Isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of ten steroid metabolites in serum
Abstract
An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T), and estrone 3-sulfate (E1-S) in serum was developed and validated. Limits of quantification, variability (inter- and intra-day), analytical range and linearity were all found to be acceptable for clinical use. Furthermore, sample stability was evaluated including the influence of freeze-thaw cycles and the effects of temperature and storage time. The method was applied to 391 serum samples from healthy, Danish boys 10-18years old. The concentration ranges of the included steroid metabolites for this population are presented. Concentrations of DHEAS, 17-OHP, Adione and T in the 391 serum samples were furthermore compared to results obtained using an existing LC-MS/MS method in our laboratory. Excellent agreement was found between the methods. Furthermore, the improved sensitivity of the new method allowed for quantification of a number of samples found to be below the LOQs of the existing method. Thus, the two instruments and their associated methods were validated as possible back-ups for each other, which we consider an extremely important issue in high-throughput laboratories analyzing clinical samples on a regular basis. The ten analytes included can be analyzed simultaneously but it is also possible only to include some of these analytes for specific diagnostic purposes which make the new method an extremely useful tool in the clinical laboratory.
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Compared to the previous method including ten steroid metabolites, the present method was improved by using an NH4F buffer with a weak basic MeOH buffer and a HESI probe in both positive and negative ionization mode, enabling inclusion of DHT, ALDO, DHEA, 21-DOC, 17-OHPreg and DOC.
“Compared to our previous method including ten steroid metabolites only [16], the present method was improved by using an NH4F buffer together with a weak basic MeOH buffer as mobile phases on the eluting column and by using a HESI probe operating in both positive and negative ionization mode. Thereby, it was possible to include mainly DHT, ALDO, DHEA, 21-DOC, 17-OHPreg and DOC.”
Steroid hormone levels in culture media were measured using a method established to quantify steroid metabolites in serum by isotype-dilution TurboFlow-LC-MS/MS as previously described.
“Steroid hormone levels were measured using a method established to quantify steroid metabolites in serum by isotype-dilution TurboFlow-LC-MS/MS as previously described (42).”
From Establishment of a novel human fetal adrenal culture model that supports de novo and manipulated steroidogenesis (2021) · cites this paper as [42]
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Self-citationMelau, C., Gayete-Mor, B., Riis, M. L., Nielsen, J. E., Dreisler, E., Aaboe, K., Brenøe, P. T., Thuesen, L. L., Hare, K. J., Mitchell, R. T., et al. (2023) · Frontiers in Endocrinology · doi:10.3389/fendo.2023.1114211 · Cited by 2 · see citing works
Self-citationFritzbøger, A. F. Ø., Aksglæde, L., Johannsen, T. H., Vanderschueren, D., Antonio, L., Claessens, F., Poulsen, K. H., Jørgensen, A., Petersen, J. H., Andersson, A., et al. (2025) · Clinical Endocrinology · doi:10.1111/cen.15249 · Cited by 1 · see citing works
Self-citationCharacterization of human adrenal steroidogenesis during fetal development
Melau, C., Nielsen, J. E., Frederiksen, H. W., Kilcoyne, K. R., Perlman, S., Lundvall, L., Thuesen, L. L., Hare, K. J., Andersson, A. M., Mitchell, R., et al. (2019) · Yearbook of pediatric endocrinology · doi:10.1530/ey.16.2.16
Self-citationSteroid determination—Purification of extracts
Honour, J. W. (2023) · Elsevier eBooks · doi:10.1016/b978-0-12-818124-9.00002-4
Quantitative analysis of steroids
Honour, J. W. (2023) · Elsevier eBooks · doi:10.1016/b978-0-12-818124-9.00010-3
Clausen, C. L., Johannsen, T. H., Skakkebæk, N. E., Frederiksen, H. W., Juul, A., & Benfield, T. (2024) · Endocrine Connections · doi:10.1530/ec-24-0093
Self-citationAbildgaard, J., Palani, N. P., Henriksen, T. I., Aleliunaite, A., Horváth, C., Svenstrup, V., Frederiksen, H. W., Juul, A., Scheele, C. C., Pedersen, B. K., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.01.21.634039
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734548
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734533
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26045562
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734554
Self-citationAdditional file 2 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.25566413
Self-citationMelau, C., Riis, M. L., Nielsen, J. E., Perlman, S., Lundvall, L., Thuesen, L. L., Hare, K. J., Hammerum, M. S., Mitchell, R. T., Frederiksen, H., et al. (2021) · Figshare · doi:10.6084/m9.figshare.16584675
Self-citationAdditional file 8 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703919
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734551
Self-citationAdditional file 4 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703907
Self-citationMelau, C., Riis, M. L., Nielsen, J. E., Perlman, S., Lundvall, L., Thuesen, L. L., Hare, K. J., Hammerum, M. S., Mitchell, R. T., Frederiksen, H., et al. (2021) · Figshare · doi:10.6084/m9.figshare.16584672
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734542
Self-citationAdditional file 3 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703904
Self-citationAdditional file 7 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703916
Self-citationMelau, C., Riis, M. L., Nielsen, J. E., Perlman, S., Lundvall, L., Thuesen, L. L., Hare, K. J., Hammerum, M. S., Mitchell, R. T., Frederiksen, H., et al. (2021) · Figshare · doi:10.6084/m9.figshare.16584678
Self-citationAdditional file 1 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.25566195
Self-citationAdditional file 5 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703910
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734527
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734530
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734539
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734524
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Thuesen, L. L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26045482
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734545
Self-citationLundgaard Riis, M., Delpouve, G., Nielsen, J. E., Melau, C., Langhoff Thuesen, L., Juul Hare, K., Dreisler, E., Aaboe, K., Tutein Brenøe, P., Albrethsen, J., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26734536
Self-citationAdditional file 6 of High incidence of imperforate vagina in ADGRA3-deficient mice
Kvam, J. M., Nybo, M. L., Torz, L., Sustarsic, R. K., Jensen, K. H. R., Nielsen, J. E., Frederiksen, H., Gadgaard, S., Spieß, K., Poulsen, S. S., et al. (2024) · Figshare · doi:10.6084/m9.figshare.26703913
Self-citationEffects of a single-dose denosumab on glucose and lipid homeostasis in young infertile men
Wriedt, E. B., Jorsal, M. J., Yahyavi, S. K., Mortensen, L. J., Eldrup, E., Juul, A., Holt, R., & Jensen, M. B. (2026) · Endocrine · doi:10.1007/s12020-026-04683-8
Self-citation
- Authors
- Søeborg, T., Frederiksen, H., Johannsen, T. H., Andersson, A.-M., & Juul, A.
- Affiliations
- University of Copenhagen (DK)
- Corresponding author
- Juul, Anders
- Venue
- Clinica Chimica Acta
- Publisher
- Elsevier BV
- Published
- 2017-05
- Volume
- 468
- Pages
- 180-186
- ISSN
- 0009-8981 (ISSN-L)
- OpenAlex
- W2594318512
- Cited by
- 83
- References
- 16
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 22 · 2023: 10 · 2022: 6 · 2021: 11 · 2020: 15 · 2019: 5 · 2018: 8 · 2017: 1
- FWCI
- 5.55
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Clinica Chimica Acta (publishedVersion)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion)
- Funders
- InterReg (F-22717-07, F-22717-26)
- Topics
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Keywords
- steroid metabolites, isotope dilution, simultaneous determination, serum, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, androstenedione, cortisol, corticosterone, 11-deoxycortisol, testosterone, estrone-3-sulfate, method validation, sample stability, pediatric reference values, method comparison
- MeSH terms
- Adolescent, Blood Chemical Analysis, Child, Chromatography, Liquid, Humans, Isotopes, Male, Steroids, Time Factors, Tandem Mass Spectrometry, Metabolomics
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