DETERMINATION OF PENTAMIDINE IN LEISHMANIA-INFANTUM PROMASTIGOTES BY ION-PAIRED LIQUID-CHROMATOGRAPHY

Pentamidine is an aromatic diamidine, which have recently been shown t o be non-competitive inhibitors of putrescine uptake in parasitic prot ozoa In order to understand the mechanism involved in Pentamidine upta ke by parasitic protozoa cells, a HPLC analytical method for determina tion of pentamidine in Leishmania infantum promastigote cultures was d eveloped. The influence on the capacity factor of various characterist ic parameters in the mobile phase such as pH, percentage of methanol a nd temperature of column is studied, taking into account that pentamid ine can form ion-pairs with sulphonic compounds, the influence of the length of the lateral chain and concentration of various compounds of this type are also studied. The results obtained in this study allow u s to know the best chomatographic conditions for the determination of pentamidine in Leishmania infantum promastigotes and they can also be used for the optimization of analytical methods in order to determine pentamidine in other biological media or other complex mixtures. The c hromatographic procedure uses a reversed-phase column Nucleosil C-18 5 mu m, the column effluent was monitored by ultraviolet-visible spectr ophotometry at 261 nm. The procedure involves a simple method of the c leaning-up Leishmania promastigote extract samples cultured with penta midine by ultrafiltration through a polysulfone membrane with 100,000 relative molecular mass cut-off. The method shows good recovery, preci sion and accuracy. The limit of detection for pentamidine is 5.70 ng/m l in Leishmania cells culture.