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Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 61
4.1. MATERIALS
Materials Source
Efavirenz Shasun labs (Pondicherry India)
Atorvastatin calcium Strides Arco Labs (Bangalore India)
Rosuvastatin Calcium Strides Arco Labs (Bangalore India)
Labrafac PG, Labrafil,
Labrasol, Capryol 90
Gattefosse, France (through Bombay
College of Pharmacy, Mumbai)
Capmul MCM, Captex 200 Abitec Group (USA)
Span 80, Triethanolamine,
PEG 800, PEG 200,
Oleic acid, Castor oil,
Propylene glycol
Merck (Mumbai)
Merck (Mumbai)
Merck (Mumbai)
Merck (Mumbai
Tween 80, Tween 20,
Ethyl oleate, Isopropyl myristate
Loba chemie pvt ltd
Loba chemie pvt ltd,
Porous polystyrene beads Thermax Limited, India
Accurel® MP 1000 Membrana, Obernburg, Germany
Colloidal silicon dioxide Loba Chemie, Mumbai, India
Lactose Loba Chemie, Mumbai, India
MCC PH 101 Loba Chemie, Mumbai, India
Sodium Starch Glycolate Malpe biotech Pvt. Ltd., Pune, India.
Polyvinyl pyrrolidone (PVP) SRL, Mumbai, India
Talc Lobachemie Pvt. Ltd., Mumbai, India.
Magnesium Stearate Lobachemie Pvt. Ltd., Mumbai, India.
All other chemicals and buffers used were of analytical grade
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 62
4.2. INSTRUMENTS AND EQUIPMENTS
Instruments Model/ Manufacturer
Digital balance Shinko Sansui, Japan
Hot air oven Tempo, India
Magnetic stirrer Remi equipments, India
Tablet punching machine Rimek, Minipress- 1(model-1674),
Karnavati, India
Micrometer screw gauge Mitutoyo, Japan
Dissolution apparatus (8 basket) Electrolab, India
UV-Visible spectrophotometer Shimadzu-1800, Japan
FT-IR spectrophotometer Shimadzu-8400 S, Japan
KBr Press Techno search instruments, India
Scanning electron microscopy (SEM) Hitachi S3400, Tokyo, Japan
Differential scanning calorimetry (DSC) Shimadzu DSC-60, Japan
X-ray powder diffractometer X-ray Diffractometer, Rigaku Corporation,
Tokyo, Japan
Zetasizer 3000 Malvern Instruments, UK
Tablet hardness tester Inweka, IHT 100, Ahmedabad , India
Digital pH meter Elico-LI120pH(type003),Hyderabad
Vortex mixer Remi motors
Viscometer Brookfield, Inc
HPLC Shimadzu LC 2010A HT
Abbe Refractometer Bausch and lomb optical, NY.
Deep freezer Labline
Sonicator Bandelin RK 100 H, Germany
Centrifuge Remi, Rajendra Electrical Industries Ltd,
Vasai, India.
Stability Chambers Thermolab humidity chambers, India.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 63
REAGENTS93
0.1 M Hydrochloric acid (HCl)
0.1 M HCl Solution was prepared by diluting 8.5ml concentrated hydrochloric
acid with distilled water to 1000 ml in a volumetric flask.
0.02M potassium dihydrogen phosphate
2.7218 g of potasium dihydrogen phosphate was dissolved in water and
diluted with distilled water to 1000 ml in a volumetric flask.
Sodium Hydroxide, 0.2 M
Dissolved accurately weighed 8.0 g of sodium hydroxide in 1000 ml of
distilled water in a volumetric flask.
Potassium Chloride, 0.2 M
Dissolved 14.911 g of potassium chloride in water and dilute to 1000 ml with
water in a volumetric flask.
Hydrochloric Acid Buffer
50.0 ml of 0.2 M potassium chloride was placed in a 200ml volumetric flask,
added 85ml of 0.2 M hydrochloric acid and volume was made up to the volume with
water.
Phosphate Buffer 7.4
50.0 ml of 0.2 M potassium dihydrogen phosphate was taken in a 200ml
volumetric flask, added 39.1 of 0.2 M sodium hydroxide and volume was made up to
the volume with water.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 64
4.3. ANALYTICAL METHODS
4.3.1. UV method
Efavirenz
Determination of max
UV Scanning Range: 200-400nm.
Accurately weighed 100 mg of efavirenz was dissolved in small amount of
methanol and the volume was made up to 100 ml using 0.1 M HCl (1000 µg/ml). The
stock solution (2 ml) was further diluted to 100 ml using 0.1 M HCl to get a
concentration of 20 µg/ml.
From the stock solution, 12 µg/ml solutions was prepared and scanned
between 200-400 nm. It was found that the sample showed a λmax of 247 nm (Figure
4.1) and this value is used for further analysis.
Figure 4.1: UV Spectra of Efavirenz in 0.1M HCl
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 65
Standard Plot of Efavirenz
From the above stock solution, appropriate aliquots were taken, so as to get
drug concentrations of 2, 4, 6, 8, 10 and 12 µg/ml. The absorbance of these solutions
was measured at 247 nm and a graph of concentration versus absorbance was plotted
(Table 4.1). The calibration curve of efavirenz in 0.1M HCl is shown in the
Figure 4.2.
Table 4.1: Calibration curve data of efavirenz in 0.1M HCl
Sl. No. Concentration
(µg/ml)
Absorbance
Mean ± SD*
1 0 0
2 2 0.127±0.001
3 4 0.262±0.003
4 6 0.386±0.001
5 8 0.496±0.002
6 10 0.636±0.002
7 12 0.740±0.003
*Standard deviation: n=6
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 66
Figure 4.2: Calibration curve of Efavirenz in 0.1M HCl
Atorvastatin calcium
Determination of max
Standard stock solution of Atorvastatin calcium was prepared by dissolving
accurately weighed 100 mg of drug in 10 ml of methanol and the volume was then
made up to 100 ml of 0.1M HCl to obtain the solution of 1000 µg/ml. From the
standard stock solution (1000 µg/ml), 10 ml solution was diluted to 100 ml using
0.1M HCl to get the solution of concentration 100 µg/ml. The suitable diluted solution
was scanned between 200-400nm. It was found that the sample showed a λmax of 246
nm (Figure 4.3) and this value was used for further analysis.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 67
Figure 4.3: UV Spectra of Atorvastatin Calcium in 0.1M HCl
Standard plot of Atorvastatin Calcium
From the above stock solution, appropriate aliquots were taken, so as to get
drug concentrations of 2.0 to 18.0 µg/ml. The absorbencies of these drug solutions
were estimated at 246 nm (Table 4.2). The calibration curve of Atorvastatin Calcium
in 0.1M HCl is shown in the Figure 4.4.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 68
Table 4.2: Calibration curve data of Atorvastatin Calcium in 0.1M HCl
Sl. No. Concentration in
µg/ml
Absorbance
Mean ± SD*
1 0 0
2 2 0.086±0.003
3 4 0.164±0.004
4 6 0.241±0.002
5 8 0.322±0.003
6 10 0.402±0.001
7 12 0.479±0.004
8 14 0.566±0.002
9 16 0.634±0.002
10 18 0.694±0.005
* Standard deviation: n=6
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 69
Figure 4.4: Calibration curve of Atorvastatin Calcium in 0.1M HCl
Rosuvastatin Calcium
Determination of max
UV Scanning Range: 200-400nm.
Standard stock solution of rosuvastatin calcium was prepared by dissolving
accurately weighed 100 mg of drug in 10ml of methanol and the volume was then
made up to 100 ml with 0.1M HCl to obtain the solution of 1000 µg/ml. From the
standard stock solution (1000 µg/ml), 10 ml solution was diluted to 100 ml using
0.1M HCl to get a solution of concentration 100 µg/ml. The suitable diluted solution
was scanned between 200-400nm. It was found that the sample showed a λmax of
243 nm (Figure 4.5) and this value was used for further analysis.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 70
Figure 4.5: UV Spectra of Rosuvastatin Calcium in 0.1M HCl
Standard plot of Rosuvastatin Calcium
From the standard stock solution (100 µg/ml), appropriate aliquots were taken,
so as to get drug concentrations of 2.0 to 20.0 µg/ml. The absorbencies of these drug
solutions were estimated at 243 nm (Table 4.3). The calibration curve of Rosuvastatin
Calcium in 0.1M HCl is shown in the Figure 4.6
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 71
Table 4.3: Calibration curve data of Rosuvastatin Calcium in 0.1M HCl
Concentration in
µg/ml
Absorbance
Mean ± SD*
0 0
2 0.066±0.003
4 0.167±0.002
8 0.341±0.004
12 0.484±0.001
16 0.655±0.003
20 0.814±0.002
* Standard deviation: n=6
Figure 4.6: Calibration curve of Rosuvastatin Calcium in 0.1M HCl
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 72
4.3.2. HPLC method
Efavirenz
The RP-HPLC method was used to estimate efavirenz in plasma. The mobile
phase consisted of acetonitrile:water (70:30) with C18 column (250mm×4.6mm×5µ).
The drug retention time of was found to be 5.61 min. Representative chromatogram of
efavirenz monitored at 247 nm is shown in Figure 4.7. A linear relationship was
observed in the concentration range of 1-100 mcg/ml (r2=0.998; n= 6). The method
was specific as there was no interference at the retention time of the analyte by the
blank solution and the LOQ i.e. the limit of quantification was with acceptable
precision. The accuracy at low (1µg/ml), moderate (50 µg/ml) and higher (100 µg/ml)
concentrations of efavirenz ranged from 97.46-101.34% and 95.74-103.72% for intra
and inter day respectively107, 108.
Figure 4.7: Typical Chromatogram of Efavirenz by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 73
Table 4.4: Standard calibration data of efavirenz by RP-HPLC
Sl No Concentration(µg/ml) Area
Mean ± SD*
1 0 0
2 1 12033.64±1357.12
3 10 110431.23±2689.95
4 20 228955.07±5344.32
5 50 575184.75±8200.74
6 70 793782.1±6275.54
7 100 1145940.79±9344.82
* Standard deviation: n=6
Figure 4.8: Standard calibration curve of Efavirenz by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 74
Atorvastatin calcium 109, 110,111
The RP-HPLC method was used to estimate AC in plasma. The mobile phase
consisted of mixture of 0.02M potassium dihydrogen phosphate, acetonitrile and
methanol (30:10:60) v/v/v at a flow rate of 1 ml/min with C-18 column
(250mm×4.6mm×5µ).
The drug retention time of was found to be 6.8min. Representative
chromatogram of AC monitored at 247 nm is shown in Figure 4.9. A linear
relationship was observed in the concentration range of 1-50 mcg/ml (r2 = 0.998; n =
6). The method was specific as there was no interference at the retention time of the
analyte by the blank solution and the LOQ i.e. the limit of quantification was with
acceptable precision. The accuracy at low (1µg/ml), moderate (25µg/ml) and higher
(50 µg/ml) concentrations of AC ranged from 98.19-103.76% and 96.18-106.42% for
intra and inter day respectively.
Figure 4.9: Typical Chromatogram of Atorvastatin Calcium by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 75
Table 4.5: Standard calibration data of Atorvastatin Calcium by RP-HPLC
Sl. No Concentration
(µg/ml)
Area
Mean ± SD*
1 0 0
2 1 72.89±1.24
3 10 732.6±3.76
4 20 1465.2±10.24
5 30 2097.8±13.98
6 40 2728.7±18.56
7 50 3360.6±22.45
* Standard deviation: n=6
Figure 4.10: Standard calibration curve of Atorvastatin Calcium by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 76
Rosuvastatin calcium112
The RP-HPLC method was used to estimate RC in plasma. The mobile phase
consisted of methanol-water (68:32 v/v; pH adjusted to 3.0 with trifluoroacetic acid)
at a flow rate of 1.5 ml/min with C18 column (250mm×4.6mm×5µ). The drug
retention time of was found to be 3.6 min. Representative chromatogram of RC
monitored at 243 nm is shown in Figure 4.11. A linear relationship was observed in
the concentration range of 1-100 mcg/ml (r2=0.999; n= 6). The method was specific
as there was no interference at the retention time of the analyte by the blank solution
and the LOQ i.e. the limit of quantification was with acceptable precision. The
accuracy at low (1µg/ml), moderate (50 µg/ml) and higher (100 µg/ml) concentrations
of RC ranged from 98.84-102.63% and 95.87-104.12% for intra and inter day
respectively.
Figure 4.11: Typical Chromatogram of Rosuvastatin Calcium by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 77
Table 4.6: Standard calibration data of Rosuvastatin Calcium by RP-HPLC
Sl. No Concentration
(µg/ml)
Area
Mean ± SD*
1 0 0
2 1 91876±1659
3 10 312592±7347
4 20 601576±9648
5 50 1536078±24471
6 70 2209383±43437
7 100 3072157±51402
* Standard deviation: n=6
Figure 4.12: Standard calibration curve of Rosuvastatin Calcium by RP-HPLC
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 78
4.3.3. Bio analytical method
Efavirenz
The HPLC bio analytical method used for efavirenz analysis was rapid and
simple. Protein precipitation method was used (Acetonitrile) for extraction, the clear
supernatant liquid was separated, filtered (0.45µ filter) and injected into the HPLC
system. Drug retention time was 5.69 min. A linear relationship was observed in the
concentration range of 1-30 µg/ml (r2 = 0.999; n = 6). The results show the linearity
between the peak area and the concentration of the analyte. The developed method
was specific as there was no interference by the matrix components at the retention
time of the analyte and the LOQ i.e. the limit of quantification was with acceptable
precision. The accuracy at low (1µg/ml), moderate (15 µg/ml) and higher (30 µg/ml)
concentrations of efavirenz ranged from 97.24-102.57% and 94.62-104.12% for intra
and inter day respectively.
Table 4.7: Standard calibration data of Efavirenz in rat plasma
Sl. No Concentration
(µg/ml) Area
Mean ± SD*
1 0 0
2 1 12011±2647
3 5 60062±3548
4 10 110416±5029
5 15 170456±7123
6 20 228947±6643
7 25 289019±5473
8 30 349097±6110
* Standard deviation: n=6
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 79
Figure 4.13: Standard calibration curve of Efavirenz in rat plasma
Atorvastatin calcium
The HPLC bio analytical method used for AC analysis was rapid and simple.
Protein precipitation method was used (Acetonitrile) for extraction, the clear
supernatant liquid was separated, filtered (0.45µ filter) and injected into the HPLC
system. Drug retention time was 6.9 min. A linear relationship was observed in the
concentration range of 1 - 30 µg/ml (r2 = 0.999; n = 6). The results show the linearity
between the peak area and the concentration of the analyte. The developed method
was specific as there was no interference by the matrix components at the retention
time of the analyte and the LOQ i.e. the limit of quantification was with acceptable
precision. The accuracy at low (1µg/ml), moderate (15 µg/ml) and higher (30 µg/ml)
concentrations of AC ranged from 98.17-101.23% and 96.05-103.27% for intra and
inter day respectively.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 80
Table 4.8: Standard calibration data of Atorvastatin Calcium in rat plasma
Sl. No Concentration
(µg/ml)
Area
Mean ± SD*
1 0 0
2 5 349.2±12.64
3 10 647.18±23.27
4 15 994.89±41.12
5 20 1287.7±52.82
6 25 1627.9±64.05
7 30 1947.64±57.64
* Standard deviation: n=6
Figure 4.14: Standard calibration curve of Atorvastatin Calcium in rat plasma
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 81
Rosuvastatin calcium
The HPLC bio analytical method used for RC analysis was rapid and simple.
Protein precipitation method was used (Acetonitrile) for extraction, the clear
supernatant liquid was separated, filtered (0.45µ filter) and injected into the HPLC
system. Drug retention time was 3.71 min. A linear relationship was observed in the
concentration range of 1 - 50 µg/ml (r2 = 0.998; n = 6). The results show the linearity
between the peak area and the concentration of the analyte. The developed method
was specific as there was no interference by the matrix components at the retention
time of the analyte and the LOQ i.e. the limit of quantification was with acceptable
precision. The accuracy at low (1µg/ml), moderate (25µg/ml) and higher (50 µg/ml)
concentrations of RC ranged from 98.56-103.19% and 93.44-105.23% for intra and
inter day respectively.
Table 4.9: Standard calibration data of Rosuvastatin Calcium in rat plasma
Sl. no Concentration
(µg/ml)
Area
Mean ± SD*
1 0 0
2 1 65876±2758
3 10 312592±4236
4 20 601576±7236
5 30 934712±9245
6 40 1203629±12547
7 50 1583028±17634
* Standard deviation: n=6
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 82
Figure 4.15: Standard calibration curve of Rosuvastatin calcium in rat plasma
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 83
4.4 PREPARATION OF S(N)EDDS
4.4.1. Selection of oils, surfactants and cosurfactants 113,114
The oils, surfactants and cosurfactants were selected based on solubility of the
drug. The study was carried out by taking 2 ml of selected oil (Labrafac PG, Capmul
MCM, Ethyl oleate etc) / surfactant (Tween 80, Tween 20, Labrasol etc) /
cosurfactant (PEG 200, PEG 400, Propylene Glycol etc) in glass vial containing
excess amount of drug. The mixtures were mixed manually for 30 min in order to
facilitate proper mixing of drug with the vehicles. The vials were sonicated for 2 h
and kept in water bath for 48 h for equilibriation. The vials were centrifuged at 3000
rpm for 20 min, followed by filtration. The filtrate was suitably diluted with methanol
and drug dissolved in various vehicles was analysed by UV spectrophotometer.
4.4.2. Compatibility Study
Interaction between the drug, oil, surfactant and cosurfactant were studied by
FT-IR technique. The blank KBr pellets were prepared, onto which oil, surfactant and
cosurfactant were dropped individually and it was pressed with another blank KBr
pellet using hydraulic press. The pure drug was mixed with KBr in the ratio of 1:3 and
punched in a hydraulic press at 5-6 ton load. The prepared pellets were scanned from
4000 to 400 cm-1 using FT-IR spectrophotometer (FT-IR 8400 S, Shimadzu). The
FT-IR spectra of the physical mixture were compared with the spectra of pure drug,
oil, surfactant and cosurfactant.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 84
4.4.3. Construction of pseudoternary phase diagram115, 116, 117
Pseudoternary phase diagrams were constructed to examine the formation of oil in
water nanoemulsions using four components: oil, surfactant, cosurfactant and aqueous
system. Phase diagrams were constructed using Chemix software.
Based on the solubility study, the oil, surfactant and cosurfactant were selected.
Drug Oil Surfactant Cosurfactant Aqueous
system
Efavirenz Labrafac PG Tween 80 PEG 200
Distilled
Water
Atorvastatin calcium Capmul MCM Tween 20 Propylene glycol
Rosuvastatin calcium Capmul MCM Tween 20 PEG 200
Surfactant and cosurfactant (Smix) in each group were mixed in different
weight ratios (1:0, 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1). These Smix ratios were chosen in
increasing concentration of surfactant with respect to cosurfactant and increasing
concentration of cosurfactant with respect to surfactant for comprehensive study of
the phase diagrams. For each phase diagram, oil and specific Smix ratio was mixed
thoroughly in different weight ratios from 1:1 to 2:1 in different glass vials. Ten
combinations of oil and Smix, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 2:1 were
made so that maximum ratios were covered for the study. Phase diagrams were
constructed using aqueous titration method. Slow titration with aqueous phase was
carried out to each weight ratio of oil and Smix, through the visual observation the
following categories were assigned
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 85
1. Transparent and easily flowable: oil/water nanoemulsions
2. Transparent gel: nanoemulsion gel
3. Milky or cloudy: emulsion
4. Milky gel: emulgel
In the phase diagrams, only nanoemulsion (NE) points were plotted (shaded
area), so that there is no overcrowding of the phases in the diagram, as for formulation
development, only the nanoemulsion region is of interest.
4.4.4. Selection of formulations from Phase diagrams
From each phase diagram constructed, different formulations were selected
from NE region so that drug could be incorporated into it on the following basis.
Efavirenz
50mg of efavirenz (dose of drug) was dissolved in oil phase. The oil phase
used was in the increment of 5% (10%, 15%, 20%, 25%, etc) from the NE
region. For each 5 % of oil selected, the formula that used the minimum
concentration of Smix for its NE formulation was selected from the phase
diagram.
Atorvastatin calcium (AC)
10mg of AC (dose of drug) was dissolved in oil phase. The oil phase used was
in the increment of 5% (10%, 15%, 20%, 25%, etc) from the NE region. For
each 5 % of oil selected, the formula that used the minimum concentration of
Smix for its NE formulation was selected from the phase diagram.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 86
Rosuvastatin Calcium (RC)
10mg of RC (dose of drug) was dissolved in oil phase. The oil phase used was
in the increment of 5% (10%, 15%, 20%, 25%, etc) from the NE region. For
each 5 % of oil selected, the formula that used the minimum concentration of
Smix for its NE formulation was selected from the phase diagram.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 87
4.5. EVALUATION
4.5.1. Thermodynamic stability tests
Selected formulations were subjected to different thermodynamic stability
tests (Centrifugation, Heating cooling cycle and Freeze thaw cycle), to overcome
selecting metastable formulation.
Centrifugation: Selected formulations from phase diagrams were centrifuged
at 3500 rpm for 30 min and observed for phase separation, creaming and
cracking. Formulations that are stable were taken for Heating cooling cycle.
Heating cooling cycle (H/C cycle): Stability of nanoemulsions on variation of
temperature was studied by H/C cycle. Six cycles between refrigerator
temperature 4oC and 45oC with storage at each temperature for not less than
48 h. Formulations, that are stable at these temperatures, were subjected to
Freeze thaw cycle.
Freeze thaw cycle: Three freeze thaw cycles between -21oC and +25oC with
storage at each temperature for not less than 48 h was carried out for the
formulations. Formulations, which passed these thermodynamic stress tests,
were further taken for the dispersibility tests for assessing the efficiency of self
emulsification118.
4.5.2. Dispersibility tests 42,55
The efficiency of dispersibility was assessed using a USP XXII dissolution
apparatus II. Each formulation (0.5ml) was added to 500 ml distilled water maintained
at 37±0.5◦C, with paddle rotating at 50rpm for gentle agitation. The in vitro
performance of the formulations was visually assessed using the grading system as
shown below.
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Dept of Pharmaceutics, JSSCP, Mysore 88
Grade A: Rapidly forming (within 1 min) nanoemulsion, having a clear or bluish
appearance.
Grade B: Rapidly forming, slightly less clear emulsion, having a bluish white
appearance.
Grade C: Fine milky emulsion that formed within 2 min.
Grade D: Dull, greyish white emulsion having slightly oily appearance that is slow to
emulsify (longer than 2 min).
Grade E: Formulation, exhibiting either poor or minimal emulsification with large oil
globules present on the surface.
The Formulations that passed the thermodynamic stability and dispersibility
tests in Grade A and B were selected for further studies.
4.5.3. Effect of pH and robustness to dilution
Formulations were subjected to 50, 100, 1000 and 3000 fold dilution with
distilled water, 0.1M HCl and simulated intestinal fluid (pH 6.8). The resultant diluted
nanoemulsions were checked manually for any physical changes such as (coalescence
of droplets, precipitation or phase separation) after 24 h storage 101, 119.
4.5.4. Globule size measurement
The mean globule size and polydispersity index (P.I.) of the resulting
nanoemulsions were determined by photon correlation spectroscopy (which analyses
the fluctuations in light scattering due to Brownian motion of the particles) using a
Zetasizer 3000 (Malvern Instruments Worcestershire, UK) Light scattering was
monitored at 25ºC at a 90ºangle120.
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 89
4.5.5. Zeta potential determination
The zeta potential of the diluted formulation was measured using a zeta meter
system. (Malvern instrument, Worcestershire, UK)120.
4.5.6. Viscosity
Brookfield DV III ultra V6.0 RV cone and plate rheometer (Brookfield
Engineering Laboratories, Inc, Middleboro, MA, spindle # CPE40) was used to
determine the viscosity of different formulations at 25±1.0°C 65.
4.5.7. Refractive index and percent transmittance
The refractive index of the system was measured using Abbe’s refractometer.
The percent transmittance of the system was measured using UV
spectrophotometer (Shimadzu, Japan) keeping distilled water as blank109.
4.5.8. Differential scanning calorimetry
The samples (about 3.00 mg) were placed in standard aluminum cups, and dry
nitrogen was used as effluent gas. All samples were scanned at a temperature ramp
speed of 5°C /min and the heat flow from 0 to 200°C.
4.5.9. Drug content estimation
Prepared SNEDDS containing drug equivalent to one dose was added in
50 mL volumetric flask containing methanol and mixed it well. The extracted solution
was suitably diluted and analysed for drug content using UV-spectrophotometer121.
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Dept of Pharmaceutics, JSSCP, Mysore 90
4.5.10. Drug release studies
Efavirenz
Efavirenz release from the prepared SNEDDS was performed using
dissolution apparatus II containing 900 mL of 0.1 M HCl as dissolution medium
maintained at 37±0.5◦C with the paddle speed of 50 rpm. SNEDDS equivalent to
50mg drug was directly introduced into the dissolution medium and a suitable aliquot
of sample was collected at 0, 5, 10, 15, 20, 30, 40 60, 80 160 and 320min. The sample
withdrawn were suitably diluted with 0.1M HCl and analyzed spectrophotometrically
at 247nm. An equivalent volume of fresh dissolution medium maintained at 37°C was
added to compensate the loss due to sampling122.
Atorvastatin calcium
Atorvastatin calcium release from the prepared SNEDDS was performed using
dissolution apparatus II containing 900 mL of 0.1 M HCl as dissolution medium
maintained at 37±0.5◦C with the paddle speed of 50 rpm. SNEDDS equivalent to
10mg drug was directly introduced into the dissolution medium and a suitable aliquot
of sample was collected at 0, 5, 10, 15, 20 and 30 min. The sample withdrawn were
suitably diluted with 0.1M HCl and analyzed spectrophotometrically at 246nm. An
equivalent volume of fresh dissolution medium maintained at 37°C was added to
compensate the loss due to sampling123.
Rosuvastatin calcium
Rosuvastatin calcium release from the prepared SNEDDS was performed
using dissolution apparatus II containing 900 mL of 0.1 M HCl as dissolution medium
maintained at 37±0.5◦C with the paddle speed of 50 rpm. SNEDDS equivalent to
Materials and Methods
Dept of Pharmaceutics, JSSCP, Mysore 91
10mg drug was directly introduced into the dissolution medium and a suitable aliquot
of sample was collected at 0, 5, 10, 15, 20 and 30 min. The sample withdrawn were
suitably diluted with 0.1M HCl and analyzed spectrophotometrically at 243nm. An
equivalent volume of fresh dissolution medium maintained at 37°C was added to
compensate the loss due to sampling124, 125.
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Dept of Pharmaceutics, JSSCP, Mysore 92
4.6. PREPARATION OF SOLID SNEDDS (S-SNEDDS)
The S-SNEDDS were prepared using adsorption technique. The solid
adsorbents, used to load prepared SNEDDS, are Aerosil 200, Porous polystyrene
beads (TULSION® ADS-600) and Accurel P 1000. A constant aliquot of SNEDDS
was initially added in increments and mixed with the adsorbent in a mortar. Addition
of SNEDDS to the adsorbent, however, was discontinued once a non-flowing
cohesive mass was formed. The granular mass obtained was passed through 250 µm
mesh to get uniformity in particle size. The samples were stored in a desiccator until
further evaluation99, 105, 126.
4.6.1. Angle of repose
The flow characteristics of prepared S-SNEDDS were measured by angle of
repose using fixed funnel method. Angle of repose is the maximum angle possible
between the surface of a pile of the powder and the horizontal plane. Relationship
between angle of repose and powder flow is given in Table 4.10. A funnel that was
secured with its tip at a given height above the graph paper was placed on a flat
horizontal surface. S-SNEDDS were carefully poured through the funnel until the
apex of the conical pile just touches the tip of the funnel. The radius and height of the
pile were determined. The angle of repose () for samples were calculated using the
following formula
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Dept of Pharmaceutics, JSSCP, Mysore 93
Table 4.10: Relationship between angle of repose and powder flow
Angle of repose () Flowability
<25 Excellent
25-30 Good
30-40 Passable
>40 Very poor
4.6.2. Loading efficiency
Loading efficiency of adsorbents was determined using following formula
Where WL is the weight of SNEDDS-loaded adsorbent, and WI is the initial
weight of adsorbent.
4.6.3. Scanning electron microscope
The surface morphology of S-SNEDDS was examined by scanning electron
microscope (Hitachi S3400, Tokyo, Japan). The powder samples were glued and
mounted on metal sample plates. The samples were gold coated with a sputter coater
using an electrical potential of 2.0 kV at 25 mA for 10 min27, 127.
4.6.4. Differential scanning calorimetry
The samples (about 3.00 mg) were placed in standard aluminum pans, and dry
nitrogen was used as effluent gas. All samples were scanned at a temperature ramp
speed of 5°C /min and the heat flow from 0 to 200°C 102.
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Dept of Pharmaceutics, JSSCP, Mysore 94
4.6.5 X-ray powder diffraction
Powder X-ray diffraction patterns on S-SNEDDS were obtained by using an
X-ray Diffractometer (Miniflex II Desktop X-ray Diffractometer, Rigaku Corporation,
Tokyo, Japan). The samples were scanned from 6° to 40° (2θ) with an increment of
0.02° and measurement time of 10 s/increment128.
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4.7. EXPERIMENTAL DESIGN129-132
A randomized, 23 full factorial design with three factors at two levels was
employed to systematically study the preparation of SNEDDS loaded tablets. A total
of eight experimental trials were performed at all possible combinations. The
independent variables, the amount of MCC [micro crystalline cellulose PH 101] (X1),
PVP [polyvinyl pyrollidone] (X2), and the amount of SSG [sodium starch glycolate]
(X3) were selected on the basis of trials taken during optimization of excipient which
were varied at two levels (low and high). The levels of the factors studied were
chosen so that their relative difference was adequate to have a measurable effect on
the response, along with the information that the selected levels are within practical
use. The hardness, disintegration and percentage cumulative drug release at 40 min
were used as dependent variables (responses). Design-Expert 8.0.6.1 software (Stat-
Ease Inc., USA) was used for generation and evaluation of the statistical experimental
design.
4.8. PREPARATION OF TABLETS
Tablets are prepared by direct compression on a rotary tablet press(Remi) with
flat plane face punches (12mm punch diameter). All the excipients, except the
lubricant, were passed through a #20 mesh screen. The drug blend was prepared by
mixing them manually in a polyethylene bag for 10-12min. The lubricant was added
to this blend and mixed again for 2 min. The formulations were prepared according to
the matrix of the 23full factorial design; varying the levels of the factors, i.e.
concentration of MCC, PVP and SSG133, 134.
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Dept of Pharmaceutics, JSSCP, Mysore 96
Table 4.11: Composition of Efavirenz loaded SNEDDS tablet
Ingredient Qty (mg)
Efavirenz loaded SNEDDS 150
Microcrystalline cellulose PH 101 (MCC) 200/300
Polyvinyl pyrollidone K 30 (PVP) 12/20
Sodium starch glycolate (SSG) 8/16
Magnesium stearate 5
Talc 5
Lactose q.s 500
Table 4.12: Composition of Atorvastatin Calcium loaded SNEDDS tablet
Ingredient Qty(mg)
AC loaded SNEDDS 100
MCC 250/350
PVP 8.75/15.75
SSG 5/14
Magnesium stearate 5
Talc 5
Lactose q.s 500
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Dept of Pharmaceutics, JSSCP, Mysore 97
Table 4.13: Composition of Rosuvastatin Calcium loaded SNEDDS tablet
Ingredient Qty(mg)
RC loaded SNEDDS 100
MCC 250/350
PVP 8.75/15.75
SSG 5/14
Magnesium stearate 5
Talc 5
Lactose q.s 500
4.9. MICROMERITIC PROPERTIES OF POWDER BLEND
4.9.1. Bulk density and Tapped density
Bulk density was determined by pouring the samples into a dried measuring
cylinder without tapping. Samples were carefully leveled without compacting and the
initial volume to the nearest graduated unit was recorded and untapped density in g/
cc was calculated. The test was carried out in triplicate.
W Bulk density Untapped (g / cc) = --------
V
Where,
W = weight of the sample in g
V = Volume occupied by the sample in cc.
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Dept of Pharmaceutics, JSSCP, Mysore 98
Tapped density was determined by placing a graduated cylinder containing a
known mass of powder on a mechanical tapper apparatus (Electro lab-tap density
tester-USP). Samples were tapped until no further reduction in volume of the sample
was observed. The test was carried out in triplicate. The tapped density was calculated
by following equation:
W Tapped Bulk density (g / cc) = -----
Vf
Where,
W = Weight of the sample (g).
Vf = Volume occupied by the sample (cc)
4.9.2. Carr’s Index
Flowability of prepared S-SNEDDS was quantified using Carr’s Index (CI).
The CI was determined from their apparent bulk density and the tapped densities. The
test was carried out in triplicate.
4.9.3. Hausner’s Ratio
Hausner’s ratio is an indication of the flowability of powder and the ratio
greater than 1.25 is considered to be an indication of poor flowability. Hausner’s ratio
was determined by the following equation. The test was done in triplicate.
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Dept of Pharmaceutics, JSSCP, Mysore 99
4.9.4. Angle of repose ()
Angle of repose was carried out as explained in the section 4.6.1
4.10. EVALUATION OF TABLETS 135, 136
4.10.1. Thickness
The thickness of the tablets was determined by using digital vernier calipers.
The average of five tablets from each formulation was taken and expressed in
millimeter.
4.10.2. Weight variation test
20 tablets from each formulation were randomly picked and weighed
individually and the average weight was calculated. The individual weights were
compared with the average weight. Percentage deviation were calculated using
following equation
% deviation
4.10.3. Hardness test
Hardness indicates the ability of a tablet to withstand mechanical shocks while
handling. The hardness of the tablets was determined using Inweka hardness tester.
Ten tablets were chosen randomly from the composite samples for each of the
tableting runs and the average value was determined.
4.10.4. Disintegration test
Six tablets were placed in a disintegration tester (Electro lab) filled with
distilled water at 37±2°C. The tablets were considered completely disintegrated when
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Dept of Pharmaceutics, JSSCP, Mysore 100
all the particles passed through the wire mesh. Disintegration times recorded are mean
of three determinations.
4.10.4. Friability
The friability of tablets was determined using Roche Friabilator. It is
expressed in percentage (%). Ten tablets were initially weighed ( ) and transferred
into friabilator. The friabilator was operated at 25rpm for 4 minutes or run up to 100
revolutions. The tablets were weighed again (W). The % friability was then calculated
by
% Friability of tablets less than 1% are considered acceptable.
4.10.5. Drug content
Five tablets were weighed individually and powdered. The powder equivalent
to one dose was weighed and drug was extracted with methanol and filtered. The
filtrate was suitably diluted with methanol and drug content was determined using UV
spectrophotometer. The results are mean of three experiments.
4.10.6. In vitro drug dissolution
In vitro drug dissolution tests were performed using dissolution apparatus II
(Electro Lab, Mumbai). The dissolution tests were carried out in 900 ml of 0.1 M HCl
maintained at 37±0.5°C with 100 rpm. One ml of sample was withdrawn from the
dissolution apparatus at predetermined time intervals. The samples were filtered and
suitably diluted with 0.1M HCl, drug content was determined using UV/Vis
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Dept of Pharmaceutics, JSSCP, Mysore 101
spectrophotometer. The sample withdrawn from the dissolution medium was replaced
with equal amount of fresh 0.1M HCl maintained at 37±0.5°C 105, 137. Results are
average of three replicate experiments
4.11. RECONSTITUTION PROPERTIES OF OPTIMIZED S-SNEDDS
TABLETS
4.11.1. Dispersibility tests and robustness to dilution
Dispersibility tests and robustness to dilution was studied as described in
section no 4.5.2. and 4.5.3.
4.11.2. Globule size measurement
Globule size measurement was made as described in section no 4.5.4
4.12. In vivo studies
In vivo study protocols were approved by the Institutional Animal Ethics
Committee (Regd. No 107/2012). The oral pharmacokinetics of drug was assessed in
Wistar rats (220-250 g) of either sex at a dose equivalent to 10 mg/kg of drug. The
(i) drug-loaded S-SNEDDS tablets (ii) extemporaneous drug suspensions were
systematically compared. A wash out period of one month was given between testing
of two products.
After collecting the zero hour blood sample (blank), the S-SNEDDS (tablets)
in the study was administered orally with 10 ml of water. No food or liquid other than
water was permitted until 4 hours following the administration of the product. Blood
samples (0.5 ml) were collected from tail vein at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours
after administration. The blood samples were collected in heparinized tubes and were
centrifuged at 10000 rpm for 10 min and the plasma separated was collected into dry
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Dept of Pharmaceutics, JSSCP, Mysore 102
tubes. All the samples were stored under refrigerated conditions prior to assay on the
same day. Plasma drug concentrations were determined by a HPLC method 29, 138. The
pharmacokinetic parameters are calculated using software Pka solution.
Efavirenz
Each of the plasma samples was brought to room temperature. One milliliter
of diethyl-ether was added and mixed using a vortex mixer for 30 s. The upper
organic phase was transferred and evaporated to dryness on a water bath at 50◦C. The
residue was reconstituted with 0.5ml mobile phase and mixed using the vortex mixer
for 10 s. 10µl of the solution was injected into the HPLC equipped with UV detector
(SPD-10A UV) and C18 column. The flow rate was 1 ml min−1 and the UV detector
was set at a absorption max of 247 nm11.
Atorvastatin calcium
The plasma sample was alkanised with 1 ml of sodium hydroxide, vortexed
for 30s and 1 ml of dichloromethane was added. The mixture is vortexed for 1 min
and centrifuged at 10,000rpm for 10 min. supernatant liquid is separated and was
reconstituted with mobile phase and 10µl of the solution was injected into the HPLC
equipped with UV detector (SPD-10A UV) and C18 column. The flow rate was 1 ml
min−1 and the UV detector was set at a absorption max of 247 nm. .
Rosuvastatin calcium
The drug was extracted from plasma by adding 1.0 ml of diethyl ether, vortex-
mixed for 5min and centrifuged at 3500rpm at 0◦C for 5min. The organic layer was
separated, evaporated to dryness under the gentle stream of nitrogen on a heating
block maintained at 40◦C. After drying, the residue was reconstituted in mobile phase,
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Dept of Pharmaceutics, JSSCP, Mysore 103
vortex-mixed for 2min and. 10µl of the solution was injected into the HPLC equipped
with UV detector (SPD-10A UV) and C18 column. The flow rate was 1.5 ml min−1
and the UV detector was set at a absorption max of 243 nm.
4.13. STABILITY STUDIES128, 139
Drug decomposition or degradation occurs during storage, because of
chemical alteration of the active ingredients or due to product instability, leading to
lower concentration of the drug in the dosage form, hence the stability of
pharmaceutical preparation needs to be evaluated. The objective of stability studies is
to predict the shelf life of a product by accelerating the rate of decomposition,
preferably by increasing the temperature and relative humidity (RH) conditions.
A drug formulation is said to be stable if it fulfills the following requirements:
It contains at least 90% of the stated active ingredient
It contains effective concentration of the added preservatives, if any
It does not exhibit discoloration or precipitation, nor develops foul odour
It does not develop irritation or toxicity
Formulations were packed in a screw capped bottle and studies were carried
out for 12 months by keeping at
25± 2°C and 60 ± 5% RH
30 ± 2 °C and 65 ± 5% RH
and for 6 months for accelerated storage condition at
40 ± 2°C and 75 ± 5% RH
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Dept of Pharmaceutics, JSSCP, Mysore 104
Samples were withdrawn on 0, 3, 6 and 12 months for long term storage
condition and 0, 3 and 6 months for accelerated storage condition and checked for
changes in FTIR, drug content and percentage cumulative drug release.
Results obtained and the conclusions derived from them are provided in the
following chapters.
Recommended