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.