5. PREFORMULATION STUDY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9500/15/15_chapter 5.pdf · Department of Pharmaceutics, SPS, SOA University 85 Page 5. PREFORMULATION

Department of Pharmaceutics, SPS, SOA University 85 Page

5. PREFORMULATION STUDY

Preformulation studies are the first step in the rational development of dosage form of

a drug substance. The work was conducted by using Losartan Potassium as a model

drug. The pure was subjected to Preformulation study. The drug was studied for its

organoleptic properties, solubility, compatibility with other excipients. A through

investigation of physicochemical properties may ultimately provide a rationale for

formulation design or support the need for molecular modification or merely confirm

that there are no significant barriers to compounds development. The goals of the

study therefore are:

� To establish the necessary physicochemical characteristics of a new drug

substance.

� To establish its compatibility with other excipients

� To establish its kinetic release profile

5.1 Analytical Method Development for Losartan Potassium

5.1.1 Preparation of Phosphate Buffer pH 6.5 solution (IP-2007)

According to specification described in Indian pharmacopoeia an accurately weighed

quantity of 2.38 gm of di-sodium hydrogen phosphate, 0.19 gm of potassium

dihydrogen phosphate and 8.0 gm of Sodium chloride was added to sufficient double

distilled purified water and it was made up to a 1000 ml clear solution. The pH of the

buffer was analyzed by digital pH meter (Hanna instruments pHep®, Model

No.PHEP).

5.1.2 Preparation of stock solution


An accurately weighed quantity of 25mg of Losartan Potassium was taken in a clean,

dry 250ml of volumetric flask. Then volume was made up to 250ml with phosphate

buffer pH 6.5 and shaken vigorously to yield a clear solution of 100 mcg/ml

concentration.

5.1.3 Determination of λmax and calibration curve:

A particular concentration from the stock solution was scanned from 200-400nm

wavelength range in UV-Visible Spectrophotometer [Jasco V 630 Double Beam UV-

Visible Spectrophotometer]. From the scanning report it was evident that the

wavelength of maximum absorbance (λmax) of Losartan Potassium was found at

248.7nm. The appearance of a peak at 204.6nm was considered as the solvent peak

so can be eliminated as the 1st peak and hence the λmax for Losartan Potassium was

found to be at 248.7nm.


Fig.6. UV Scan Report of LP in PBS 7.4

From the stock solution (100 mcg/ml) different amount of solution was withdrawn and

transfer to the volumetric flask and volume was made up to 10ml with phosphate

buffer pH 6.5. This gives a concentration ranging from 4 mcg/ml-16 mcg/ml. The

absorbances of 9 different concentrations were obtained at λmax. The different

concentrations and corresponding absorbance were given on in Table 3. Absorbance

data (Y) were plotted against concentration(X) and Regression analysis was done.


The linear equation found in the form of Y = mX + c, where m=slope, c = intercept.

The corresponding standard curve generated by linear Regression analysis along

with the mathematical representing the curve is given Fig. 7.

Concentration (mcg/ml) Absorbance λmax

4 0.1290 248.7

5 0.1603 248.7

6 0.1895 248.7

7 0.2244 248.7

8 0.2630 248.7

10 0.3132 248.7

12 0.3808 248.7

14 0.4461 248.7

16 0.5166 248.7

Table 3: Calibration Plot of LP in PBS 7.4


CALIBRATION CURVE OF LP IN PBS 7.4

y = 0.032x - 7E-05

R2 = 0.9994

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20Concentration (mcg/ml)

absorbance

Fig.7. Calibration Curve of LP in PBS 7.4

5.1.4 Preparation of 0.1M HCl

According to specification described in Indian pharmacopoeia an accurately

measured quantity of 8.4 ml of concentrated hydrochloric acid was added to sufficient

double distilled purified water and it was made up to a 1000 ml clear solution. The pH

of the buffer was analyzed by digital pH meter (Hanna instruments pHep®, Model

No.PHEP).

5.1.5 Preparation of stock solution

An accurately weighed quantity of 25mg of Losartan Potassium was taken in a clean,

dry 250ml of volumetric flask. Then volume was made up to 250ml with 0.1M HCl

and shaken vigorously to yield a clear solution of 100mcg/ml concentration.

5.1.6 Determination of λmax and calibration curve:


A particular concentration from the stock solution was scanned from 200-400nm

wavelength range in UV-Visible Spectrophotometer [Jasco V 630 Double Beam UV-

Visible Spectrophotometer]. From the scanning report it was evident that the

wavelength of maximum absorbance (λmax) of Losartan Potassium was found at

248.8nm. The appearance of a peak at 204.6nm was considered as the solvent peak

so can be eliminated as the 1st peak and hence the λmax for Losartan Potassium was

found to be at 248.7nm.

From the stock solution (100mcg/ml) different amount of solution was withdrawn and

transfer to the volumetric flask and volume was made up to 10ml with 0.1 M HCl.

This gives a concentration ranging from 6mcg/ml-20mcg/ml. The absorbances of 9

different concentrations were obtained at λmax. The different concentrations and

corresponding absorbance were given on in Table 4. Absorbance data (Y) were

plotted against concentration(X) and Regression analysis was done. The linear

equation found in the form of Y = mX + c, where m=slope, c = intercept. The

corresponding standard curve generated by linear Regression analysis along with the

mathematical representing the curve is given Fig.8


Fig.8 Scan report of LP in pH 1.2

From the stock solution (100mcg/ml) different amount of solution was withdrawn and

transfer to the volumetric flask and volume was made up to 10ml with 0.1M HCl pH

1.2. This gives a concentration ranging from 6mcg/ml-20mcg/ml. The absorbances of

9 different concentrations were obtained at λmax. The different concentrations and

corresponding absorbance were given on in Table 4. Absorbance data (Y) were

plotted against concentration(X) and Regression analysis was done. The linear

equation found in the form of Y = mX + c, where m=slope, c = intercept. The


corresponding standard curve generated by linear Regression analysis along with the

mathematical representing the curve is given Fig.9.

Concentration

(mcg/ml)

Absorbance λ max

6 0.1822 248.7

8 0.2422 248.7

10 0.3111 248.7

12 0.3622 248.7

14 0.4238 248.7

16 0.4752 248.7

20 0.6124 248.7

Table 4: Calibration plot of LP in ph 1.2

CALIBRATION CURVE OF LP IN 0.1M HCl

y = 0.0303x + 0.0005

R2 = 0.9992

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20 25Concentration(mcg/ml)

Absorbance

Fig.9. Calibration Curve of LP in pH 1.2

5.2 Preparation of Losartan Potassium loaded Microspheres


Various encapsulation techniques are readily available for microencapsulation of

drugs and one of the most commonly employed is the solvent evaporation method.

The method can be performed via various protocols and the selection for best option

readily depends on the property of the compounds that are intended to be

encapsulated. Particularly for water-soluble compounds, the most often employed

method is the double emulsion type, in which aqueous phase containing the

dissolved compounds is entrapped into water insoluble matrices. The state of art falls

on the unique inter-phase formation between immiscible aqueous and organic layers.

Emulsion occurs when aqueous solution containing the dissolved compounds is

suspended within organic solvent containing dissolved polymer. This emulsion

mixture is then dispersed into a secondary aqueous solution forming secondary

emulsion; commonly known as water–oil–water (W1/O/W2) double emulsion. Under

such process, the first aqueous solution will be entrapped in the core of the polymer

while physically the polymer is shaped into small fine spheres due to the secondary

dispersion. In oil-in-water (O/W) emulsion techniques, drug is dispersed with the

polymer in an organic phase, which is again dispersed into an outer aqueous phase.

Upon contact, the organic solvent diffuses into the external water phase and

evaporates at its surface. Consequently the polymer precipitates and entraps the

drug.


5.3 Design and Formulation of Losartan Potassium loaded Microspheres by

Double Emulsion Solvent Evaporation Technique (W1/O/W2 Method)

For the formulation of Losartan potassium loaded microspheres by W1/O/W2 method,

1 ml double distilled purified water containing 100 mg Losartan potassium was

emulsified under vigorous ultra-sonication (350 watt, 2 min) into 10 ml methylene

chloride containing 4:1 blend of different amounts of Eudragit RS:RL (400mg, 500mg,

600mg). This O/W emulsion was then added drop wise into 500 ml aqueous PVA

solution (0.25% w/w) to yield the secondary emulsion (W1/O/W2). Then the

secondary emulsion was stirred continuous with a mechanical stirrer with a blade

fitted with a four-blade “butterfly” propeller with a diameter of 50 mm (Lab Digital

Stirrer, Remi) for 2 hours at 750 rpm in room temperature to allow microsphere

hardening.

The microspheres were separated by vacuum filtration using a Wattman filter paper

and simultaneously washed three times with double distilled purified water and dried

in a desiccator at room temperature for 48 h. Repeated (triplicate) batches were

prepared to obtain reproducible results. It was observed that all the formulations

produced a reproducible yield (Pe´reza et al., 2000). The different batch

specifications of LP loaded microspheres are given in Table 5.


Fig. 10: Schematic Representation of Preparation of Losartan potassium

loaded microspheres by W1/O/W2 method

Amount of Polymer

taken in ratio

4:1(mg)

Method of

Preparation

Formulation

Code

D : P

Amount of

Drug taken

(mg)

ERS ERL

Amount of

Methylene

Chloride

(ml)

F 1 1 : 4 100 320 80 10

F 2 1 : 5 100 400 100 10

W1/O/W2

METHOD

F 3 1 : 6 100 480 120 10

Table 5: Batch specification of Losartan potassium loaded microspheres by

W1/O/W2 method


5.4 Design and Formulation of Losartan Potassium loaded Microspheres by

Emulsion Solvent Evaporation Technique (O/W Method)

For the formulation of Losartan potassium loaded microspheres by O/W method, 100

mg Losartan potassium was dispersed under vigorous ultra-sonication (350 watt, 2

min) into 10 ml methylene chloride containing 4:1 blend of different amounts of

Eudragit RS:RL (400mg, 500mg, 600mg). This resulting dispersion was then added

drop wise into 250 ml aqueous PVA solution (0.25% w/w) to yield the emulsion

(O/W). Then the primary emulsion was stirred continuous with a mechanical stirrer

with a blade fitted with a four-blade “butterfly” propeller with a diameter of 50 mm

(Lab Digital Stirrer, Remi) for 2 hours at 750 rpm in room temperature to allow

microsphere hardening.

The microspheres were separated by vacuum filtration using a Wattman filter paper

and simultaneously washed three times with double distilled purified water and dried

in a desiccator at room temperature for 48 h. Repeated (triplicate) batches were

prepared to obtain reproducible results. It was observed that all the formulations

produced a reproducible yield (Pe´reza et al., 2000). The different batch

specifications of LP loaded microspheres are given in Table 6.


Fig. 11: Schematic Representation of Preparation of Losartan potassium

loaded microspheres by O/W method

Amount of Polymer

taken in ratio

4:1(mg)

Method of

Preparation

Formulation

Code

D : P

Amount of

Drug taken

(mg)

ERS ERL

Amount of

Methylene

Chloride

(ml)

F 4 1 : 4 100 320 80 10

F 5 1 : 5 100 400 100 10

O/W

METHOD F 6 1 : 6 100 480 120 10

Table 6: Batch specification of Losartan potassium loaded microspheres by

O/W method


5.5 Process Optimization during Production of Batches of Microspheres

a) Polymer concentration and Drug concentration

When 1:1 (w/w) polymer ratios were used for both the Eudragit RS and RL polymers,

the quality of microspheres formed was poor. These were irregularly shaped, not

flowing, and presented with lots of indentation. Microspheres were only formed when

the polymer ratio was increased to ratios of between 4:1. Discrete, spherical, and

uniform microspheres were obtained within a ratio of 1:4 to 1:6 (w/w) drug/polymer

ratios for both the manufacturing techniques as can be seen in SEM analysis. Drug

concentration was optimized according to the dose of the drug.

b) Choice and Amount of Organic Solvent

The particle size and the drug loading efficiency of the microspheres appeared to

show dependency on the type of organic solvent used. As a reference examination,

the value of an interfacial tension between the solvent used and water phase was

cited in literatures. The values of interfacial tension of Dichloromethane (DCM) (20.4)

and Chloroform (CR) (31.4) were relatively higher than those of Acetone (AC) (0) and

Ethyl Acetate (EA) (6.78). Apparently, the particle size of the microspheres increased

with the interfacial tension in the water phase. The solvent with a low interfacial

tension with water phase produced smaller-sized droplets during the preparation of

the emulsion. Amount of organic solvent depends upon viscosity of polymeric

solution. The polymeric solution should be less viscous and a clear solution

completely solubilizing the polymers.


c) Speed of stirrer

During processing, it was found that when the stirring speed was kept at 500 rpm, the

shapes of particles were found to be irregular for all formulations because the stirring

speed was not fast enough to disperse the inner phase in outer phase and a huge

coalesced mass was obtained. This is due in part to inadequate agitation of the

media to disperse the inner phase in discrete droplets within the bulk phase. At

stirring speeds above 1000 rpm, the turbulence caused frothing and adhesion of the

microspheres to the container walls and propeller blade surfaces, resulting in high

shear and a smaller size of the dispersed droplets. When stirring speed was raised to

750 rpm the best spherical particles with good surface characteristics were obtained

with all formulations which is exhibited in the scanning electron micrograph analysis.

5.6 Characterization Process of Losartan potassium Loaded Microspheres

a) % Yield Value of Microspheres:

The prepared microspheres were assessed for the yield value. The batch was

weighed after total drying and the yield % was calculated using the formula give

below. Each batch was formulated in triplicate batches (n=3) to get a reproducible

yield (Lim et al., 2000).

Results are given in Table 7

b) Flow properties of prepared microspheres:


Flow properties of prepared microspheres were determined by bulk density, tapped

density, Carr’s index and Hausner Ratio or Packing factor (Jain et al.,2005).

i) Determination of Bulk Density and Tapped Density:

Accurately weighed quantities of prepared microspheres were carefully poured into

the graduated cylinder (10ml). The initial volume was measured. The graduated

cylinder was tapped for 100 times. After that the volume was measured.

OV

W Density Bulk =

FW

WDensity Tapped =

Where W = weight of the formulation

VO = Bulk Volume

WF = Tapped Volume

Bulk and Tapped density expressed in gm/ml.

The results were given in Table 8

ii) Carr’s Index or Compressibility Index:

100×=density Tapped

density bulk -density Tappedindex sCarr'

Grading of the powders for their Flow properties according to the Carr’s index.

CARR’S INDEX (%) FLOW

5–15 Excellent


12–16 Good

18–21 Fair to passable

23–25 Poor

33–38 Very poor

>40 Very very poor

The results were given in Table 8.

iii) Hausner Ratio:

It indicates the flow properties of the microspheres and measured by the ratio of

tapped density to bulk density.

density Bulk

density Tapped Ratio Hausner =

Hausner Ratio Properties

0–1.2 Free flowing

1.2–1.6 Cohesive powder

The results were given in Table 9.

c) Drug Loading and Encapsulation efficiency

Microspheres were thoroughly triturated in a mortar with a pestle. An equivalent

accurately weighed amount of 50 mg of powdered microspheres was extracted with

50 ml of 0.1 M HCl by extensive stirring for 24 h at 150 rpm. The solution was then

filtered through a 0.45 micron membrane filter (Millipore Millex-HV); a sample of 1mL

was withdrawn from this solution, diluted to 10 mL with 0.1 M HCl. The resulting


acidic solution containing the extracted drug was clarified by centrifugation at 2000 g

for 20 min and assayed using UV spectroscopy at 248.7 nm to find out the actual

drug content of microspheres. The measured absorbance was converted to drug

concentration using a standard curve for the known concentration of the drug in 0.1M

HCl. The experiment was carried out in triplicate for each sample.

The % drug loading (Yang et al., 2003) was calculated using the following equation:

Study was performed in triplicate (n=3) to get reproducible results.

Results are given in Table 10.

Encapsulation efficiency (Haznedar et al., 2003) was calculated using the following

equation: Study was performed in triplicate (n=3) to get reproducible results.

Results are given in Table 10.

d) Particle Size Distribution Study

The size distribution study was carried out using an optical microscope, and the

mean particle size was calculated by measuring 300 particles with the help of a

calibrated ocular micrometer. Calibration of the optical microscope was carried out

using following procedure (Martin et al., 1993):

2 divisions of stage micrometer coincided with 3 division of eye-piece micrometer


Each division of stage = 0.01 mm

1 division of eye-piece will correspond to = 2/3 X 0.01 X 1000 = 6.66 µm

The microspheres are mounted on a slide and placed on a mechanical stage. The

microscope eye-piece is fitted with a micrometer by which the size can be

determined. The field is projected on to the screen and the particles are measured

along an arbitrary chosen fixed line horizontally across the center of the particles. A

size-frequency distribution curve is plotted as shown in Fig 12.


e) Scanning electron microscope analysis

The external and internal morphology of the microspheres were studied by scanning

electron microscopy (SEM). The samples for SEM were prepared by lightly sprinkling

the powder on a double adhesive tape stuck to an aluminum stub. The stubs were

then coated with gold to a thickness of about 300 Å under an argon atmosphere

using a gold sputter module in a high-vacuum evaporator. The coated samples were

then randomly scanned and photomicrographs were taken with a scanning electron

microscope (Jeol JSM-1600, Tokyo, Japan). SEM images are shown in Fig. 13-18

(Kılıcarslan et al., 2003)

f) Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR spectra were taken on JASCO FT-IR (Model 4100, Japan) to confirm cross

linking and to investigate chemical interactions between drug and polymer matrix. 2%

of samples were crushed with KBr to get transparent pellets by applying a pressure

of 6 Ton. FT-IR spectra of pure LP (Losartan potassium), pure polymers (ERS 100 &

ERL 100) and Losartan potassium loaded microspheres prepared by both the

techniques were scanned in the range between 500 and 4000 cm-1. FT-IR spectra

are shown in Fig. 19-27 (Chopra et al., 2007)


g) Proton NMR Spectroscopy

Approximately 200 mg of the sample was introduced into the probe rotor. Solid state

1H NMR spectra were recorded on a Bruker MSL-100 NMR spectrometer operating

at 25.18 MHz with a magnetic field strength of 2.35 T. A ninety degree pulse 1H

decoupling field of 15 gauss with a pulse duration of 4 ms and a spinning frequency

rate of 4.2 kHz was used to record the cross-polarization magic angle spinning

(CP:MAS) spectra. More than 2400 scans were acquired for each resulting spectrum.

All observations were made at temperatures between 20 and 22°C (Omari et al.,

2004). 1H NMR spectra are shown in Fig. 28-32.

h) Thermal Analysis of Drug crystallinity (Differential Scanning Calorimetry)

Differential scanning calorimetry (DSC) thermograms were obtained by a Mettler

Toledo DSC 822e Stare 202 System (Mettler Toledo, Switzerland) equipped with a

thermal analysis automatic program. Aliquots of about 5mg of each sample were

placed in an aluminium pan of 40µl capacity and 0.1mm thickness, press-sealed with

a perforated aluminium cover of 0.1mm thickness. An empty pan sealed in the same

way was used as reference. DSC curves of pure API (LP), pure polymers (ERS &

ERL) and LP loaded microspheres prepared by both the techniques were recorded.

Conventional DSC measurements were performed by heating the sample from 20 to

300 OC at the rate of 5 OC/min under a nitrogen flow of 50 cm3/min. The starting

temperature was 20 OC. Indium (99.99% purity) was used as a standard for

calibrating the temperature (Yfiksel et al., 1996). DSC Thermograms are shown in

Fig.33-38.

i) Crystallographic characterization using Powder – XRD

Powder XRD patterns were obtained with a Philips XRD (Model: 1730/10) with CuKα

radiation (0.154 nm) at 35 kV and 20 mA over a 2θ range of 2-60 °. Diffraction

patterns for pure API (LP), pure polymers (ERS & ERL) and LP loaded microspheres


prepared by both the techniques were recorded. The samples were grounded before

analysis (Aso et al., 1993). X-Ray Diffraction spectra are shown in Fig. 39-44.

j) In-Vitro Drug Release Study

i) Release Kinetics

Data obtained from In-vitro release studies were fitted to various kinetics models to

find out the mechanism of drug release from microspheres (Li W et al., 1994).

Kinetics models used for In-vitro drug release from microspheres:

• Zero order release kinetic model.

• First order release kinetic model [1897].

• Hixon-Crowell Model [1931].

• Higuchi Model [T.Higuchi 1963 & W.I. Higuchi 1967].

Zero order Kinetics:

According to this model, under standard condition of temperature and agitation, the

dissolution medium, the dissolution rate model can be described by the equation.

dq/dt = K0

or, Expressing in an integrated form

q = K0t

Where, q = amount of drug released per unit surface area.

K0 = zero order release rate constant

t = time

A plot of q vs. t gives a straight line.

First order Release Kinetics (Noyes Whitney’s Equation):


According to Noyes Whiteny, under standard condition of agitation and temperature,

the dissolution rate process for solids can be described by the equation.

dq/dt = K1(Cs–Ct)

Under Sink condition, i.e. when Ct < 0.15 Cs, the equation becomes,

dq/dt = K1Cs

or in an integrated form

ln q0/q = K1t

Where, q=amount of drug released per unit surface area.

K1=First order release rate constant

q0= initial amount.

Cs= Saturation solubility

Ct = Concentration at time t.

A plot of log % amount remaining to be released VS time gives a straight line with a

negative slope.

Hixon-Crowell Model Kinetics:

As solid dissolved the surface area S changes with time. The Hixon-Crowell cube

root equation for dissolution kinetics is based on the assumption that:

• Dissolution occurs normal to the surface of the soluble particles.

• Agitation is uniform on the overall exposed surface and there is no stagnation.

• The particles of solute retain its geometric shape.

• For a non-dispersed powder with spherical particles a bit mathematical

derivation leads to the Kinetics equation.


tK wW HC1/31/3

0 =−

Where, W0 = Initial weight of the particles.

W = Weight of the particles at t.

KHC = Hixon-Crowell Release Rate constant.

t = time

A plot of 1/31/3

0 wW − Vs time gives a straight line with a negative slope since W

increases with time.

Higuchi Model Kinetics:

For coated or matrix type dosage form, the dissolution medium enters the dosage

form in order for the drug to be released and diffused into the bulk solution.

In such cases, often the dissolution follows the equation proposed by Higuchi.

0.5

ssE t

C)ECAD Q

−= 2(

or, Q = KHG t 0.5

Where, Q= Amount of drug released per unit surface area of the dosage

form

D = Diffusion Co-efficient of the drug.

E = Porosity of the matrix.

T = Tortuosity of the matrix.

Cs = Saturation solubility of the drug in the surrounding liquid.

KHG = Higuchi Release Rate constant.


t = time

Fitness of the data into various kinetics models were assessed by determining the

correlation co-efficient, the rate constants, for respective models were also calculated

from slope.

ii) Experimental Procedure for In vitro drug release study

The release studies of pure drug and prepared microspheres were carried out in

accordance to compendia specification given in USP-XXIV / NF XVIV. For this

purpose USP Apparatus 1 (Basket type) method has been used while the instrument

used is an 8 basket dissolution apparatus (Electrolab Tablet Dissolution Tester,

Mumbai Model NO. TDT-06P, Sr. No. 0807019). The dissolution profiles of pure

drug and the drug release rate from the microspheres were studied at pH 1.2 (0.1 M

HCl) and 7.4 (PBS) under sink conditions. Accurately weighed samples of

microspheres equivalent to 10mg of Losartan potassium were added to 900 ml of

dissolution medium kept at 37 ± 0.5 OC. The rotational speed of the Basket was fixed

at 100 rpm throughout the study. The basket was covered with a nylon screen to

prevent the coming out of particles during the progress of dissolution. Sample

aliquots of 5 ml were withdrawn at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8 upto 12 h. A

volume of dissolution medium equal to what had been removed for analysis was

replaced to maintain sink condition. Samples for analysis were monitored

spectrophotometrically using UV Visible Spectrophotometer (Jasco-V-630) at 248.7

nm. In order to understand the mechanism and kinetics of drug release the results of

in-vitro dissolution studies were fitted to various kinetics equation like zero order, first

order, Higuchi and Hixon-Crowell Model. Correlation co-efficient values were

calculated for linear curves obtained by regression analysis of above plots. The

experiment was carried out in triplicate for each sample (n=3) to get reproducible

results (Pe´reza et al., 2000 & Hazendar et al., 2003).


In vitro dissolution results are shown in Table 13(a) & (b) for PD and Plots are shown

in Fig. 45 (PD), Fig. 46-51 for dissolution of optimized formulation in 0.1M HCl and

Fig. 52-57 for PBS 7.4. Correlation coefficients are shown in Table 14.

5.6 Physical Characterizations of API for tablet preparation

5.6.1 Organoleptic Evaluation

Organoleptic characters like colour, order and taste of drug were observed and

recorded using descriptive terminology.

5.6.2 Solubility Study

The solubility of the Losartan Potassium was evaluated in various media by

quantitatively assessing the amount of drug, which is solubilized in the respective

media.


5.6.3 Bulk Density

Bulk Density and tapped density were measured using 100 ml graduated measuring

cylinder as measure of packability of powders by tapping method. Bulk density and

tapped density were calculated by following formula.

Bulk density = weight of powder/ bulk volume

Tapped density = weight of powder/tapped volume

5.6.4 Carr’s compressibility Index

Compressibility is a parameter used to evaluate the flow property of powder by

comparing the bulk density and tapped density. High density powders tend to posses

free flowing properties. Carr’s index was calculated by using following formula.

Carr’s index = (Tapped density – Bulk density/ Tapped density) X 100

5.6.5 Hausner’ Ratio

Huasner’s ratio provides an indication of the degree of densification which could

result from variation of the feed hopper. A lower value of indicates better flow and

vice versa.

Hausner’ Ratio = Tapped density/ Bulk density

5.6.6 Angle of Repose

Angle of repose is usually determined by Fixed Funnel Method and is the measure of

the flowability of powder/granules. Angle of repose was calculated from radius of the

base using the following formula.

θ = tan -1 (h/r) = tan-1 (Height of pile/ 0.5 base)


(All the results are given in table No. 15 & 16)


M i c r o m e r i t i c s t u d yPreformulation before compaction of tablet

Parameters LP SSG MCC Mag.St PVP MS

Bulk

Density(gm/ml)

0.65 0.53 0.51 0.21 0.375 1.25

Tapped

Density(gm/ml)

0.73 0.78 0.67 0.36 0.43 1.53

Carr’s Index (%) 12.63 0.92 23.31 42.01 14.3 18.85

Hausner’s Ratio 1.12 0.82 1.28 1.74 1.13 1.32

Angle of Repose 24.60 22.34 29.23 36.35 32.24 32.2

5.6.7 Moisture content

Moisture content of LP was carried out in a vacuum oven (Alpha Scientific and Lab.

Equipment,ASLE/OO). In this method first the temperature of the oven was set i.e.

140 0C and the crucible was kept to be dried completely. Then vaccume was applied

at less than 5mm Hg and 1 gm of sample was taken in the crucible and the oven was

closed. After 30 min. dried samples were removed from the oven and kept in

dessicator. Then the weight of the crucible was taken

Loss on Drying (LOD) = W2-W3/W2-W1

Where, W1 = weight of the empty crucible

W2 = weight of the empty crucible along with sample

W3 = weight of the empty crucible along with sample after drying


5.6.8 Loss on Drying

Lp and other excipents 1-2 gm were taken and analyzed in an electronic moisture

balance (Sartorius MA 150) at 105 0C for five min.

Sl. No INGREDIENTS LOD (%W/W) at 1050C

1 LP 2.32

2 SSG 4.32

3 MCC 1.23

4 Mag.St 3.13

5 PVP 2.34

6 MS 0.223


5.7 Formulation and Development of Bi-layer Solid Dosage Form of Losartan

Potassium Containing an Immediate Release Layer and a Slow Release Layer

The bi-layered tablets of losartan potassium were prepared by the direct

compression method. The drug, polymers and other excipients (batch size 50 tablets)

used for both immediate (IR) and controlled release (CR) layers were passed through

sieve # 80 and used as per formulation Table. 6.i

Stage I: The Sustained release layer containing microspheres, diluents, binder and

lubricants were mixed uniformly and passed through sieve # 80, then compressed on

single station tablet machine using 8 mm round and flat punches with hardness

between 4-5 kg/cm.

Stage II: IR layer containing drug, super disintegrating agent, diluents and lubricant

were mixed uniformly and compressed over CR layered tablet with hardness

between 5-7 kg cm-2 to obtain bi-layer tablet.

Ingredients of IR layer (mg) Ingredients of SR layer (mg) For.

code Drug SSG MCC Mag.St MS MCC PVP Talc Mg.St Total

Wt.

B1 10 3 35 0.5 204.3 169.2 20 4 4 450

B2 10 3 35.5 0.5 204.3 168.7 20 4 4 450

B3 10 3 36 0.5 204.3 168.2 20 4 4 450

B4 10 3 36.5 0.5 204.3 167.7 20 4 4 450

B5 10 3 37 0.5 204.3 167.2 20 4 4 450

B6 10 3 37.5 0.5 204.3 166.7 20 4 4 450


Table 6.i: Batch specification of Losartan potassium bi-layer tablet by direct

compression method

5.8 Evaluation of Tablets

a) Weight Variation Test

Twenty tablets were weighed and the average weight was calculated. The individual

weight was compared with the average weight. The tablets pass the test if not more

than two tablets are outside the percentage limit and if no tablet differs by more than

two times the percentage limit. The following percentage deviation in weight variation

is allowed according to USP.

Weight Variation allowed as USPXX-NF XV.

Average weight of tablet Percentage weight variation

130 mg or less 10 %

More than 130 mg and less than 324 mg 7.5 %

324 mg or more 5 %

b) Length, Width and Thickness Test


Ten tablets were picked from formulations randomly and length, width and thickness

were measured indivisually using “Vernier-caliper” (Mitutoyo Digimatic, CD-8 CDX).It

is expressed in millimetre and average was calculated.

c) Hardness Test

Hardness indicates the ability of a tablet to withstand mechanical shocks while

handling. The hardness of the tablet was determined using (Dr. Schleuniger

Hardness Tester, 8M). It was expressed in Kilopound (Kp). Ten tablets were

randomly picked from each formulations and hardness of the tablets were

determined. The average value was also calculated.

d) Friability Test

The friability of tabets was determined using Roche Friabilator (Electrolab,EF-1W). It

is expressed in percentage (%). Ten tablets were initially weighed (W initial) and

transferred into friabilator. The fribilator was operated at 25 rpm for 4 minutes. %

friability of tablets less than 1% are considered acceptable. The tablets were weighed

again (W final). The % friability was calculated by,

% Friability= ( Initial Wt- Final Wt)/Initial Wt X 100

% Friability of tablets less than1% are considered acceptable.

e) Disintegration Time

It was determined by using USP device (Electrolab, ED-2 SAPO) which consists of 6

glass tubes that are 3 inches long. To perform disintegration test, one tablet was

placed in each tube and the basket arch was positioned in a 900 ml beaker of water

at 37C=+-2C.A standard motor driven device was used to move the basket assembly


up and down. To be in compliance with the USP standard, all tablets must

disintegrate and all particles must pass through the 10 mesh in the time specified.

f) Drug excipients compatibility study

API, MS and excipients were thoroughly mixed in predetermined ratio given in table

No.18 and passed through the sieve # 40. The blend was filled in glass vials and

closed with gray rubber stoppers and sealed with aluminium seal and charged in to

stress conditions like at 25 0C/60 % RH, 400C/75 %RH, and control (2-8 0C). The

samples were observed in different time interval for Physical observation and

Chemical observation were carried out by FTIR spectroscopy.

g) Dissolution study

The release studies of pure drug and prepared microspheres were carried out in

accordance to compendia specification given in USP-XXIV / NF XVIV. For this

purpose USP Apparatus 1 (Basket type) method has been used while the instrument

used is an 8 basket dissolution apparatus (Electrolab Tablet Dissolution Tester,

Mumbai Model NO. TDT-06P, Sr. No. 0807019). The dissolution profiles of pure

drug and the drug release rate from the bi-layer tablets were studied at pH 1.2 (0.1 M

HCl) and 7.4 (PBS) under sink conditions as mention in the microspheres. Samples

for analysis were monitored spectrophotometrically using UV Visible

Spectrophotometer (Jasco-V-630) at 248.7 nm. In order to understand the

mechanism and kinetics of drug release the results of in-vitro dissolution studies

were fitted to various kinetics equation like zero order, first order, Higuchi and Hixon-

Crowell Model. Correlation co-efficient values were calculated for linear curves

obtained by regression analysis of above plots. The experiment was carried out in

triplicate for each sample (n=3) to get reproducible results.

h) Stability study


Stability study was carried out by exposing the formulation to different conditions

including stress conditions of temperature and pressure as per ICH guidelines.

Generally stability study was done at initial, 30 0C/65 % RH (for 1,2,3, 6 months).

After the study the samples were checked for physical and chemical properties for

any changes and to be within the limit.

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5. PREFORMULATION STUDY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9500/15/15_chapter 5.pdf · Department of Pharmaceutics, SPS, SOA University 85 Page 5. PREFORMULATION