Analytical approaches for the determination of deferiprone and its iron (III) complex: Investigation of binding affinity based on liquid chromatography-mass spectrometry (LC-ESI/MS) and capillary electrophoresis-frontal analysis (CE/FA)

Abstract

Two rapid and highly sensitive separation techniques were developed for the determination of free and metal-bound 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 r2 > 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 (Ltot/Leff), 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 μM, with regression coefficient r2 > 0.998. The binding parameters have been estimated for both methods in terms of binding constant (LogKa) and number of binding sites (ni). The values LogKa and ni8.3 ± 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 LogKa10.4 ± 0.12 and ni 3.0 ± 0.10.