Targeted metabolic profiling of urinary steroids with a focus on analytical accuracy and sample stability

Nora Vogg; Tobias Müller; Andreas Floren; Thomas Dandekar; Oliver Scherf-Clavel; Martin Fassnacht; Matthias Kroiss; Max Kurlbaum

doi:10.1016/j.jmsacl.2022.07.006

Targeted metabolic profiling of urinary steroids with a focus on analytical accuracy and sample stability

J Mass Spectrom Adv Clin Lab. 2022 Jul 25:25:44-52. doi: 10.1016/j.jmsacl.2022.07.006. eCollection 2022 Aug.

Authors

Nora Vogg^{1

2}, Tobias Müller³, Andreas Floren³, Thomas Dandekar³, Oliver Scherf-Clavel⁴, Martin Fassnacht¹, Matthias Kroiss^{1

5}, Max Kurlbaum^{1

2}

Affiliations

¹ Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital Würzburg, Germany.
² Central Laboratory, Core Unit Clinical Mass Spectrometry, University Hospital Würzburg, Germany.
³ Department of Bioinformatics, Biocenter, Am Hubland, University of Würzburg, Germany.
⁴ Institute for Pharmacy and Food Chemistry, Am Hubland, University of Würzburg, Germany.
⁵ Department of Internal Medicine IV, University Hospital Munich, Ludwig-Maximilians-Universität München, Munich, Germany.

Abstract

Introduction: Preoperative diagnostic workup of adrenal tumors is based on imaging and hormone analyses, but charged with uncertainties. Steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 24-h urine has shown potential to discriminate benign and malignant adrenal tumors. Our aim was to develop and validate a specific and accurate LC-MS/MS method for the quantification of deconjugated urinary marker steroids, to evaluate their pre-analytical stability and to apply the method to clinical samples of patients with adrenal tumors.

Methods: A method for the quantification of 11 deconjugated steroids (5-pregnenetriol, dehydroepiandrosterone, cortisone, cortisol, α-cortolone, tetrahydro-11-deoxycortisol, etiocholanolone, pregnenolone, pregnanetriol, pregnanediol, and 5-pregnenediol) in human urine was developed and validated based on international guidelines. Steroids were enzymatically deconjugated and extracted by solid phase extraction before LC-MS/MS quantification in positive electrospray ionization mode.

Results: Excellent linearity with R² > 0.99 and intra- and inter-day precisions of < 10.1 % were found. Relative matrix effects were between 96.4 % and 101.6 % and relative recovery was between 98.2 % and 115.0 %. Sufficient pre-freeze stability for all steroids in urine was found at 20-25 °C for seven days and at 4-6 °C for up to 28 days. Samples were stable during long-term storage at -20 °C and -80 °C for 6 months.

Conclusions: A sensitive and robust LC-MS/MS method for the quantification of 11 urinary steroids was developed and validated according to international guidelines. Pre-analytical matrix stability was evaluated and the suitability of the method for the analysis of clinical samples and prospective validation studies was shown.

Keywords: 5-PD, 5-pregnen-3β,20α-diol; 5-PT, 5-pregnen-3β,17,20α-triol; ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; Adrenal tumors; Adrenocortical carcinoma; DHEA, dehydroepiandrosterone; Etio, etiocholanolone; IS, internal standard; LC-MS/MS, liquid chromatography tandem mass spectrometry; LC–MS/MS; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; Mass spectrometry; PD, 5β-pregnan-3α,20α-diol; PT, 5β-pregnan-3α,17,20α-triol; QC, quality control; R2, coefficient of determination; SPE, solid phase extraction; Steroid profiling; ULOQ, upper limit of quantification; Urinary steroids.