A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry

Scott G Denham; Joanna P Simpson; Federico Diez; Patricia Lee; Catriona Kyle; Ruth Morgan; Natalie Zm Homer

doi:10.1016/j.mex.2024.102728

A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry

MethodsX. 2024 May 4:12:102728. doi: 10.1016/j.mex.2024.102728. eCollection 2024 Jun.

Authors

Scott G Denham¹, Joanna P Simpson¹, Federico Diez^{2

3}, Patricia Lee¹, Catriona Kyle⁴, Ruth Morgan^{4

3}, Natalie Zm Homer^{1

4}

Affiliations

¹ Mass Spectrometry Core, Edinburgh Clinical Research Facility, Centre for Cardiovascular Sciences, University of Edinburgh, Edinburgh, UK.
² Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK.
³ SRUC, Roslin Institute Building, Easter Bush Campus, EH25 9RG, UK.
⁴ BHF/University Centre for Cardiovascular Sciences, Queen's Medical Research Institute, 47 Little France Crescent, University of Edinburgh, Edinburgh, EH16 4TJ, UK.

Abstract

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17β-estradiol and estriol). •SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

Keywords: Automation; Chromatography; Comparative endocrinology; Mass spectrometry; Steroid profiling; Supported liquid extraction and LC-MS/MS analysis of multiple steroids in 200 µL plasma; Targeted.