Physical stability
All pertinent procedures to establish the physical stability of the
formulations were carried out following the specifications of National
Drug Formulary
(19)
and the Guide to Good Practice of Preparation of Medications in Hospital
Pharmacy Services
(17).
pH, uniformity, extensibility, absence of crystals and absence of phase
separations were evaluated on a transparent surface according to 3
levels: level 1, the least favorable and level 3, the most favorable, at
t=0 days for each formulation.
Only for liposomes was determined mean particle size, zeta potential and
encapsulation efficiency (EE%) by triplicate at t=0 days. Mean particle
size and zeta potential was determined using particle size analyzer
which uses laser diffraction method (Malvern Instruments,
Worcestershire, UK). The EE% were determined via ultra-filtration,
using Amicon® ultra-centrifugal filters (Merck Millipore, Ireland) with
a molecular weight cut off of 10,000 Daltons. An aliquot of 500 μL of
the liposomal formulation was added to the sample reservoir and
centrifuged for 15 min at 14,000 g. Then 50 μL was aliquoted by
duplicate and after 1/20 dilution with methanol was analyzed with HPLC
method to determine the concentration of free drug in the filtrate. The
following equations were used for the
calculations(13,20):
Where Wt and Wf represent the total amount of the drug and the free
amount of the drug, respectively.