Heart‐Cutting Two‐Dimensional Liquid Chromatography‐Isotope Ratio Mass Spectrometry for Compound‐Specific δ ¹³ C 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 δ 13 C 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 R 2 ≥ 0.9999, repeatability of ≤ 0.13‰, and isotope‐stability‐based method detection limits of 5 mgC L −1 . Analyzed δ 13 C 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 δ 13 C 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.