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 [1] is one of the most common contraceptive hormones under concern. Despite the high sensitivity of the fluorometric methods, detection of LEV 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 LEV, 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 LEV estimation in breast milk samples with linearity of (0.4-80 ng.mL-1) and low detection limit of 0.18 ng.mL-1 without 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 LEV bioanalysis in different matrices.