Development and clinical application of a novel urinary cortisol extraction agent

Dichloromethane (DCM), a traditional extractant for 24-h urine free cortisol (UFC) testing, poses significant occupational hazards. This study aimed to explore a novel extractant, evaluate its performance in replacing DCM, and establish the biological reference range for UFC detection using the new extractant. Comparative analyses of extraction efficiency were conducted between ethyl acetate (EA) and DCM. A stabilized EA-based formulation (named EEG) was developed by incorporating anhydrous ethanol and gallic acid as anti-hydrolysis and volatility-reducing agents. Recovery rates were assessed using a control product. Stability was tested through delayed detection. Biological reference intervals were determined via chemiluminescence immunoassay (CLIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) following EEG extraction, and determined the reference interval using mean ± 1.96 standard deviation. EA demonstrated superior extraction efficiency, yielding significantly higher median cortisol levels (6.27 (IQR 4.68-8.68) µg/dL) versus DCM (3.96 (IQR 2.41-5.07) µg/dL, p = 0.002). Recovery tests confirmed EA's enhanced performance (mean recovery 95.3% vs. DCM's 65.4%, p = 0.011). The EEG formulation exhibited improved stability with unchanged extraction efficacy. Established reference intervals (95% range) were 14.58~136.62 µg/24 h (CLIA) and 0.81~60.55 µg/24 h (LC-MS/MS). EA extraction surpasses DCM in recovery and safety. The optimized EEG formulation demonstrates enhanced stability while maintaining analytical reliability. The newly established reference intervals provide method-specific benchmarks for clinical UFC assessment. This innovation offers a safer, more efficient alternative for routine laboratory practice. The findings underscore the feasibility of replacing hazardous solvents in endocrine diagnostics without compromising analytical performance.