Sensitive Rapid Fluorescence Polarization Immunoassay for Free Mycophenolic Acid Determination in Human Serum and Plasma.

ABSTRACT

In this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near-infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λexcmax) and shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 µg/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7-39 ng/mL (20%-80% binding inhibition) in a PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart-transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus, and the results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).