Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Background
Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride.
Methods
Following liquid–liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation.
Results
Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122–140 % for 3βOH-Δ5 steroids and 477–1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range −14 % to 11.9 % for 21/23, −21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from −29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured.
Conclusions
UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.
Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride.
Methods
Following liquid–liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation.
Results
Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122–140 % for 3βOH-Δ5 steroids and 477–1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range −14 % to 11.9 % for 21/23, −21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from −29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured.
Conclusions
UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.
Date Issued
2022-10-15
Date Acceptance
2022-08-06
Citation
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2022, 1209
ISSN
1570-0232
Publisher
Elsevier
Journal / Book Title
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume
1209
Copyright Statement
© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/35988498
PII: S1570-0232(22)00317-8
Subjects
11-oxygenated androgens
Androgens
Biochemical Research Methods
Biochemistry & Molecular Biology
Chemistry
Chemistry, Analytical
DIRECT ASSAY
Glucocorticoids
HUMAN SERUM
LC-MS/MS
Life Sciences & Biomedicine
Liquid chromatography-tandem mass spec trometry
METABOLIC PHENOTYPE
Mineralocorticoids
PERFORMANCE LIQUID-CHROMATOGRAPHY
Physical Sciences
QUANTIFICATION
ROUTINE MEASUREMENT
Science & Technology
Steroid quantification
TANDEM MASS-SPECTROMETRY
TESTOSTERONE
URINE
Publication Status
Published
Coverage Spatial
Netherlands
Article Number
ARTN 123413
Date Publish Online
2022-08-09