Chapter 7 Experimental work

EXPERIMENTAL METHOD21

PRE-FORMULATIONSTUDY
The received sample of Famotidine was subjected to pre-formulation study.

1. Organoleptic Properties
The drug sample was evaluated for its colour, odour.

2. Melting Point Determination

Melting point of drug sample was determined by using microprocessor based-
programmable melting point apparatus (veego).

3. UV Spectroscopy21,22
Reagents and chemicals
Pure drug sample of FAM was supplied from Swapnroop Agency, Sambhaji Nagar
India. Sample was used without further purification. All chemicals and reagents were
of analytical grade, and double-distilled water was used throughout the investigation to
develop spectral characteristics.

Selection of solvent
0.1N HCl, pH 1.20 was selected as a solvent to study the spectral characteristics of
FAM. It was prepared according to the Indian Pharmacopoeia .

Preparation of standard stock solution

Accurately weighed 10 mg pure drug sample of FAM was transferred to 100 ml (100
µg/ml) calibrated volumetric flask, dissolved, and made up to the mark with 0.1 N HCl,
pH 1.20. It was the stock solution of FAM (100 µg/ml) in 0.1 N HCl, pH 1.20.
Using the stock solution of 100 µg/ml, subsequently dilution was carried out by
withdrawing different aliquots (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml) from standard solution
were transferred into a series of 10 ml calibrated volumetric flasks and all were made
up to the mark with 0.1 N HCl, pH 1.20 to prepare working standard solutions of
different concentrations (5-30 µg/ml)23.
Selection of detection wavelength 24

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 Chapter 7 Experimental work

Solution of FAM in concentration of 10 µg/ml was scanned in the range of wavelength

200-400 nm . It was observed that the FAM showed considerable absorbance at
wavelength of 266 nm. The absorption spectrum was found to be sharp and maximum
at wavelength of 266 nm, therefore, it was selected as the wavelength for detection in
0.1 N HCl, pH 1.20
Preparation of calibration curve
Solution of FAM in 0.1 N HCl, pH 1.20 in different concentrations (5, 10, 15, 20, 25,
and 30 µg/ml) absorbance of these solutions was measured against solvent 0.1 N HCl,
pH 1.20 as blank at wavelength of 266 nm. A calibration curve was plotted from the
absorbance values so obtained . From the calibration curve, it was found that FAM
obeys Beer’s law in concentrations of 5-30 µg/ml25,26.

Figure 10: Calibration Curve of Famotidine

Preparation of sample solution

Ten tablets of FAM (Famocid 20) were weighed accurately and powdered finely. An
accurately weighed quantity of tablets powder equivalent to 100 mg of FAM was
transferred to a 100 ml volumetric flask and diluted with 0.1 N HCl, pH 1.20, and the
ontent was ultrasonicated for 20 minutes. The volume was made up to the mark with
solvent and mixed well. The solutions were further filtered using Whatman No. 1 filter
paper to remove any unwanted particulate matters. The filtered solutions were further
appropriately diluted with respective solvent to finally produce sample solution of

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 Chapter 7 Experimental work

concentration 10 µg/ml for analysis. The amount of FAM present in the sample
solution was determined using the calibration curve of standard drug27,29.
Determinationofλmax

A stock solution of drug (1000 μg/ml) was prepared by taking accurately

weighed 100 mg of model drug and dissolved in sufficient quantity of Glacial
Acetic Acid in 100 ml volumetri

c flask and volumewasmadeupto100mlmarkwithGlacial Acetic

Acid.Further1mlofabovesolutionwasdiluted to100mlwith Glacial Acetic
Acidtoget10μg/ml.UVspectrumwastakenusingShimadzu(UV-1800)
spectrophotometer. The solution was scanned in the range of 263 nm30.

PreparationofMicroballoons35

Preparation of Microballoons by solvent evaporation method

Table 3: Formula for Preparation of Microballoons.

F
Batch F1 F2 F3 F4 F5 F6 F7 F8
9

Drug(mg) 40 40 40 40 40 40 40 40 40

Ethyl Cellulose(mg) 10 15 20 - - - - - -

HPMC(mg) - - - 10 15 20 - - -

Magnesium
- - - - - - 10 15 20
Trisilicate(mg)

Polyvinyl alcohol(mg) 2 2 1 3 2 1 2 2 1

Dichloromethane(ml) 3 2 1 3 2 1 3 2 1

Distilled water(ml) 90 90 90 90 90 90 90 90 90

Microballoons can be prepared by using different proportions of ethyl cellulose and

HPMC with
Famotidine.Thedispersedphasecontainingethylcelluloseanddrugwaskeptintheultra

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 Chapter 7 Experimental work

probesonicator for 5 min.The drug is dissolved in Ethanol and then dissolved in

polymer and resulting solution in added to aqueous phase containing 0.2% sodium
of PVP as emulsifier, this mixture was stirred at 500 rpm then the drug and polymer
(Ethyl Cellulose) was transformed in to fine droplet which solidified in to rigid
microballoons by solvent evaporation and then collected by filtration and washed
with demineralized water and desiccated at room temperature for 24 hrs39.

Evaluation of Microballoons42,45

Micromeritics

Micromeritics are characterized for their micromeritics properties such as particle size,
angle of repose, compressibility index, and Hausner’s ratio. Prior to filling
microballoons into capsules, the micromeritic properties of the microspheres must be
considered in order to analyze their flow properties.

Particle Size

The particle size of the microballoons is measure using an optical, microscopic method,
and then mean microballoons size is calculated by measuring 100 particles with the
help of a calibrated ocular micrometer. Particle size is influenced by process parameters
and formulation parameters such as solvent composition, amount of polymer,
emulsifier concentration, temperature and stirring rate.

Bulk Density

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 Chapter 7 Experimental work

10g of microballoons is to be placed in to 25 ml graduated measuring cylinder. The

volume occupied by the microballoons is observed without disturbing the cylinder, and
the bulk density is calculated using the equation,

Bulk density = weight of sample/volume of sample

Tapped Density

About 10 g of microballoons is placed in 25 ml measuring cylinder. The cylinder is

dropped at 2 s intervals on to a hard-wooden surface 100 times, from a height of one
inch. The final volume is recorded, and the tapped density is calculated by the
following equation (the value expressed in gm/cm3),

Tapped density = weight of sample/tapped volume

Carr’s Index

It is frequently used as an indication of the flowability of a powder. Flow property of

blend depends on compressibility index. The Carr’s index is an indication of the
compressibility of a powder. The propensity to shape bridges between particles is
indicated by a high Carr's index. Smaller the Carr’s index better will be the flow
properties. It is calculated by the formula,

Carr’s index (%) = tapped density-bulk density/tapped density X 100

Angle of Repose (Ɵ)

The angle of repose shows the substance's flowability. A funnel is fixed to a burette
stand in such a way that the stem of the funnel lies 2.5 cm above the horizontal surface.
The sample is allowed to flow from the funnel, until the height of the pile just touches
the tip of the funnel. The radius of the pile is determined by drawing a boundary along
the circumference of the pile and taking the average of the radius of the circumference
from three trails. The angle of repose can be calculated by,

Angle of repose (ɵ) = tan-1h/r

Scanning Electronic Microscopy (SEM)48

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 Chapter 7 Experimental work

SEM techniques are used for determining the surface morphology of the microballoons.
The SEM sample is prepared by sprinkling the powder on the tape stuck attached to an
aluminum stub. The stub is coated using the mixture of gold and palladium at a
thickness of 250-450 Ả under an argon atmosphere in a high vacuum evaporator at a
voltage of 20 KV, Current 10 mA, and low pressure. Photomicrographs are taken on
the random screening coated samples using SEM.

Percentage Yield48,49

Percentage yield of floating microballoons is calculated by dividing the actual weight

of the product to the total amount of all non-volatile components that are used in the
preparation of floating microballoons and is represented by formula,

Percentage yield = Actual weight of product / Total weight of drug excipients

In vitro buoyancy50

Microspheres (300mg) were spread over the surface of a USP dissolution apparatus
type II filled with 900 mL of 0.1 N hydrochloric acid containing 0.02% tween 80. The
medium was agitated with a paddle rotating at 100 rpm for 12 h. The floating and the
settled portions of microspheres were recovered separately. The microspheres were
dried and weighed. Buoyancy percentage was calculated as the ratio of the mass of the
microspheres that remained floating and the total mass of the microspheres

In vitro drug release studies: The drug release was studied using a USP 24 dissolution
apparatus type I (at 100 rpm in 0.1N Hcl as dissolution medium (900 mL) maintained
at 37±1°C. A sample (10 mL) of the solution was withdrawn from the dissolution
apparatus hourly and the samples were replaced with fresh dissolution medium. The
samples were filtered through a 0.45 μ membrane filter and diluted to a suitable
concentration with 0.1 N Hcl. Absorbance of these solutions was measured at 292 nm
using a UV/visible spectrophotometer. Cumulative percentage drug release was
calculated using an equation obtained from a standard curve.

Drug entrapment efficiency52,53

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 Chapter 7 Experimental work

Estimation of drug entrapment efficiency (DEE) in floating microballoons was

carriedout by dissolving the weight amount (100 mg) of crushed microballoons in
required quantity of 0.1N HCl, pH 1.20 and analyzed spectrophotometrically at a
wavelength of 266 nm using the calibration curve. The polymer debris was removed by
filtering through Whatman filter paper (No. 40). Each batch should be examined in a
tripletmanner. The entrapment efficiency of floating microballoons was calculated by
dividing the actual drug content by the theoretical drug content of microballoons . The
% DEE of floating microballoons was calculated using this following formula:

In vitro buoyancy study

This study was carried out by USP type II dissolution test apparatus by spreading the
microballoons on a simulated gastric fluid (900 ml of 0.1 N HCl, pH 1.20) containing
0.02% w/v Tween 20 as a surfactant. The media is stirred at 100 rpm at 37±0.5оС for
12 h. After a specific interval of time, both the fractions of microballoons (floating and
settled microballoons) were collected and buoyancy of the floating microballoons
wasdetermined by using formula54,56.

Where Qf is the weight of floating microballoons and Qs is the weight of settled

microballoons respectively.

In vitro dissolution study57,59

The in vitro dissolution studies were carriedout using USP type II dissolution apparatus
(Paddle Type). The study was carried out in 900 ml of 0.1N HCl (pH 1.20).
Thedissolution medium was kept in a thermostatically controlled water bath,
maintained at37±0.5°C. Microballoons containing drug equivalent to 40 mg were
spread over the surface of 900 ml of dissolution media (0.1N HCl, pH 1.20). The
paddle was rotated at 50 rpm. At predetermined time intervals i.e. 1, 2, 4, 6, 8, 10, 12
and 15 h 5 ml of sample was withdrawn from the dissolution apparatus and replaced

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 Chapter 7 Experimental work

with fresh media tomaintain sink conditions. The drug concentration was analyzed
byusing UVspectrophotometer at 266 nm

USPDissolution apparatus :Type 1I(Basket Type).

Volumeofdissolution medium : 900ml.

Speedofbasketrotation :50 rpm.

Temperature :37ºC ± 0.5ºC.

Dissolution medium :0.1N HCL pH6.8.

Testtime :12 minutes.

Samplinginterval : 1,2,4,6,8,10,12 minutes

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