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.