CHAPTER- VIII EXPERIMENTAL WORK: PART I
8.1 Drug Authentication
a) Physical appearance: The sample of progesterone was observed for its colour
and texture.
b) Solubility: The solubility of progesterone was determined in water and
ethanol.
c) Melting point: Melting point determination provides the description about purity of
the drug. Capillary method is used to determine the melting point of progesterone.
Progesterone was placed in capillary tube that was in connection with thermometer.
Further it was positioned in oil bath and heated. The temperature was monitored and
eventually the temperature at melting was noted. The experiment was repeated in
triplicate.
d) IR spectra: Progesterone was scanned in the region of 4000-400 cm-1.
8.2 Development of UV-Vis Spectrophotometric method
8.2.1.2 Selection and Optimization of Solvent
0.25% Sodium lauryl sulphate (SLS) in distilled water was used as a solvent.
8.2.1.3Preparation of Standard Stock Solution
Progesterone (10 mg) was dissolved in 0.25% aqueous solution of sodium lauryl
sulphate (SLS) in the 100 ml volumetric flask and volume was adjusted up to
mark.
8.2.1.4: Selection of Wavelength (Determination)
The solution of drug (100µg/ml) was scanned from 400 to 800nm in the
spectrophotometer.
Linearity Study
The stock solution was diluted to yield a range of concentrations from 2 to 18
µg/ml by diluting 1, 2, 3, 4, 5, 6, 7, and 8 ml stock solution to 50 ml separately.
The absorbance of the solutions was measured at 241 nm and plotted the graph
absorbance vs concentration.
44
CHAPTER- VIII EXPERIMENTAL WORK: PART I
Preparation of solid dispersion
Selection of carrier
PEG6000, Polaxomer 188, and gelucire 50/13 were used for the study in the ratio
1:0.5, 1:1 and 1:1.5 with drug.
Solid dispersion methods
Three methods namely kneading, spherical crystallization and melt granulation
were used for Solid dispersion formulations. The drug solubility of each
formulation was determined.
8.4.1Physical Kneading
A mixture of drug and polymer (1:0.5, 1:1 and 1:1.5 by weight) were moistened
with water and kneaded thoroughly for 30 minutes in a glass mortar. The paste
formed was dried for 24 hours. The dried powder was passed through sieve No.
60 and stored in a desiccator until further evaluation. Similar procedure was
applied for other polymers.[65].
Kneading-SD PG Poloxamer PEG- Gelucire
(mg) 188 (mg) 6000 (mg) 50/13 (mg)
Formulation
KD-B1 250 00 00
KD-B2 500 00 00
KD-B3 750 00 00
KD-B4 00 250 00
KD-B5 500 00 500 00
KD-B6 00 750 00
KD-B7 00 00 250
KD-B8 00 00 500
KD-B9 00 00 750
45
CHAPTER- VIII EXPERIMENTAL WORK: PART I
8.4.2 Spherical crystallization
A mixture of drug and polymer (1:0.5, 1:1 and 1:1.5 by weight) were dissolved in
25 mL boiling di-chloromethane (good solvent) to make quasi-saturated solution.
Then this solution was poured into cold water (5°C) (poor solvent) while stirring
at 1000 rpm. Then 44 mL of isopropyl acetate (wetting agent) was added
thermally controlled under agitation with a propeller type agitator with four
blades. The agglomerates were separated from the solution through filtration
under vacuum and then were placed in a thin layer in an oven at 50°C for 12
h[66,67].
SC-SD PG Poloxamer PEG-6000 Gelucire
Formulation (mg) 188 (mg) (mg) 50/13 (mg)
SC-B1 250 00 00
SC-B2 500 00 00
SC-B3 750 00 00
SC-B4 00 250 00
SC-B5 500 00 500 00
SC-B6 00 750 00
SC-B7 00 00 250
SC-B8 00 00 500
SC-B9 00 00 750
8.4.3 Melt granulation (ADD REF)
250/500/750 mg of each polymer was melted in the beaker separately then 500
mg of the drug was added in each beaker with stirring using a magnetic stirrer
until homogenization. Solidification was allowed to occur at room temperature.
The product was stored in desiccator for 24 hr and then passed through 80# sieve.
46
CHAPTER- VIII EXPERIMENTAL WORK: PART I
MG-SD PG Poloxamer PEG-6000 Gelucire
Formulatio (mg) 188 (mg) (mg) 50/13 (mg)
n
MG-B1 250 00 00
MG-B2 500 00 00
MG-B3 750 00 00
MG-B4 00 250 00
MG-B5 500 00 500 00
MG-B6 00 750 00
MG-B7 00 00 250
MG-B8 00 00 500
MG-B9 00 00 750
8.5 Solubility study
Drug-polymer systems prepared using different methods were screened for their
ability to improve solubility of drug. Briefly, SDF equivalent to 25 mg of drug
were taken in separate volumetric flasks and volume adjusted to 10 ml with water.
The mixture was kept for shaking for 3 h and filtered through Whatman filter. The
00 ml filterate is diluted to 50 ml and the absorbance was recorded. The conc of
each was calculated.
The data was collected in triplicates and plotted in the excel sheet. Demonstrating
the superior solubility of drug was selected further for optimization. Based on the
results, method and ratio of drug: polymer showing better results were selected for
the optimization.
8.6 Micromeritic properties
One optimum solid dispersion batch which exhibited better solubility
enhancement property for progesterone from each method i.e. kneading method,
47
CHAPTER- VIII EXPERIMENTAL WORK: PART I
spherical crystallization and melt granulation was subjected micromeritic
properties evaluation. Different flow properties were estimated as follows,
8.6.1 Bulk density
Apparent bulk density (ρb) was determined by pouring the blend into a graduated
cylinder. The bulk volume (Vb) and weight of powder (M) was determined. Bulk
density apparatus was used for tapping (Lab Hosp, Mumbai, Maharashtra, India).
The bulk density was calculated using the formula [68].
M
ρb = Eq. 8.1
Vb
8.6.2 Tap density
The measuring cylinder containing known mass of blend was tapped for a fixed
time. The minimum volume (Vt) occupied in the cylinder and weight (M) of the
blend was measured. The tap density (ρb) was calculated using the following
formula [68],
M
ρt = Eq. 8.2
Vt
8.6.3 Angle of Repose
Flowability of Powdered Blend of all the batches was assessed by the angle of
repose, determined using fixed funnel free-standing cone method. Angle of repose
was determined in triplicate for batches by using the formula,
1
θ=tan−¿ ( H /R) ¿ Eq. 8.3
Where, ‘θ’ is angle of repose; ‘H’ is height between lower tip of the funnel and
the base of heap of powder; and ‘R’ is radius of the base of heap formed.
8.6.4 Carr’s Compressibility Index (CCI) and Hausner’s Ratio (HR)
Granules of all the batches were evaluated for Carr’s compressibility index (CCI)
and Hausner’s ratio (HR) [69].
(TD−BD)
CCI= X 100 Eq. 8.4
TD
48
CHAPTER- VIII EXPERIMENTAL WORK: PART I
TD
HR= Eq. 8.5
HD
Where, TD and BD are tapped density and bulk density respectively.
8.7 Optimization of formulation
8.7.1 Experimental Design
The impact of factors on melt granulates' stability and integrity was initially
screened for in a series of preliminary investigations. The concentration of
Poloxamer 188 was shown to be a critical formulation parameter, while stirrer
speed was found to be a critical process parameter. Using a 3 2-factorial design, we
systematically tested the effects of varying three levels of key process and
formulation parameters (-1, 0 and +1) on the critical quality attributes of the
optimized formulation. Both the quantity of drug used and the time for which it
was stirred were constant throughout the trial. Total 9 batches were generated
using factorial design as shown in Table 8.1.
Table 8.1:Preparation of melt granulates with 32 factorial design
Independent Variables
Formulation Variable
Formulation Code Conc. of
Poloxamer Stirring Speed
X1 X2
188 (rpm)
(mg)
MG-B1 ̶ 1 250 +1 3000
MG-B2 0 500 +1 3000
MG-B3 +1 750 +1 3000
MG-B4 ̶ 1 250 0 2000
49
CHAPTER- VIII EXPERIMENTAL WORK: PART I
MG-B5 0 500 0 2000
MG-B6 +1 750 0 2000
MG-B7 ̶ 1 250 -1 1000
MG-B8 0 500 -1 1000
MG-B9 +1 750 -1 1000
8.8 Characterization of solid dispersion
8.8.1 FTIR analysis
The FTIR spectra were captured using an IR Spectrophotometer (Alpha T
Bruker). Using a dry potassium bromide mixture, around 2 mg samples were
scanned between 4000 and 500 cm-1[70].
8.8.2 XRD analysis
Powder x-ray diffraction (PXRD) patterns of pure drug, polymer, physical
mixture and optimized formulation were recorded on diffractometer (Miniflex 600
x-ray diffractometer, Rigaku Corporation, Japan). Samples were scanned from 10
to 80°C 2θ at a scan rate of 2°C/min[71].
8.8.3 DSC analysis
Progesterone, physical mixture, and the best melt granulation batch was screened
through DSC analysis. DSC (Mettler Toledo) thermograms were taken at a rate of
10° per minute over the temperature range of 40°C to 360°C with a flow rate of
30 ml/min of nitrogen[72].
8.8.4 In vitro dissolution study
The in vitro dissolving rate of progesterone from the melt granulates were
evaluated in comparison to marketed formulation (Progesterone Capsule) using
the basket method (Disso-Labindia). The experiment was conducted in a 900 ml
vesselat 37±0.5°C, constant-speed 75 rpm, 0.1 N HCl as dissolution media. The
aliquot of test sample (5 ml) were collected manually at the regular intervals of
50
CHAPTER- VIII EXPERIMENTAL WORK: PART I
time for up to 1 hour. The withdrawn samples were replaced with fresh media
with an equal volume at same temperature to maintain the sink condition. The
removed samples were properly diluted and tested for progesterone content [73].
The amount of dissolved progesterone was detected at 241 nm on a UV
spectrophotometer and computed for percent cumulative drug release. Data
obtained from dissolution study was subjected to different drug dissolution kinetic
models in order to find best fit model [74]..
8.9 Statistical Analysis
Data obtained from the experiments were analyzed by ANOVA, lack-of-fit tests,
and multilinear regression analysis. Student's t-test was used to test the statistical
significance wherever applicable and all data were expressed as mean ± SD (n=3).
51
Table no.(??) :
Preparation of Linearity Solutions
Sr. No. Dilution
1 1.0 ml in 50 ml
2 2.0 ml in 50 ml
3 3.0 ml in 50 ml
4 4.0 ml in 50 ml
5 5.0 ml in 50 ml
6 6.0 ml in 50 ml
6 7.0 ml in 50 ml
7 8.0 ml in 50 ml
8 9.0 ml in 50 ml
70
