4-azido-7-nitrobenzoxadiazole as Innovative Clickable Fluorescence Probe for Trace and Selective Quantification of Ethinylestradiol in Human Plasma.

Abstract

Quantification of Ethinylestradiol (EE) in biological matrices is challenging since it is a very potent drug with a very low Cmax (75 pg.mL-1 ). Despite the high sensitivity of fluorometric methods, detection of EE by them is confined because its structure exhibited very limited fluorescence. Therefore, it must be derivatized first using a fluorogenic agent to produce more potent fluorescent derivative to achieve the desired ultra-sensitive bioanalysis. Herein, For the first time, we proposed a promising click fluorescent probe, 4-azido-7-nitrobenzoxadiazole (NBD-AZ) to react with the alkyne group of EE, by the help of copper sulphate and L-ascorbic acid to give a highly fluorescent and stable 1,2,3- triazole derivative. Density functional theory calculation revealed how the triazole formation affects the quantum yield and fluorescence of click reaction product when comparing to NBD-AZ. The resulting triazole exhibited strong signal at wavelength of 540 nm after excitation at 470 nm. Reaction parameters impacting the intensity of fluorescence were cautiously studied and optimized. The suggested approach has shown outstanding performance, high linearity (25-300 pg.mL-1 ) and low detection limit of 7.5 pg.mL-1 . The enhanced sensitivity and selectivity were exploited for analysing EE in plasma using liquid-liquid extraction for samples cleaning up without interference from any biological components and with mean % recovery of 100.13±0.39. Accuracy, sensitivity, selectivity, simplicity, and cost-effectiveness make this approach a convincing, promising, and appealing alternative over the reported analytical methods for EE bioanalysis in different matrices.