A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry

François Fraissinet; Tony Pereira; Alizée Violin; Guillaume Feugray; Kalyane Bach-Ngohou; Valéry Brunel

doi:10.1016/j.jmsacl.2022.12.004

A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry

J Mass Spectrom Adv Clin Lab. 2022 Dec 15:27:24-32. doi: 10.1016/j.jmsacl.2022.12.004. eCollection 2023 Jan.

Authors

François Fraissinet¹, Tony Pereira¹, Alizée Violin², Guillaume Feugray^{1

3}, Kalyane Bach-Ngohou^{2

4}, Valéry Brunel¹

Affiliations

¹ Department of General Biochemistry, Rouen University Hospital, 76000 Rouen, France.
² Nantes Université, CHU Nantes, Department of Biology, Laboratory of Biochemistry, F-44000 Nantes, France.
³ Normandie University, UNIROUEN, INSERM U1096 Rouen, France.
⁴ Nantes Université, CHU Nantes, INSERM, The Enteric Nervous System in Gut and Brain Disorders, IMAD, F-44000 Nantes, France.

Abstract

Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients.

Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase.

Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione.

Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

Keywords: 11DF, 11-deoxycortisol; 17OHP, 17-hydroxyprogesterone; 2D-UHPLC-MS/MS, Two dimensional ultra-high performance liquid chromatography-tandem mass spectrometry; Automation; D4, delta4-androstenedione; DHEA, dehydroepiandrosterone; EMA, European Medicine Agency; GC–MS/MS, Gas chromatography tandem mass spectrometry; LC-MS/MS, Liquid chromatography tandem mass spectrometry; LLE, Liquid-liquid extraction; LLOQ, Lower limit of quantification; Liquid chromatography tandem mass spectrometry; MRM, Multiple reaction monitoring; PTFE, Polytetrafluoroethylene; QC, Quality control; RIA, Radioimmunoassay; Radioimmunoassay; SLE, Supported liquid extraction; SPE, Solid phase extraction; SRM, Standard reference material; Steroids; T, Testosterone; Testosterone; UHPLC, Ultra-high performance liquid chromatography.