Selective fluorescence turn-on detection of methylglyoxal using N-substituted NBD derivatives

Methylglyoxal (MGO) is a reactive endogenous α-dicarbonyl involved in glycation, oxidative stress, and metabolic disease. Its measurement in biological samples remains challenging because conventional assays generally require derivatization, chromatographic separation, and costly instrumentation. Fluorescence detection offers a simpler alternative, but currently available probes often lack selectivity for MGO over other biologically relevant carbonyl compounds. We therefore developed a series of N-substituted 4-hydrazino-7-nitrobenzofurazan derivatives for fluorimetric detection of MGO. Alkyl groups were introduced on the hydrazine nitrogen to modulate probe reactivity, steric accessibility, and the fluorescent properties of the resulting hydrazones. The probes were characterized by fluorescence spectroscopy, HPLC with fluorescence detection, LC-MS, and density functional theory calculations. Compared with unsubstituted NBD-H, most N-substituted derivatives produced stronger and more stable responses to MGO, while showing weaker responses to competing carbonyl compounds. Chromatographic and mass spectrometric analyses confirmed formation of the expected probe-MGO hydrazones, and calculations supported preferential condensation at the aldehyde group of MGO. In human plasma, most N-substituted probes retained a strong and highly linear fluorescence response to MGO, even in the presence of biologically relevant carbonyl interferents such as glyoxal, acrolein, acetaldehyde, and formaldehyde. Compared with unsubstituted NBD-H, these derivatives markedly improved analytical sensitivity toward MGO, supporting their use for fluorimetric detection in complex biological matrices. These findings support a microplate-compatible fluorimetric assay for MGO requiring only a protein-precipitation step and avoiding chromatographic separation or extensive sample clean-up.