A novel high-throughput liquid chromatography assay for Carbohydrate-Deficient transferrin (CDT) based on flow-modulated isocratic elution and terbium-induced fluorescence.

Transferrin is a glycoprotein containing two bi- or tri-antennary carbohydrate chains ending with sialic acid. Its glycosylation is reduced in chronic alcohol abuse and in inborn glycosylation pathologies, where the carbohydrate-deficient fraction of the protein (CDT) increases significantly. The current methods require a gradient chromatographic separation and time-consuming sample preparation. In comparison, the proposed approach uses a novel flow-modulated liquid chromatography technique (fmLC) and a highly selective and sensitive fluorescence derivatization reaction with terbium ion. A fmLC-FLD method using isocratic anion exchange separation was optimized and validated to resolve disialo-transferrin and trisialo-transferrin from other transferrin glycoforms. Detection took place by recording fluorescence at 550 nm wavelength (excitation at 298 nm). The chromatographic separation needed 5 min, allowing seriate injection every 7.5 min. The method was validated according to the current guidelines of analytical chemistry showing adequate accuracy and precision for the quantitative determination of CDT. The proposed method proved also to be suitable to analyse haemolyzed sera which, because of interference by haemoglobin, fail the standard HPLC-Vis analysis. The method was tested in parallel with HPLC-Vis on 131 sera showing an excellent correlation of results proved by a correlation coefficient of 0.995 (Pearson's r). The proposed approach proved much simpler than the current methods and cheaper in terms of instrumental costs offering a ground-breaking analytical tool that could likely make available the characterization of CDT outside specialized laboratories, such as in occupational medicine centres, doctor's offices, small laboratories, alcohol rehabilitation centres, and in developing countries.