Presenter Bio: Clinical biochemist with a background in LC-MS-based metabolomics and CMC biopharma R&D
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction
The analysis of steroid hormones is an important part of the diagnostic workup of many conditions caused by disorders in the hypothalamic-pituitary-adrenal (HPA) axis and gonads. Examples include Cushing’s syndrome characterized by cortisol excess, and congenital adrenal hyperplasia resulting from enzyme deficiencies leading to altered production of glucocorticoids, mineralocorticoids and sex steroids, female hyperandrogenism and male hypogonadism.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive methodology suited for rapid analysis of multiple analytes in a clinical laboratory. The simultaneous detection of multiple steroid hormones in one sample has the advantage of increased precision and cost-efficiency.
Objective
To develop an LC-MS/MS multi-assay for the quantification of seven endogenous steroid hormones and precursors in serum: 17α-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone, cortisol, cortisone and 11-deoxycortisol, for use in our routine clinical laboratory.
Method
Automated sample preparation was performed using a robotic liquid-handling system (Hamilton). Starting with 200 µl of serum, samples were mixed with deuterated internal standards followed by hydrophilic-lipophilic solid phase extraction (Oasis HLB, Waters). The steroids in a 20 µl aliquot of eluate were then separated by reversed phase ultra-high performance LC (Acquity UPLC HSS T3 column, Waters) and detected by positive mode MS (TQS, Waters) with a total runtime of 20 min per sample.
Results
The limit of quantification was in the low nmol-range or below for all seven compounds, with acceptable linearity (R2 > 0.99) within their respective measuring range, which included each reference range. The total coefficient of variation of the assay was < 7%.
Conclusion
Our newly developed LC-MS/MS-based multi-assay of seven steroid hormones and precursors in serum will aid in the diagnostic process of disorders of the HPA axis and gonads. It also has the potential to be of use in patient follow-up care.
= Discovery stage.
= Translation stage.
= Clinically available.
