Enhanced δD measurability in low-abundance compound-specific isotope analysis using controlled isotope dilution with mass-balance correction

Compound-specific hydrogen isotope (δD) analysis is frequently limited by low analyte abundance, which can lead to disproportionately elevated analytical uncertainty and incomplete environmental records. Here, we evaluate an isotope dilution approach designed to improve the measurability of low-abundance compounds through controlled mixing with an isotopically characterized spike. Using n -alkane isotope reference mixtures, we systematically assess the influence of spike-sample mixing ratios and injection strategies on recovered δD values. Comparison of inlet introduction methods shows that pre-mixed injections outperform autosampler co-injections, reducing RMSE by approximately a factor of two. Uncertainty analyses indicate that variability in the measured isotope ratios is the dominant contributor to propagated uncertainty, whereas mixing-ratio variability contributes a smaller but non-negligible component. Our results define a practical operating window in which the injected spike concentration may reach up to 2.8 times that of the sample, increasing total injected analyte abundance nearly fourfold while maintaining reliable isotope recovery. Under these conditions, long-chain homologs (C 27 and C 29 ) achieve RMSE values of approximately 5–6 ‰, within ~1.5 ‰ of those obtained from independent high-abundance measurements. These results demonstrate that isotope dilution provides a practical means of extending compound-specific δD measurements to substantially lower analyte abundance, expanding analytical access to low-concentration biomarkers in paleoclimate and environmental archives.