Simultaneous analysis by LC–MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue
Open accessAbstract
This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 μl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11β-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11β-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.
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Several sensitive and robust LC-MS/MS methods for multiple steroids have been developed in recent years and many methods meet the requirements for clinical applications.
“Several sensitive and robust LC-MS/MS methods for multiple steroids have been developed in recent years and many methods meet the re quirements for clinical applications [5 -16].”
From Sex- and age-specific reference intervals of 16 steroid metabolites quantified simultaneously by LC-MS/MS in sera from 2458 healthy subjects aged 0 to 77 years (2024) · cites this paper as [9]
The Introduction describes published LC-MS methods for measuring multiple steroids, including sensitive methods, and the Conclusions describe this paper’s validated method as usable in clinical trials. The paper does not establish that many of the published methods are robust or meet requirements for clinical applications; it notes that some have higher limits of quantification. (LLM, conf=0.83)
Experimental — accuracy not yet measuredPreviously published methods including multiple steroid metabolites used larger sample volumes (150-500 μL) and/or longer total run-times (13.5-17 min).
“Previously published methods including multiple steroid metabolites used either larger sample volumes from 150-500 µ L and/or longer total rum-times from 13.5-17 min. [5,7,9,14,22].”
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- Authors
- Häkkinen, M. R., Murtola, T., Voutilainen, R., Poutanen, M., Linnanen, T., Koskivuori, J., Lakka, T., Jääskeläinen, J., & Auriola, S.
- Affiliations
- University of Eastern Finland (FI)
- Tampere University Hospital (FI)
- University of Turku (FI)
- Corresponding author
- Häkkinen, Merja R.
- ORCID
- Venue
- Journal of Pharmaceutical and Biomedical Analysis
- Publisher
- Elsevier BV
- Published
- 2019-02
- Volume
- 164
- Pages
- 642-652
- ISSN
- 0731-7085 (ISSN-L)
- OpenAlex
- W2901588957
- Cited by
- 74
- References
- 34
- Citations by year
- 2026: 5 · 2025: 11 · 2024: 8 · 2023: 6 · 2022: 13 · 2021: 18 · 2020: 10 · 2019: 3
- FWCI
- 4.83
- Type
- journal article
- Language
- en
- Open access
- green
- erepo.uef.fi
- Licence
- cc-by-nc-nd
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- Locations
- Journal of Pharmaceutical and Biomedical Analysis (publishedVersion)
- PubMed (publishedVersion)
- UEF eRepo (University of Eastern Finland) (submittedVersion, cc-by-nc-nd)
- Funders
- Kuopion Yliopistollinen Sairaala
- Biocenter Finland
- Topics
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Physical Sciences › Chemistry › Spectroscopy › Analytical Chemistry and Chromatography
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
- Keywords
- ketosteroids, androgens, estrogen, progestins, human plasma, serum, prostate tissue, isotope-labeled internal standards, testosterone, dehydroepiandrosterone, dihydrotestosterone, estrone, pregnenolone
- MeSH terms
- Chromatography, High Pressure Liquid, Estradiol, Humans, Hydroxylamines, Isotope Labeling, Ketosteroids, Male, Prostate, Tandem Mass Spectrometry
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