Analytical Methods
Total amikacin concentrations were measured by high-performance liquid chromatography (HPLC) using a Hypersil GOLD C18 column (175 Å, 3 µm, 150 mm × 4.6 mm) with a mobile phase of 65% methanol, 35% water, and 0.3% pentafluoropropionic acid (PFPA). An aliquot of each liposome suspension was centrifuged in an Amicon Ultra-0.5 centrifugal filter unit with Ultracel-30 KDa membrane to separate free amikacin, and the amount of unencapsulated amikacin was then determined by HPLC.
Lipid concentrations were measured by HPLC using an XBridge C8 column (130Å, 3.5µm, 150mm × 4.6mm) with a mobile phase A consisting of 49.9% acetonitrile, 49.9% water, 0.1% acetic acid, and 0.1% triethylamine and mobile phase B consisting of 44.9% acetonitrile, 45% isopropyl alcohol, 10% water, 0.1% acetic acid, and 0.1% triethylamine.
Residual ethanol or ethanol concentrations were measured by using gas chromatography (GC) using a Duraguard DB.