Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 11:46:e00484.
doi: 10.1016/j.plabm.2025.e00484. eCollection 2025 Sep.

Comparative evaluation of four new immunoassays and LC-MS/MS for the measurement of urinary free cortisol in Cushing's syndrome diagnosis

Qi Zhang  1 , Danni Mu  1 , Yichen Ma  1 , Yuemeng Li  1 , Yumeng Gao  1 , Yingying Hu  1 , Kui Zhang  1 , Fang Zhao  1 , Ran Gao  1 , Liangyu Xia  1 , Huijuan Zhu  2 , Songlin Yu  1 , Ling Qiu  1   3 , Xinqi Cheng  1
Affiliations

Comparative evaluation of four new immunoassays and LC-MS/MS for the measurement of urinary free cortisol in Cushing's syndrome diagnosis

Qi Zhang et al. Pract Lab Med. .

Abstract

Objectives: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by four new immunoassays using Autobio A6200, Mindray CL-1200i, Snibe MAGLUMI X8 and Roche 8000 e801 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, we evaluated the value of 24-h UFC measured by four direct immunoassays for diagnosing CS.

Methods: Residual 24-hr urine samples of 94 CS and 243 non-CS patients collected from previous cohort were used. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by immunoassays using Autobio, Mindray, Snibe and Roche platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for each assay and corresponding sensitivities and specificities were calculated by ROC analysis.

Results: All four immunoassays showed strong correlations with LC-MS/MS (Spearman coefficient r = 0.950, 0.998, 0.967, and 0.951, respectively). All immunoassays showed proportionally positive bias. The areas under the curve were 0.953 for Autobio, 0.969 for Mindray, 0.963 for Snibe, and 0.958 for Roche. The cut-off values varied from 178.5 to 272.0 nmol/24 h). Assay sensitivity and specificity ranged from 89.66 % to 93.10 % and from 93.33 % to 96.67 %, respectively.

Conclusions: Four newly available direct immunoassays for measuring UFC show good analytical consistency compared to LC-MS/MS. The elimination of organic solvent extraction simplifies workflows while maintaining high diagnostic accuracy. Additionally, they exhibited similarly high diagnostic accuracy for CS identification. Future multi-center studies are needed to validate our findings and establish method-specific UFC cut-off values to enhance clinical utility.

Keywords: Cushing's syndrome; Immunoassay; Liquid chromatography-tandem mass spectrometry; Method comparison; Urinary free cortisol.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Passing–Bablok regression curves of the immunoassays vs. LC-MS/MS (as the comparison method). (a) Autobio vs. LC-MS/MS. (b) Mindray vs. LC-MS/MS. (c) Snibe vs. LC-MS/MS. (d) Roche vs. LC-MS/MS.
Fig. 2
Fig. 2
Bland-Altman plots for (a) Autobio vs. LC-MS/MS, (b) Mindray vs. LC-MS/MS, (c) Snibe vs. LC-MS/MS, and (d) Roche vs. LC-MS/MS.
Fig. 3
Fig. 3
ROC curves for 24-hr UFC levels measured by the five methods for the diagnosis of Cushing's syndrome.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Pivonello R., Isidori A.M., De Martino M.C., Newell-Price J., Biller B.M., Colao A. Complications of Cushing's syndrome: state of the art. Lancet Diabetes Endocrinol. 2016;4(7):611–629. - PubMed
    1. Webb S.M., Valassi E. Morbidity of cushing's syndrome and impact of treatment. Endocrinol Metab. Clin. N. Am. 2018;47(2):299–311. - PubMed
    1. Fleseriu M., Auchus R., Bancos I., Ben-Shlomo A., Bertherat J., Biermasz N.R., Boguszewski C.L., Bronstein M.D., Buchfelder M., Carmichael J.D., Casanueva F.F., Castinetti F., Chanson P., Findling J., Gadelha M., Geer E.B., Giustina A., Grossman A., Gurnell M., Ho K., Ioachimescu A.G., Kaiser U.B., Karavitaki N., Katznelson L., Kelly D.F., Lacroix A., McCormack A., Melmed S., Molitch M., Mortini P., Newell-Price J., Nieman L., Pereira A.M., Petersenn S., Pivonello R., Raff H., Reincke M., Salvatori R., Scaroni C., Shimon I., Stratakis C.A., Swearingen B., Tabarin A., Takahashi Y., Theodoropoulou M., Tsagarakis S., Valassi E., Varlamov E.V., Vila G., Wass J., Webb S.M., Zatelli M.C., Biller B.M.K. Consensus on diagnosis and management of Cushing's disease: a guideline update. Lancet Diabetes Endocrinol. 2021;9(12):847–875. - PMC - PubMed
    1. Galm B.P., Qiao N., Klibanski A., Biller B.M.K., Tritos N.A. Accuracy of laboratory tests for the diagnosis of cushing syndrome. J. Clin. Endocrinol. Metab. 2020;105(6) - PubMed
    1. 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. - PMC - PubMed

LinkOut - more resources