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Based on the nature of work each heading has been subcategorized under the subheading: Preparation and Characterization of the crystal forms; Solubility and Dissolution Studies and Formulation studies.
8.1 PREFORMULATION STUDIES:
The drug was dissolved in very slightly soluble in water and methanol, Soluble in 0.1 mol/L HCl, Practically insoluble in chloroform. The melting point of the drug was found to be 185oC
8.2 DETERMINATION OF λ max OF GATIFLOXACIN:
On the basis of preliminary identification test it was concluded that the drug complied the preliminary identification. From the scanning of drug, it was concluded that the drug had λmax of 292 nm, which was equal to 292 nm as reported.
8.3 PREPARATION OF STANDARD CURVE OF GATIFLOXACIN WITH 0.1 N HCL PH 1.2:
From the standard curve of Gatifloxacin (Table No.6, Graph 1), it was observed that the drug obeys beer's law in concentration range of 1 - 10 µg/ml in 0.1 N HCL pH 1.2. The slope Y = 0.0914x, the correlation coefficient R2 =0.9999. It follows fit curve since R2 <1
8.4 PREPARATION OF CRYSTALS FORMS FROM DIFFERENT SOLVENTS:
Gatifloxacin was obtained as a raw material; it was used as such for the preparation of crystal forms. The salt showed appreciable solubility only in methanol, ethanol, isopropanol (70%) and ethanol: water (1:1) to be used as solvents for crystallization.
8.5 CHARACTERIZATION OF CRYSTALS FORMS:
8.5.1 Optical Microscopy
Photomicrographs showed a distinct difference in the morphology of different solid-states crystals forms of Gatifloxacin obtained from different solvents. The morphology of the crystals obtained may be described as follows:
From GATI - I are Prismatic crystals forms; GATI - II is Spongy opaque like crystal forms; GATI - III are thin pole like crystal forms; and GATI - IV are Rod shaped like crystal forms. It shown in Figure No. 6, 7, 8, 9.
8.5.2 Particle Size Determination
Table No. 8 was shown the particle size distributions of Gatifloxacin crystals obtained from different solvents. There is a large difference between the size distributions of these samples. Particle size of GATI - I is 90 μm, GATI - II is 85 μm, GATI - III is 94 μm and GATI - IV is 101 μm.
8.5.3 Determination of True Density:
Prepared crystals forms of GATI - I, GATI - II, GATI - III and GATI- IV was shown in Table No.8. Obtained crystal forms true densities were 1.110 g/ml, 1.070 g/ml, 1.125 g/ml, 1.140 g/ml respectively. True density of all crystal forms are more than pure drug true density.
8.5.4 FT-IR Spectroscopy
FTIR analysis was carried out to confirm the purity and quality of the drug. The results of these studies confirmed the purity of the drug. On comparing these spectra, differences in the peak pattern were observed in the region of 1400cm-1 to 500cm-1.
Infrared (IR) spectroscopy, another important technique for characterization of polymorphs, was also utilized. On comparing the IR spectra of the crystals, differences in the peak patterns were observed for some of the crystal forms GATI - I, GATI - II, GATI - III and GATI -IV to Gatifloxacin between the ranges of 1800-1100 cm-1 (Fig .7). Forms GATI - II and GATI - III are slightly similar in spectra but between the 1800-1100 cm-1 region obtained peaks are different. Peaks at 1719.81 cm-1, 1557.60 cm-1 1395.39 cm -1 appeared in form GATI - II where as it shifted to 1716.12 cm-1, 1616.58 cm-1, 1391.70 cm-1 respectively. Peak at 1321.65 cm-1 appeared in Form GATI - III and 1321.65 cm-1 appeared in Form GATI - II, and disappeared in Form GATI - IV. Peaks at 1391.70 cm-1, 1277.41 cm-1, 1716.12 cm-1 appeared in case of Form GATI - III, where as it shifted to 1395.39 cm-1 and 1719.81 cm1 in GATI - IV, and peak at 1277.41 cm-1 it disappeared in Form GATI - IV. Peak at 1616.58 cm-1 is appeared in GATI - I, where as it shifted to 1557.60 cm-1 in GATI - II. Peak at 1317.95 cm-1 appeared in GATI - I, where as it shifted to 1278.60 and 1277.60 in GATI - III and GATI - IV respectively. Peak at 1240.65 cm-1 appeared in GATI - IV, where as it shifted in all other crystal forms. Peaks in pure drug of Gatifloxacin (GATI - P) are different in all other crystal forms as GATI - I, GATI - II, GATI - III and GATI - IV.
Categories of crystal forms of Gatifloxacin characterized on the basis of IR spectroscopy and FT-IR Spectra comparison study and interpretations of crystals forms and pure drug (GATI - P) was shown in Spectra No. 7 and Table No.9 respectively.
8.5.5 Powder X-ray Diffraction Studies (PXRD)
Considering powder X-ray diffraction (PXRD) to be the ideal technique for characterizing polymorphs, all the crystal forms were submitted for PXRD studies. In PXRD (Fig.6), Form GATI - I and Form GATI - II gave some what similar peak intensities spectra while rest of other crystal forms (GATI - III, and GATI - IV) afforded different patterns. Spectra of these forms (GATI-I and GATI - II) were neither matching with one another nor with Form GATI - III nor Form GATI - IV. On this base it was concluded that these crystals existed in three different polymorphic forms. It is interesting to note that the superimposable Form GATI - I and Form GATI - II were prepared from aprotic polar solvents while rests of the other crystals were obtained from polar protic solvents.
Gives the PXRD data obtained for the four crystal forms as well as the commercial Gatifloxacin in terms of lattice spacing and the relative intensities. X-ray diffraction patterns for all of the three reported polymorphs are given in Figure 6. All the high intensity lines (relative intensity) observed in the powder pattern of the polymorphs were observed in the pure form. High intensity line at 19.78o2 theta as appeared in case of Form GATI - II was absent in remaining crystal forms as well as commercial Gatifloxacin XRD spectrum report. Other forms did not show any intense line in the region 19.78 o2 theta indicating Form GATI - II to be a different polymorph. Form GATI-I showed intense lines which were somewhat similar intense lines as Form GATI - II but o2 theta values different to Form GATI - II. Form GATI - III showed characteristic intense lines at 9.12, 11.23, 16.70, 19.61o2, 20.94 and 21.69 o2 theta, which were absent in rest of the forms as well as commercial Gatifloxacin XRD spectrum report. Form GATI - IV intense peaks at 9.17, 9.83, 12.91, 21.05 and 25.34 o2 theta in case of absent in remaining crystal forms but only one intense peak (9.17 o2 theta) matched with commercial Gatifloxacin and this type of pattern was not observed in rest of the polymorphs.
Above PXRD data was compared with the reported PXRD data of commercial Gatifloxacin. Commercial Gatifloxacin showed entirely different pattern as well as line intensities to prepared crystals. Commercial Gatifloxacin showed 9.20, 12.95, 12.95, 13.49, 19.92, 21.06 and 24.31 o2 theta. Lines were different in other forms of polymorphs. So where as polymorphs reported here showed different intense lines in this region, indicating them to be different from commercial Gatifloxacin. Powder X-ray Diffraction Studies (PXRD) of four crystal forms of Gatifloxacin and pure drug was shown in Table No. 10.
8.5.6 Thermal Analysis:
Gatifloxacin crystal forms pure drug (GATI - P), GATI - I, GATI - II, GATI - III and GATI - IV DSC thermograms are shown in Figure No(s), 16-20 . DSC thermogram of GATI - I, GAT - III shown small difference in endothermic melting points as sharp peaks at 186.35oC, 186.42oC and heat of fusion at 70.48j/g, 65.76j/g respectively. GATI - I and GATI - III decomposes at 318.36oC and 282.38oC respectively. Polymorphic form GATI - II shown sharp peaks at 183.96oC, heat of fusion at 70.67 j/g. In GATI - I and GATI - II forms having transitions at 161.18oC, 160.80oC respectively. GATI - II having low melting point than the pure drug (GAT I - P), GATI - I, GATI - III and GATI - IV. So, Form GATI - II said to be a metastable Form. GATI - I, GATI - II and GATI - III forms are enantiotropic and GATI - IV and Pure Drug (GATI - P) are monotropic, so GATI- IV was more stable, proofed by Heat of fusion rule of Burger and Ram Berger's rules. Forms GATI - I, GATI - III and GATI - IV are having high melting point and stable than GATI - P (Pure Drug) and GATI - II having low melting point and metastable form. Form GATI - IV is more stable than other three forms. DSC data of Crystal forms and pure drug (GATI - P) of Gatifloxacin was shown in Table No. 11.
TGA shown very interesting results, form of GATI - I weight losses start from temperature 61.01oC after that upon increasing temperature it losses weight at 231.82oC and weight losses 1.88%, 9.82% respectively. Then weight loss is rapidly occurred and final residue was obtained 58.54%. GATI - II I weight losses start from temperature 36.78oC after that it losses weight at 228.54oC and weight losses 1.69%, 8.23% respectively. Then weight loss is rapidly occurred and final residue was obtained 68.11%. Crystal form of GATI - III shown weight losses from temperature range of 37.61oC, 69.63oC, 234.29oC and weight losses 2.29%, 7.22%, 9.08% in orderly in TGA thermogram, and residue was obtained 63.30%. GATI - IV form weight losses from temperature range of 34.32oC and then 253.17oC. Percentage weight losses as 1.99% and 9.58% respectively. Final residue was obtained 56.67%. Pure drug (GATI - P) weight losses start from temperature from 39.25oC, 257.28oC and weight losses 1.88% 11.76% respectively. Final residue was collected 58.07%. DTA results also shown impressive results and similar to DSC results.
8.5.7 SOLUBILITY STUDIES
Solubility of GATI - P (Gatifloxacin) was found to be 22.15 mg/ml. Improvement in solubility was observed with crystal form of GATI - II. Where GATI - I, GATI - III and GATI - IV crystal forms solubility decrease slightly. Solubility of crystal forms follows in order of GATI - IV < GATI - III< GATI - I < AGTI - P < GATI - II. Solubility of crystal forms shown in Table No. 12, Graph No. 2.
8.5.8 DISSOLUTION STUDIES
The result of the in vitro dissolution study is shown in table IV. The results had shown a marked difference in their dissolution behavior of the crystals forms of pure drug and pure drug. Results shown that the amount of crystal form of GATI - II dissolution rate was considerably higher than others crystal forms. The highest dissolution rate was observed in the crystal form of GATI - II 99.47% than other crystal forms GATI - I, GATI - III, GATI - IV and GATI - P (pure drug) dissolution rates 93.73%, 92.70%, 90.22% and 95.02% respectively at the end of 60 min. percentage drug release follows in order of GATI - IV < GATI - III < GATI - I < GATI - P < GATI - II. Percentage of drug release of prepared crystals shown in Table No. 13, Graph No.3.
8.6 Evaluation of Tablets:
The prepared crystals forms of Gatifloxacin tablets were subjected to preliminary characterization such as hardness, thickness, % weight variation, friability and disintegration. The evaluated parameters are within the acceptable range for all the formulations of tablets.
There were no marked variations in the thickness of tablets within each formulation indicating uniform behavior of crystal forms throughout the compression process.
The hardness of the tablet was found to be in the range of 4.6 - 5.0kg/cm2.
Friability of all the formulation showed tablets was found to be in the range of 0.33 - 0.51 %.
No significant differences were observed in the weight of individual tablets from the average weight. It had shown that all the formulations fell within the range of ± 5%. The average weight of tablets within each formulation was found to be uniform. This indicates uniform feeling of the die cavity during tablet compression process.
All prepared crystal forms GATI - I, GATI - II, GATI - III and GATI - IV shown disintegration time below 8 minutes.
In Vitro Drug Release Profile:
The dissolution test for in vitro drug release of all formulations was carried out by using of USP Type -II (paddle). A revolving at 100 rpm with phosphate buffer pH - 7.4 dissolution media was used. The study carried out at 37±0.5° drug release as shown in Table No. 15.
The results are observed as crystal form of Gati - II shown highest dissolution rate 98.28% than other crystal forms GATI - I, GATI - III, GATI - IV and GATI - P (pure drug) dissolution rates 92.34 %, 91.01 %, 88.62 % and 93.58 % respectively at the end of 90 min. But other crystal forms as GATI - I, GATI -II and GATI - IV shown slowest dissolution in their tablet drug release with constant release than pure drug (GATI - P) and crystal form of GATI - II. Prepared crystal forms tablets percentage drug release shown in the order of GATI - IV < GATI - III < GATI - I < GATI - P < GATI - II. Dissolution tare profile of prepare crystals tablets shown in Graph No. 4.