Heart-Cutting Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry for Compound-Specific δ13C Analysis of Water-Soluble B Vitamins in Complex Supplement Matrices.

Compound-specific stable carbon isotope analysis by one-dimensional liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is fundamentally constrained by the requirement for fully aqueous mobile phases, which limits chromatographic selectivity and prevents effective separation of analytes from complex sample matrices. Here, a heart-cutting two-dimensional LC-IRMS method (2D-LC-IRMS) is presented for the first compound-specific δ13C analysis of the water-soluble vitamins B5 (pantothenic acid) and B9 (folic acid) in commercial dietary supplements and fortified beverages. An organic-modified reversed-phase separation in the first dimension achieved matrix reduction, while a fully aqueous second-dimension separation ensured IRMS compatibility and delivered baseline-resolved analyte peaks for precise isotope determination. Validation demonstrated linear calibration over 2-100 mgC L-1 with R2 ≥ 0.9999, repeatability of ≤ 0.13‰, and isotope-stability-based method detection limits of 5 mgC L-1. Analyzed δ13C values were independent of chromatographic configuration, confirming that heart-cutting transfer does not introduce isotope fractionation. Application to 12 commercial products, including tablets, effervescent formulations, powdered supplements, and an energy drink, yielded δ13C ranges of -20.6‰ to -32.9‰ for vitamin B5 and -20.4‰ to -36.4‰ for vitamin B9, reflecting differences in synthetic production routes across manufacturers. The presented workflow extends LC-IRMS to the compound class of water soluble vitamins that were so far inaccessible to this technique and provides a broadly applicable strategy for compound-specific isotope analysis of polar analytes in challenging matrices.