A modified HPLC method for monensin analysis in liposomes and nanocapsules and its comparison with spectrophotometric...

Monensin is a carboxylic ionophore which can potentiate the immunotoxin activity against human tumors in vitro and in vivo. Currently monensin is being encapsulated in liposomes and nanocapsules in our laboratory. The reported methods for monensin analysis by spectrophotometric and HPLC lack the required sensitivity. We have developed a sensitive HPLC method for analysis on monensin. Separation was achieved on a Beckman C18 reverse phase column with methanol-acetonitrile-methylene chloride-water-acetic acid (45:20:25:9.5: 0.5) as the mobile phase. The eluent was reacted with vanillin reagent in the post column reactor at 70 degrees C. The reagent reacted with monensin and formed a pink color, which was detected at 520 nm. The retention time of monensin was found to be 6 min. By using this method it was possible to quantify monensin down to 100 ng ml-1, with a signal to noise ration of > 17:1. Linearity was observed within the range of 10 to 100 ng (r2 > 0.99). Inter-day standard deviations for monensin samples of 20, 50 and 80 ng were 0.675, 0.543 and 0.736 respectively. Alternative methods of analysis include using radioactive [3H]monensin in liposomes which can be quantified by scintillation counter. The results from the HPLC, spectrophotometric and radioactive method were compared and were found to be within acceptable limits. The HPLC method is being utilized in our laboratory for quantitative analysis of monensin in liposomes and nanocapsules.