Development and validation of a LC method for the separation and determination of the anticancer-active Fe^III(4-methoxy-salophene) using the new second-generation monolith

Abstract

LC method with the newly introduced second-generation monolithic silica RP-18e column has been developed for the separation of Fe(III) (salophene) and four methoxy-substituted Fe(III) (salophene) complexes. The method has been validated for the quantitation of Fe(III) (4-OMe-salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high-resolution continuum-source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first- and second-generation HPLC monolithic columns have been compared for this purpose. The newly introduced second-generation monolithic silica column Chromolith(®) HighResolution RP-18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe(III) (4-OMe-salophene) was found to be between 1 and 200 μg/mL. Detection and quantitation limits were 0.3 and 1 μg/mL, respectively.

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