Temperature-Responsive Liquid Chromatography for One- and Two-Dimensional Separations in Compound-Specific Isotope Analysis

Temperature-responsive liquid chromatography (TRLC) enables the possibility of retention modulation under fully aqueous isocratic conditions through incorporation of smart polymers such as poly(<i>N</i>-isopropylacrylamide) (PNIPAAm) into the stationary phase. This feature seems to be particularly advantageous for liquid chromatography isotope ratio mass spectrometry (LC-IRMS), which is constrained by its requirement for fully aqueous eluents and limited stationary phase options. In this study, we demonstrate for the first time the coupling of TRLC with LC-IRMS for compound-specific carbon isotope analysis. Using testosterone and related steroids as a model substance class, we show that PNIPAAm-based TRLC columns provide stable δ¹³C measurements across isothermal and temperature gradient programs without introducing isotopic bias (σ ≤ 0.5‰ across all measurements). One-dimensional TRLC-IRMS enabled separation of several steroid standards under fully aqueous conditions and the determination of testosterone in an ethanol-based gel. A heart-cut two-dimensional (2D) LC-IRMS configuration, combining reversed phase LC in the first dimension with TRLC in the second dimension, achieves baseline separation of otherwise coeluting steroids and accurate δ¹³C determination in complex, lipid-rich pharmaceutical matrices. This work establishes TRLC as a powerful addition to the LC-IRMS toolkit, expanding method flexibility and selectivity without sacrificing isotopic accuracy and opening new avenues for compound specific isotope analysis via LC-IRMS.