Coupling Liquid Chromatography to Orbitrap Isotope Ratio Mass Spectrometry: Overcoming Isotope Effects of Chromatography and Amount-Dependency by Peak Homogenization

Electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS) has recently proven to be a powerful tool for compound- and position-specific stable isotope analysis, targeting isotopologues of multiple elements (H, C, N, O, S) in polar analytes. However, studies have so far mainly focused on pure analyte solutions via direct infusion. Here, we present the online coupling of liquid chromatography (LC) to ESI-Orbitrap-MS for stable isotope analysis of sulfamethoxazole (SMX), a synthetic antimicrobial, used as a model compound. Our study explored the fidelity of isotope values with two strategies for capturing and broadening chromatographic peaks after LC. When capturing the target analyte in a capillary (0.7 mm inner diameter), peak shape was retained, resulting in systematic deviations in isotope values caused by isotope effects from chromatography of up to 70‰ and amount-dependency of up to 65‰. A dynamic mixing chamber was used to homogenize the target analyte upon elution, removing these deviations and resulting in stable carbon, nitrogen, and sulfur isotopologue ratios in various fragments of sulfamethoxazole across the chromatographic peak. Comparison with data from conventional magnetic sector isotope ratio mass spectrometry successfully calibrated the setup for carbon and sulfur isotope ratio analysis. This resulted in a precision for δ¹³C within the isoxazole moiety from SMX of 1.5‰ (95% confidence intervals CIs derived from uncertainties of sample (n = 4) and reference (n = 5)), and for δ³⁴S in the SO₂ fragment of 0.9‰ (95% CI, derived again from uncertainties of sample (n = 4) and reference (n = 5)). This work paves the way for the online coupling of LC to ESI-Orbitrap-MS in future compound- and position-specific stable isotope analyses across various research fields.