Abstract
Two rapid and highly sensitive separation techniques were developed for the determination of free and metalbound deferiprone for binding affinity studies. The chromatographic method was carried out on a high-resolution monolithic column using gradient elution with a mobile phase consisting of ammonium formate solution (pH 7.4; 10 mM), H2O, and methanol pumped at a flow rate of 1.0 mL min(-1). Identification was performed on single quadrupole mass spectrometer using electrospray ionization interface at positive polarity. The assay was found to be linear in the concentration range of 0.5-17.5 mM with regression coefficient of r(2) > 0.999. The electrophoretic method has been developed to achieve sufficient separation between free drug and the complex using capillary length of 48.5/8.5 cm (L-tot/L-eff), 10 kV of applied voltage in negative mode, and large plug of sample injected hydrodynamicallyat10 mbar for 50 s. The linear range of the drug concentrations was 40-240 mu M, with regression coefficient r(2) > 0.998. The binding parameters have been estimated for both methods in terms of binding constant (LogK(a)) and number of binding sites (n(i)). The values LogK(a) and n(i)8.3 +/- 0.17 and 3.4 +/- 0.65 respectively were obtained from the data of liquid chromatography while, the data obtained from capillary electrophoresis resulted in LogK(a)10.4 +/- 0.12 and n(i) 3.0 +/- 0.10.