Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry Opens New Possibilities in the Field of Compound-Specific Stable Isotope Analysis

Compound-specific stable isotope analysis (CSIA) using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is a powerful tool for determining the isotopic composition of carbon in analytes from complex mixtures. However, LC-IRMS methods are constrained to fully aqueous eluents. Previous efforts to overcome this limitation were unsuccessful, as the use of organic eluents in LC-IRMS was deemed impossible. In our study, we developed a two-dimensional (2D) LC-IRMS method that, for the first time, enables the use of organic-containing eluents in an LC-IRMS setting. Initial experiments with caffeine were performed using a sample loop modulator with 20% methanol in the mobile phase of the first dimension, while separating the organic fraction from the analyte in the second dimension. Comparing results with one-dimensional (1D) LC-IRMS methods showed high precision with δ¹³C values in 2D measurements (-34.98 ± 0.04 ‰) closely matching 1D results (-34.95 ± 0.12 ‰). In the next step, incorporation of an at-column dilution (ACD) modulator allowed for the successful use of methanol concentrations up to 40% in the first dimension, with the ACD modulator effectively mitigating both peak fronting and carbon background interference, without losing any precision or accuracy of the measurements (δ¹³C_Caffeine = -34.92 ± 0.03 ‰). All developed methods showed a method detection limit lower than 5 mg of carbon L^-1 (mgC L^-1), which is a major improvement compared with previous studies on caffeine analysis with LC-IRMS. This proof-of-concept study on 2D-LC-IRMS opens vast new possibilities for future CSIA research across diverse fields, including environmental science, pharmaceuticals, and food chemistry.