The 40th Minute Cortisol Measurement is the Key Time-Point in the Low-Dose Synacthen Stimulation Test: A Large, Assay-Specific Pediatric Validation Study

Background: Low-dose synacthen stimulation test (LDSST) is widely used to assess central adrenal insufficiency (CAI). With the adoption of monoclonal antibody (mAb) cortisol immunoassays, lower basal and peak cortisol concentration thresholds require external validation under real-world clinical conditions. Objective: To externally validate previously defined LDSST sampling strategies, basal cortisol thresholds, and gray-zone cut-offs in a large real-world cohort using mAb immunoassays. Methods: This single-center retrospective study analyzed 646 LDSSTs in patients with suspected CAI, measuring baseline, 40th, and 60th minute cortisol levels after administration of 1 μg of synthetic ACTH. The diagnostic performance of single and combined sampling strategies and previously defined basal cortisol thresholds and grey-zone cut-offs were evaluated across different peak cortisol criteria. Results: Cortisol measurement at 40th minute provided the most reliable single time-point assessment, with significantly fewer false-negative results than at 60th minute (p<0.0001). At the basal cortisol threshold of ≥6.5 μg/dL identified in our previous prospective study, sensitivity decreased from 68% to 57.5% (p=0.21) and specificity remained comparable (73.8% vs. 75.1%, p=0.74), while negative predictive value declined significantly from 91% to 78.4% (p=0.02) and positive predictive value increased to 53% in this retrospective cohort. Validation of basal cortisol gray-zone thresholds confirmed high diagnostic accuracy across different peak cortisol cut-offs. Conclusions: This study provides robust real-world external validation of LDSST sampling strategies and basal cortisol thresholds. Cortisol measurement at 40th minutes, combined with assay-specific basal cortisol interpretation and a gray-zone framework, offers a practical approach for individualized clinical decision-making in suspected CAI.