Development and clinical application of a novel urinary cortisol extraction agent

doi:10.1007/s00216-025-05998-7

. 2025 Jul 3.

doi: 10.1007/s00216-025-05998-7. Online ahead of print.

Development and clinical application of a novel urinary cortisol extraction agent

Chengxue Wang^#¹, Qian Wang^#², Wenting Yuan¹, Zichao Jia¹, Lizhen Dong¹, Yamin Chai¹, Qinzhen Cai³, Chunhui Yuan⁴, MingFeng Cao⁵, Wei Luo⁶

Affiliations

¹ Department of Clinical Laboratory, General Hospital of Tianjin Medical University, Tianjin, 300352, China.
² Department of Pathology, Prevention for State Administration of Traditional Chinese Medicine, Haihe Hospital, Tianjin, 300052, China.
³ Department of Laboratory Medicine, Wuhan Children's Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430016, China.
⁴ Department of Laboratory Medicine, Wuhan Children's Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430016, China. chunhuii.yuen@whu.edu.cn.
⁵ Tianjin Jinyinuo Biotechnology Co Ltd, Tianjin, 300050, China.
⁶ Department of Clinical Laboratory, General Hospital of Tianjin Medical University, Tianjin, 300352, China. weiluo1028@tmu.edu.cn.

^# Contributed equally.

PMID: 40610812
DOI: 10.1007/s00216-025-05998-7

Development and clinical application of a novel urinary cortisol extraction agent

Chengxue Wang et al. Anal Bioanal Chem. 2025.

. 2025 Jul 3.

doi: 10.1007/s00216-025-05998-7. Online ahead of print.

Authors

Chengxue Wang^#¹, Qian Wang^#², Wenting Yuan¹, Zichao Jia¹, Lizhen Dong¹, Yamin Chai¹, Qinzhen Cai³, Chunhui Yuan⁴, MingFeng Cao⁵, Wei Luo⁶

Affiliations

¹ Department of Clinical Laboratory, General Hospital of Tianjin Medical University, Tianjin, 300352, China.
² Department of Pathology, Prevention for State Administration of Traditional Chinese Medicine, Haihe Hospital, Tianjin, 300052, China.
³ Department of Laboratory Medicine, Wuhan Children's Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430016, China.
⁴ Department of Laboratory Medicine, Wuhan Children's Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430016, China. chunhuii.yuen@whu.edu.cn.
⁵ Tianjin Jinyinuo Biotechnology Co Ltd, Tianjin, 300050, China.
⁶ Department of Clinical Laboratory, General Hospital of Tianjin Medical University, Tianjin, 300352, China. weiluo1028@tmu.edu.cn.

^# Contributed equally.

PMID: 40610812
DOI: 10.1007/s00216-025-05998-7

Abstract

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.

Keywords: Biological reference interval; Dichloromethane; Ethyl acetate; Extraction; Urinary free cortisol.

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Conflict of interest statement

Declarations. Research ethics: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards. This study was approved by the Medical Ethics Committee of Tianjin Medical University General Hospital: IRB2023-KY-244. Competing interests: The authors declare no competing interests.

References

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[1] Jimeno B, Rubalcaba JG. Modelling the role of glucocorticoid receptor as mediator of endocrine responses to environmental challenge. Philos Trans R Soc Lond B Biol Sci. 2024;379(1898):20220501. https://doi.org/10.1098/rstb.2022.0501 . - DOI - PubMed - PMC

[2] Jimeno B, Rubalcaba JG. Modelling the role of glucocorticoid receptor as mediator of endocrine responses to environmental challenge. Philos Trans R Soc Lond B Biol Sci. 2024;379(1898):20220501. https://doi.org/10.1098/rstb.2022.0501 . - DOI - PubMed - PMC

[3] Luo J, Zhou C, Wang S, Tao S, Liao Y, Shi Z, Tang Z, Wu Y, Liu Y, Yang P. Cortisol synergizing with endoplasmic reticulum stress induces regulatory T-cell dysfunction. Immunology. 2023;170(3):334–43. https://doi.org/10.1111/imm.13669 . - DOI - PubMed

[4] Luo J, Zhou C, Wang S, Tao S, Liao Y, Shi Z, Tang Z, Wu Y, Liu Y, Yang P. Cortisol synergizing with endoplasmic reticulum stress induces regulatory T-cell dysfunction. Immunology. 2023;170(3):334–43. https://doi.org/10.1111/imm.13669 . - DOI - PubMed

[5] El-Farhan N, Rees DA, Evans C. Measuring cortisol in serum, urine and saliva - are our assays good enough? Ann Clin Biochem. 2017;54(3):308–22. https://doi.org/10.1177/0004563216687335 . - DOI - PubMed

[6] El-Farhan N, Rees DA, Evans C. Measuring cortisol in serum, urine and saliva - are our assays good enough? Ann Clin Biochem. 2017;54(3):308–22. https://doi.org/10.1177/0004563216687335 . - DOI - PubMed

[7] Choi MH. Clinical and technical aspects in free cortisol measurement. Endocrinol Metab (Seoul). 2022;37(4):599–607. https://doi.org/10.3803/EnM.2022.1549 . - DOI - PubMed

[8] Choi MH. Clinical and technical aspects in free cortisol measurement. Endocrinol Metab (Seoul). 2022;37(4):599–607. https://doi.org/10.3803/EnM.2022.1549 . - DOI - PubMed

[9] Mu D, Fang J, Yu S, Ma Y, Cheng J, Hu Y, Song A, Zhao F, Zhang Q, Qi Z, Zhang K, Xia L, Qiu L, Zhu H, Cheng X. Comparison of direct and extraction immunoassay methods with liquid chromatography-tandem mass spectrometry measurement of urinary free cortisol for the diagnosis of Cushing’s syndrome. Ann Lab Med. 2024;44(1):29–37. https://doi.org/10.3343/alm.2024.44.1.29 . - DOI - PubMed

[10] Mu D, Fang J, Yu S, Ma Y, Cheng J, Hu Y, Song A, Zhao F, Zhang Q, Qi Z, Zhang K, Xia L, Qiu L, Zhu H, Cheng X. Comparison of direct and extraction immunoassay methods with liquid chromatography-tandem mass spectrometry measurement of urinary free cortisol for the diagnosis of Cushing’s syndrome. Ann Lab Med. 2024;44(1):29–37. https://doi.org/10.3343/alm.2024.44.1.29 . - DOI - PubMed

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Development and clinical application of a novel urinary cortisol extraction agent

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Development and clinical application of a novel urinary cortisol extraction agent

Authors

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Abstract

Conflict of interest statement

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