4 Materials and Methods - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32498/10/10_chapter 4.p… · Rimek, Minipress- 1(model-1674), Karnavati, India Micrometer screw gauge

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



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.



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.



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



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



Figure 4.2: Calibration curve of Efavirenz in 0.1M HCl

Atorvastatin calcium


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.



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.



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



Figure 4.4: Calibration curve of Atorvastatin Calcium in 0.1M HCl

Rosuvastatin Calcium


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.



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



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


Figure 4.6: Calibration curve of Rosuvastatin Calcium in 0.1M HCl



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



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


Figure 4.8: Standard calibration curve of Efavirenz by RP-HPLC



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



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


Figure 4.10: Standard calibration curve of Atorvastatin Calcium by RP-HPLC



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



Table 4.6: Standard calibration data of Rosuvastatin Calcium by RP-HPLC


(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


Figure 4.12: Standard calibration curve of Rosuvastatin Calcium by RP-HPLC



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


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


(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




Figure 4.13: Standard calibration curve of Efavirenz in rat plasma


The HPLC bio analytical method used for AC analysis was rapid and simple.

Protein precipitation method was used (Acetonitrile) for extraction, the clear



concentration range of 1 - 30 µg/ml (r2 = 0.999; n = 6). The results show the linearity





concentrations of AC ranged from 98.17-101.23% and 96.05-103.27% for intra and

inter day respectively.



Table 4.8: Standard calibration data of Atorvastatin Calcium in rat plasma


(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


Figure 4.14: Standard calibration curve of Atorvastatin Calcium in rat plasma



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



concentration range of 1 - 50 µg/ml (r2 = 0.998; n = 6). The results show the linearity




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




Figure 4.15: Standard calibration curve of Rosuvastatin calcium in rat plasma



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.



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



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.



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.



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.



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.



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.



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 release from the prepared SNEDDS was performed using

dissolution apparatus II containing 900 mL of 0.1 M HCl as dissolution medium



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 release from the prepared SNEDDS was performed

using dissolution apparatus II containing 900 mL of 0.1 M HCl as dissolution medium





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.



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



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.



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.



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.



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


Talc 5

Lactose q.s 500



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


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.



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.



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



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



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



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.


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. .


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,



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



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.

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