Optimisation and Validation of a UHPLC-MS/MS Assay Using Timed Post-Column Ammonium Fluoride Infusion for Sensitive Serum Steroids Quantitation
- 1MRC Laboratory of Medical Sciences
Abstract
Steroid metabolomics has potential for identifying biomarkers and improving disease diagnosis. However, poor ionisation efficiency and low circulating concentrations of some steroids limit the broader clinical applications. Here, we developed a ultra-high-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) assay for quantitation of 18 steroids in serum encompassing the mineralocorticoid, glucocorticoid and androgen biosynthesis pathways, including both classic and 11-oxygenated androgens. Steroid extraction from 100 L of serum was performed using supported liquid extraction with ethyl acetate elution. Ionisation efficiency of steroids was investigated using formic acid in the mobile phase and post-column infusion of ammonium fluoride. Ammonium fluoride enhanced the signal intensity of 3-keto-{Delta}4 steroids up to ninefold, however A-ring reduced steroids exhibited greater ionisation with formic acid. Thus, to optimise sensitivity, a timed post-column infusion of 2 mmol/L ammonium fluoride was employed during chromatographic separation. The lower limits of quantitation ranged from 0.04 to 2.1 nmol/L. Accuracy ranged from 82 to 113% and intra- and inter-assay imprecision was <10% and <13%, respectively. The assay was applied to analyse morning serum steroid concentrations in healthy individuals (55 women, 44 men; age-matched). Serum progesterone, 11-deoxycorticosterone, dehydroepiandrosterone and androstenedione were significantly higher in women; in contrast, testosterone and 5-dihydrotestosterone were higher in men. In summary, we have successfully developed and validated a UHPLC-MS/MS assay for multi-steroid profiling ad applied it to healthy individuals of both sexes.
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BibTeX
@misc{dosedelova2026optimisation,
title = {{Optimisation and Validation of a UHPLC-MS/MS Assay Using Timed Post-Column Ammonium Fluoride Infusion for Sensitive Serum Steroids Quantitation}},
author = {V. Dosedelova and J. M. Hawley and E. Melson and L. Schiffer and K.-H. Storbeck and W. Arlt},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.27.762247},
url = {https://www.biorxiv.org/content/10.64898/2026.10.27.762247v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Optimisation and Validation of a UHPLC-MS/MS Assay Using Timed Post-Column Ammonium Fluoride Infusion for Sensitive Serum Steroids Quantitation
AU - V. Dosedelova
AU - J. M. Hawley
AU - E. Melson
AU - L. Schiffer
AU - K.-H. Storbeck
AU - W. Arlt
PY - 2026
DA - 2026/10/27
PB - bioRxiv
DO - 10.64898/2026.10.27.762247
UR - https://www.biorxiv.org/content/10.64898/2026.10.27.762247v1
AB - Steroid metabolomics has potential for identifying biomarkers and improving disease diagnosis. However, poor ionisation efficiency and low circulating concentrations of some steroids limit the broader clinical applications. Here, we developed a ultra-high-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) assay for quantitation of 18 steroids in serum encompassing the mineralocorticoid, glucocorticoid and androgen biosynthesis pathways, including both classic and 11-oxygenated androgens. Steroid extraction from 100 L of serum was performed using supported liquid extraction with ethyl acetate elution. Ionisation efficiency of steroids was investigated using formic acid in the mobile phase and post-column infusion of ammonium fluoride. Ammonium fluoride enhanced the signal intensity of 3-keto-{Delta}4 steroids up to ninefold, however A-ring reduced steroids exhibited greater ionisation with formic acid. Thus, to optimise sensitivity, a timed post-column infusion of 2 mmol/L ammonium fluoride was employed during chromatographic separation. The lower limits of quantitation ranged from 0.04 to 2.1 nmol/L. Accuracy ranged from 82 to 113% and intra- and inter-assay imprecision was <10% and <13%, respectively. The assay was applied to analyse morning serum steroid concentrations in healthy individuals (55 women, 44 men; age-matched). Serum progesterone, 11-deoxycorticosterone, dehydroepiandrosterone and androstenedione were significantly higher in women; in contrast, testosterone and 5-dihydrotestosterone were higher in men. In summary, we have successfully developed and validated a UHPLC-MS/MS assay for multi-steroid profiling ad applied it to healthy individuals of both sexes.
ER -