Quantification of Diethyl Phthalate by a Rapid and Homogenous Fluorescence Polarization Immunoassay

Abstract

A rapid and homogenous fluorescence polarization immunoassay (FPIA) for diethyl phthalate is reported. Two haptens and three immunogens for diethyl phthalate were synthesized to generate polyclonal antibodies and fluorescein isothiocyanate labeled diethyl phthalate was prepared as the tracer. The antibodies, methanol concentration, and incubation time were optimized. The cross-reactivities of the optimized antibodies to other phthalate esters were characterized. The antibody shows excellent sensitivity with a limit of detection of 6.0 ng mL−1 and a half-inhibition concentration of diethyl phthalate of 40.4 ng mL−1. The immunoassay had a linear dynamic range from 10 to 200 ng mL−1. The mean coefficient of variation was lower than 11.3% for intra-assay and lower than 14.6% for inter-assay measurements. The limit of detection was 1.46 ng mL−1 in bottled drinking water, 0.34 µg mL−1 in juice, and 50.0 µg g−1 in pharmaceutical capsules. The recovery was from 85.9 to 114.9%, and the coefficients of variation were 4.3 to 17.0%. The developed immunoassay provided good sensitivity, specificity, accuracy, and may serve as an alternative for diethyl phthalate determination for field screening of food, pharmaceuticals, and environmental materials.