Phospholipid is the main component of the liposome formulation and these liposomes can be prepared by different methods such as the thin film method, ethanol based liposomes and particulate based proliposomes. Proliposomes can be converted into liposomes by using simple hydration method. The formation of liposomes depends on variety of factors but one of them is the phospholipid itself. Phospholipid can be quantified by using Stewart method. In this method a samples of liposome were prepared and placed in a glass tube or beaker and then organic solvent was added into the glass vial to make the preparation as alcoholic liposome. These vials are needed to dry up to get the dry formulation from the liquid dispersion medium using oven. The solvent was evaporated completely because phospholipid can be quantified due to their property of developing colour and the dry formulation was therefore reacted with a mixture of ferric chloride and ammonium thiocyanate. This mixture was prepared in deionised water. These beakers or vials in which the preparation was prepared were properly covered for any leakage and the whole mixtures were shaken manually or can be shaken vigorously by using vortex mixer to have the proper distribution of the formulation. This mixture of dried formulation with ferric chloride and ammonium thiocyanate was centrifuged. The organic layer from the centrifuge tubes was removed from each sample and the complex formed between the phospholipid reacted with the liquid mixture was checked and estimated using UV spectrophotometer. This method is very important in a sense that we can entrap the maximum amount of drug in different types of natural and synthetic phospholipids like soya phosphatidyl choline, egg phosphatidylcholine, dipalmitoylphosphatidyl choline and dimyrestoyl phosphatidylcholine. Each phospholipid has their own characteristics of converting into the liposomes and so entrapment of drug within the internal core of liposomes or in the bilayers of multilamellar or single layer liposomes occurs. This test can be used for the entrapment efficiency of the drug because when we check the entrapment of drug it needs to be separated means the entrapped and unentraped drug. So for this purpose we use centrifuge and the phospholipid separation plays an important role of separation.
Stewart method for quantifying liposomes