Solvent-assisted sensing ability of ¹⁹F reporter in simultaneous discrimination and quantification of proline and...

Quantitative analysis of metabolites with very similar chemical structures in complex biological matrices remains challenging. Although ¹⁹F nuclear magnetic resonance (NMR) spectroscopy offers high sensitivity and minimal background interference in analytical chemistry, limitations in quantification of analytes with highly similar structures still remain. Here, we report a solvent-assisted ¹⁹F chemical shift separation strategy that enables direct distinction and quantification of multiple analytes with similar chemical structures, for example, proline (Pro) and 4-hydroxyproline (4-Hyp) isomers. We found that mono-fluorinated tags exhibit substantially greater sensitivity to solvent polarity than trifluoromethyl-containing tags, enabling finer modulation of chemical shifts and improved spectral resolution of ¹⁹F-NMR signals. Simultaneous discrimination and quantification of Pro and 4-Hyp isomers were achieved by ¹⁹F NMR in the presence of a certain amount of organic solvent in aqueous solution. This method was directly applied to in-situ quantification of Pro and 4-Hyp isomers in commercially available fetal bovine serum (FBS), human plasma and gelatin samples, demonstrating solvent modulation is an effective approach for enhanced chemical shift sensing of important analytes with very similar chemical structures in complex biological environments using ¹⁹F NMR.