 318 667	-	-
2	Crystals with Ethyl Acetate by room temperature method (B5)	22.175 24.375 25.925	250 199 1577	Large difference in intensities between 21 to 26 2θ	May be a polymorphism or reduction in crystallinity
3	Crystals with Acetone by low temperature cooling method (C1)	22.175 24.425 25.375	348 435 1027	No considerable difference observed between 21 to 26 2θ	No indication of polymorphism
4	Crystals with Acetone by deep freezing method (D1)	22.125 24.375 25.825	570 361 1058	No considerable difference observed between 21 to 26 2θ	No indication of polymorphism

Table 10 : DSC thermograms of the crystals of Aceclofenac obtained from different solvents and processing conditions.

Sr. No.	Samples of crystals	Fussion Temperature (°C)	ΔH_f(mJ)	Inference
1	Commercial sample (A)	159.49	-1110.0	-
2	Crystals with ethyl acetate by room temperature method (F5)	157.15	-741.20	No indication of polymorphism
3	Crystals with acetone by low temperature cooling method (E1)	156.80	-774.67	No indication of polymorphism
4	Crystals with Acetone by deep freezing method (C1)	155.62	-176.32	No indication of polymorphism

Fig. 9: X-ray diffraction patterns of the crystals of Aceclofenac obtained from different solvents and processing conditions

Fig. 10: DSC thermograms of the crystals of Aceclofenac obtained from differentsolvents and processing conditions

Fig.11: SEM photographs of (A) Aceclofenac pure drug sample (F5) Aceclofenac crystals obtained from ethyl acetate by Room Temperature method.

CONCLUSION:

No change was found in analytical data. So there was no different crystal structure observed. Solubility and dissolution profiles of crystals obtained from Ethyl acetate by room temperature method (B5), Acetone by low temperature cooling and Acetone by Deep freezing (D1) were found higher which was again supported by decreased crystallinity according to XRD result. The crystals obtained from acetone by deep freezing techniques (D1) showed highest solubility as well as dissolution among all other crystals. It was failed to show any difference in DSC study as compared to pure drug but it was proved to be one of the best combination of solvent and processing condition for recrystallization of aceclofenac. Thus, change in the polarity and processing conditions can affect the physico-chemical properties of aceclofenac crystals.

ACKNOWLEDGEMENTS:

The authors are very much grateful to Department of Physics and Department of Chemistry, Saurashtra University, Rajkot for providing facilities for XRD and FTIR study respectively. The authors are also thankful to Metallurgy Department of M.S. University, Vadodara for providing facilities for SEM study of the samples.

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Received on 22.07.2013 Accepted on 01.08.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 3, Pg 122-132

Sr. No.	Processing Conditions	Melting Point (°C) (Mean ± SD)⁺
Sr. No.	Processing Conditions	Acetone	Methanol	Ethanol	2-Propanol	Ethyl Acetate
1	Room Temperature	151.9 ± 0.2	147.8 ± 0.3	149.0 ± 0.5	151.1 ± 0.4	151.3 ± 0.5
2	Low Temperature Cooling	151.5 ± 0.5	150.3 ± 0.1	146.7 ± 0.2	150.1 ± 0.4	150.8 ± 0.5
3	Deep Freezing	151.9 ± 0.2	148.1 ± 0.5	148.8 ± 0.3	148.3 ± 0.6	149.0 ± 0.2

Sr. No.	Processing Conditions	*Solubility (µg/ml) (Mean ± SD⁺⁾**
Sr. No.	Processing Conditions	Acetone	Methanol	Ethanol	2-Propanol	Ethyl Acetate
1	Room Temp.	37.0±0.2	36.0±0.4	25.6±0.4	33.1±0.4	30.9±0.7
2	Low Temp. Cooling	44.5±0.7	43.1±0.5	41.4±0.9	39.8±0.5	44.8±0.5
3	Deep Freezing	53.1±0.9	46.9±0.4	33.7±0.7	40.1±0.7	50.1±0.3

Sr. No.	Processing Conditions	*Percent drug dissolved at 2hrs. (Mean ± SD⁺⁾**
Sr. No.	Processing Conditions	Acetone	Methanol	Ethanol	2-Propanol	Ethyl Acetate
1	Room Temperature	5.85 ± 0.30	9.37 ± 0.61	16.0 ± 0.29**	8.42 ± 0.19	15.4 ± 0.39
2	Low Temperature Cooling	7.72 ± 0.49	3.78 ± 0.51	5.19 ± 0.41	8.72 ± 0.48	7.16 ± 0.40
3	Deep Freezing	15.3 ± 0.05**	11.64 ± 0.23	7.58 ± 0.38	12.92 ± 0.56^#	8.76 ± 0.38

Sr. No.	Sample Selected	Sample Code
1	Aceclofenac Pure Drug	A
2	Crystals from Ethyl Acetate by Room temperature	B5
3	Crystals from Acetone by Low temperature cooling	C1
4	Crystals from Acetone by Deep freezing	D1

Description of crystals

Description of crystals

Description of crystals