Evaluation of different types of isotope-labelled steroids based on liquid chromatography-mass spectrometry

Stable isotope-labelled reagents, as internal standards (IS), are critical for ensuring the accuracy of mass spectrometry-based quantitative analysis, and their quality directly affects the reliability of detection results. ¹³C-labelled and deuterium (D)-labelled isotopic reagents are the most commonly used, but systematic evaluation of these reagents is still lacking. In this study, four clinically representative steroids were selected to systematically compare the performance of ¹³C-labelled and D-labelled internal standard reagents. Key parameters including isotope abundance, isotopic distribution, stability, and specificity were investigated, followed by validation in real serum samples. Results showed that both ¹³C-labelled and D-labelled reagents exhibited good stability. However, ¹³C-labelled reagents outperformed D-labelled ones in isotopic distribution and specificity. With the extension of analysis time, D-labelled reagents showed a significant separation trend from their corresponding unlabelled compounds, while the retention time difference between ¹³C-labelled IS and unlabelled analyses was ≤ 0.02 min, ensuring excellent co-elution. Serum spiking recovery experiments further demonstrated that ¹³C-labelled reagents (such as ¹³C-progesterone) with superior performance in this evaluation system, especially in methods involving large panels or long analysis times, achieved higher quantitative accuracy and precision.