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

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Temperature-responsive liquid chromatography (TRLC) coupled with liquid chromatography isotope ratio mass spectrometry (LC-IRMS) offers stable, isotope-accurate carbon analysis for steroids. This novel method enhances compound-specific isotope analysis flexibility.

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Mass Spectrometry

Background:

  • Liquid chromatography isotope ratio mass spectrometry (LC-IRMS) requires fully aqueous eluents, limiting stationary phase choices.
  • Temperature-responsive liquid chromatography (TRLC) uses smart polymers for retention modulation in aqueous conditions.
  • PNIPAAm is a smart polymer suitable for TRLC stationary phases.

Purpose of the Study:

  • To couple TRLC with LC-IRMS for compound-specific carbon isotope analysis.
  • To evaluate the performance of PNIPAAm-based TRLC columns for stable isotope measurements.
  • To explore the application of 1D and 2D TRLC-IRMS for steroid analysis in complex matrices.

Main Methods:

  • Coupling of a PNIPAAm-based TRLC column with LC-IRMS.
  • Analysis of testosterone and related steroids using isothermal and temperature gradient programs.
  • Development of a heart-cut two-dimensional (2D) LC-IRMS system combining reversed-phase LC and TRLC.

Main Results:

  • Stable and accurate δ13C measurements (σ ≤ 0.5‰) were achieved with PNIPAAm-based TRLC-IRMS.
  • One-dimensional TRLC-IRMS separated steroid standards and quantified testosterone in a gel matrix.
  • Two-dimensional TRLC-IRMS resolved coeluting steroids and enabled accurate δ13C determination in lipid-rich pharmaceutical samples.

Conclusions:

  • TRLC is a viable and powerful addition to the LC-IRMS analytical toolkit.
  • TRLC-IRMS expands method flexibility and selectivity for compound-specific isotope analysis without compromising accuracy.
  • This approach opens new possibilities for analyzing complex samples using LC-IRMS.