Detecting Submicromolar Analytes in Mixtures with a 5 min Acquisition on 600 MHz NMR Spectrometers

Amino compounds are widely present in complex mixtures in chemistry, biology, medicine, food, and environmental sciences involving drug impurities and metabolisms of proteins, biogenic amines, neurotransmitters, and pyrimidine in biological systems. Nuclear magnetic resonance (NMR) spectroscopy is an excellent tool for simultaneously identifying and quantifying these in-mixture compounds but has a limit-of-detection (LOD) over several micromolarities (>5 μM). To break such a sensitivity barrier, we developed a sensitive and rapid method by combining the probe-induced sensitivity enhancement and nonuniform-sampling-based ¹H-¹³C HSQC 2D-NMR (PRISE-NUS-HSQC). We introduced two ¹³CH₃ tags for each analyte to respectively increase the ¹H and ¹³C abundances for up to 6 and 200 fold. This enabled high-resolution detection of 0.4-0.8 μM analytes in mixtures in 5 mm tubes with a 5 min acquisition on 600 MHz spectrometers. The method is much more sensitive and faster than traditional ¹H-¹³C HSQC methods (∼50 μM, >10 h). Using sulfanilic acid as a single reference, furthermore, we established a database covering chemical shifts and relative-response factors for >100 compounds, enabling reliable identification and quantification. The method showed good quantitation linearity, accuracy, precision, and applicability in multiple biological matrices, offering a rapid and sensitive approach for quantitative analysis of large cohorts of chemical, medicinal, metabolomic, food, and other mixtures.