Evaluation of formulated microspheres

1. Micrometric studies7,8
The microspheres were evaluated for micromeritic properties such as bulk density, tapped
density, angle of repose, Carr’s index and Hausner’s ratio.

a. Bulk density
Apparent bulk density (ρb) was determined by pouring the powder blend into a graduated
cylinder. The bulk volume (Vb) and weight of the powder (M) were determined.
ρb = M / Vb
b. Tapped density
The measuring cylinder containing a known mass of blend (M) was tapped for a fixed time
(100 tapping). The minimum volume (Vt) occupied in the cylinder and weight of the blend
was measured. The tapped density (ρt) was calculated using the following formula.
ρt = M / V t

c. Carr’s compressibility index

The simplest way for measurement of free flow of powder is compressibility, an indication
of the ease with which a material can be induced to flow is given by compressibility index.
Carr’s Index = ρb -
ρt / ρb * 100
Where ρt = tapped density ρb = bulk density
d.Hausner’s ratio
Hausners ratio is an indirect index of ease of powder flow. It is calculated by the
following formula:
Hausner’s ratio (H) = ρt/ρb
Where ρt = tapped density
ρb = bulk density
e. Angle of repose
Angle of repose was determined using funnel method .The blend was poured through a
funnel that can be raised vertically until a maximum cone height (h) is obtained. Radius of
the heap (r) was measured and angle of repose (θ) was calculated using the following
formula.
θ = tan-1 h/r

2. Drug excipients compatibility studies

The infrared absorption spectrum of repaglinide, excipients and the formulation were
evaluated using an FTIR spectrophotometer, wherein 2 – 4 mg of drug sample was used.
The resultant spectrum of the drug was compared with the reference spectrum of
repaglinide.
3. Physical characterization
The surface and inner part of the microspheres were observed through the scanning electron
microscopy (SEM). The samples were prepared by lightly sprinkling the microspheres
powder on a double-side adhesive tape which was already shucked on aluminium stubs. The
stubs were then placed into a fine coat ion sputter for gold coating. After gold coating, the
samples were randomly scanned for particle size and surface morphology.
4. Percentage yield
The prepared floating microspheres were weighed after drying for all formulations. Then
percentage yield was calculated using following formula
5. Drug entrapment efficiency9
Microspheres equivalent to repaglinide dose were taken for evaluation. The amount of drug
entrapped was estimated by crushing the microspheres. The powder was transferred to a
100 ml volumetric flask and dissolved in 10ml of methanol and the volume was made up to
100ml with 0.1N HCl. Kept it for sonication about 1 hour. Then solution was filtered
through Whatmann filter paper and the absorbance was measured after suitable dilution

spectrophotometrically at respective wavelength. The amount of drug entrapped in the

microspheres was calculated by the following formula
6. in- vitro evaluation of floating ability
Floating microspheres (equivalent to 2 mg) were dispersed in 100ml of 0.1 N Hydrochloric
acid solution (pH 1.2) to simulate gastric fluid at 37°C. The mixture was stirred with a paddle
at 50 rpm and after 12 hr, the layer of buoyant microspheres (Wf) was pipetted and separated
by filtration simultaneously sinking microsphere (Ws) was also separated. Both microspheres
type were dried at 40°C overnight. Each weight was measured and buoyancy was determined
by the weight ratio of the floating microspheres to the sum of floating and sinking
microsphere.
Buoyancy (%): Wf / (Wf+Ws) × 100
Where Wf and Ws = the weights of the floating and settled microspheres respectively. All
the determinations were made in triplicate.
7. In- vitro wash-off test for microspheres
The mucoadhesive property of the microspheres is evaluated on goat intestinal mucosa by
using phosphate buffer, as per monograph. Weighed microspheres are spread onto wet
rinsed tissue specimen and immediately thereafter the slides are hung onto the arm of a
USP tablet disintegrating test machine with suitable support at 37 oC. The weight of
microspheres leached out at different intervals is measured. The % mucoadhesion is
calculated by the following equation,

Where,
Wa is the weight of microspheres applied
W1 is the weight of microspheres leached out
7. In- vitro drug release studies
The dissolution study of floating microspheres were performed over a 12 hr period using
USP type I (Basket) Dissolution Testing Apparatus (Lab India) 900ml of 0.1N HCl was
used as dissolution medium agitated at 100 RPM, at temperature of 37 o± 0.5oC. Sink
conditions were maintained during the study. 5 ml samples were withdrawn at 1 hour time
interval, pass through 5 µm membrane filter and the samples were analyzed by UV
Spectrophotometry at 237 nm.
8. Release kinetics studies
Release data of optimized formulation were fitted to different mathematical models to
reveal the release mechanism from the microspheres: Zero order (% cumulative drug
release vs. time), first order (log % drug release vs. time), Higuchi model (% cumulative
drug release vs. square root of time) and Peppas exponential equation (log % drug release
vs. log time). All curve fitting, simulation and plotting were performed using
commercially available Microsoft excel solver and regression coefficient (r 2) values were
calculated.