*9.2.4 Phospholipids per-oxidation*

Most oxidation products are further subjected to degradation and at least two separate tests should be performed for estimation of oxidation. Gas liquid chromatography (GLC) and UV absorbance are most quantitative methods to estimate oxidation. This UV method is based on the absorbance of conjugated dienes and trienes at 233 nm 270 nm and phospholipids do not absorb at these wave lengths. TBA (thiobarbituric acid) method is widely used lipid peroxidation assay. In this method the samples are heated with an aqueous TBA solution. Under these conditions the lipid oxidation product malondialdehyde reacts with TBA gives a pink chromophore and spectrophotometrically quantified at 533 nm.

#### *9.2.5 Phospholipids hydrolysis*

The phospholipids in liposomes will hydrolyse to free fatty acids and 2-acyl- and 1-acyl-lysophospholipids. The lysophospholipids further hydrolysed to glycerol phosphor compounds. The hydrolysed products can be analyzed by using HPLC method or TLC method and glycerol phosphor compounds can be analyzed by total phosphate analysis of the supernatant (methanol/water phase) after lipid extraction.

#### **9.3 Biological characterization**

Biological characterization identifies safety of liposomes formulations when *in vivo* studies are done [16]. The liposomes characterization depends on selection of phospholipid, size characteristics and charge behavior [92, 93]. Sterility of liposomes can be identified by preparing aerobic or anaerobic cultures and pyrogenicity can be known by pyrogen test on rabbits.
