Novel fluorescence approach for trace quantification of levonorgestrel in breast milk based on click reaction with benzonitrofurazan azide (NBD-AZ).

Although the great importance of oral contraceptive agents in birth control, their existence in breast milk became a cause for concern, since infant exposure to these hormones is associated with many health problems. Consequentially, developing a sensitive bioanalytical method for monitoring their concentrations in breast milk is an urgent demand to examine the safety or the risk of these compounds on infants. Levonorgestrel is one of the most common contraceptive hormones under concern. Despite the high sensitivity of the fluorometric methods, detection of Levonorgestrel by them is confined because its structure does not exhibit any fluorescence. For the first time, we proposed a promising click fluorescent probe, 4-azido-7-nitrobenzoxadiazole to react with the alkyne group of Levonorgestrel, to give a highly fluorescent triazole derivative that exhibited strong signal at wavelength of 544 nm after excitation at 470 nm. Reaction parameters impacting the fluorescence were cautiously studied and optimized. The suggested approach has been successfully applied in Levonorgestrel estimation in breast milk samples with linearity of (0.4-80 ng.ml-1) and low detection limit of 0.12 ng.ml-1without interferences from any biological components and with mean % recovery of 97.84 ± 2.73. Accuracy, sensitivity, selectivity, simplicity, and low-cost makes this approach a convincing, promising, and appealing alternative over reported analytical methods for Levonorgestrel bioanalysis in different matrices.

4-Azido-7-nitrobenzoxadiazole as innovative clickable fluorescence probe for trace and selective quantification of ethinylestradiol in human plasma.

Quantification of ethinylestradiol (EE) in biological matrices is challenging as it is a very potent drug with a very low Cmax (75 pg.ml-1 ). Despite the high sensitivity of fluorometric methods, the detection of EE was confined because its structure exhibited very limited fluorescence. Therefore, it must be derivatized first using a fluorogenic agent to produce a more potent fluorescence derivative to achieve the desired ultrasensitive bioanalysis. Here, 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, with 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 compared with NBD-AZ. The resulting triazole exhibited a strong signal at a 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 a low detection limit of 7.5 pg.ml-1 . The enhanced sensitivity and selectivity were exploited for analyzing EE in plasma using liquid-liquid extraction for samples cleaning up without interference from any biological components and with a mean % recovery of 100.13 ± 0.39. Accuracy, sensitivity, selectivity, simplicity, and cost-effectiveness make this approach a convincing, promising, and appealing alternative to the reported analytical methods for EE bioanalysis in different matrices.