Work

Multicenter comparison of LC-MS/MS, radioimmunoassay, and ELISA for assessment of salivary progesterone and estradiol

Year: 2026

Type: article

Abstract: INTRODUCTION: For psychoneuroendocrinology, accurate measurement of progesterone (P4) and estradiol (E2) in saliva is crucial for understanding the menstrual cycle phase and its impact on physiology and behavior. Although enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) are the preferred methods and are more often found in research labs, liquid chromatography-mass spectrometry (LC-MS/MS) may provide a more valid salivary steroids assessment. In this study, we aimed to compare the performance of LC-MS/MS, ELISA, and RIA to measure salivary P4 and E2. METHOD: Samples were collected from 120 participants, 81 men and 39 women, in the morning and evening. Additionally, women provided samples during the early follicular (cycle days 3-5) and luteal cycle phases (cycle days 21-23) using the forward-count method. The study considered natural hormone fluctuations (e.g., the diurnal and menstrual cycles) and quality control samples as validity criteria for method evaluation. A total of 336 samples and quality control samples were analyzed using one RIA, two ELISA, and two LC-MS/MS methods across four labs. Correlational analyses were performed to assess inter-lab x inter-method reliability, intra-lab x inter-method reliability, and inter-lab x intra-method reliability. RESULTS: For P4, natural hormone fluctuations in menstrual cycles were detected by all methods. However, in contrast to both LC-MS/MS methods, all immunoassays (IAs) detected an unexpected diurnal decline of P4. For E2, they were found only by LC-MS/MS and RIA. In terms of means, for P4 and E2, ELISA and RIA produced higher values compared to LC-MS/MS. For P4, the inter- and intra-method convergence was r ≥ .92. As for E2, inter-method correlations were between r = -.12 and r = .23, while the ELISA intra-method comparison showed a correlation coefficient of r = .85. DISCUSSION: Our findings suggest that while LC-MS/MS, RIA, and ELISA were capable of meeting most of the specified criteria for P4, only LC-MS/MS and RIA were able to perform similarly sufficiently at low E2 levels. LC-MS/MS provided more accurate and reliable measurements for P4 and E2, especially at low concentrations, but encountered challenges, too. Although RIA showed comparable performance to LC-MS/MS, it still suffered partly from cross-reactivity. ELISA often overestimated hormone levels and exhibited greater divergence. These differences highlight the importance of carefully selecting methods and considering their limitations in research applications.

Source: Psychoneuroendocrinology

DOI: 10.1016/j.psyneuen.2026.107744