MSACL 2025 Abstract

Overcoming Matrix Challenges in Steroid Detection: Combining Derivatization and Selective Extraction Strategies with LC-MS/MS

Lekha Sleno, Nathan Ghafari, Sherin Nawaito, Catherine Mounier, Nicolas Pilon
University of Quebec in Montreal (UQAM)/CERMO-FC, Montreal, Canada

Lekha Sleno, PhD (Presenter) UQAM

Presenter Bio: I received my PhD in 2006 from Dalhousie University working with Prof. Dietrich Volmer on mass spectrometry methods for elucidating small molecule structures. I then went on to the University of Geneva and University or Toronto for two post-doc stays in pharmaceutical analytical mass spectrometry and proteomics. I started my position as a professor at the University of Quebec in Montreal in 2008 and am now full professor and co-director of our Centre for Excellence for Research on Orphan Diseases. My research group continues to develop and apply mass spectrometry based methods for metabolomics, proteomics, exposomics and drug metabolism applications.

Relevant Financial Disclosures (within past 24 months, reported on Jun 09, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Steroids are biologically active molecules derived from cholesterol that regulate key physiological processes, including metabolism, immune response, and reproduction. However, their analysis by LC-MS, particularly in complex matrices, presents several challenges. Due to their nonpolar structure, steroids are difficult to extract and analyze selectively, as they are often co-extracted with lipids and phospholipids, leading to high background interference and signal degradation. Additionally, poor ionization efficiency results in low sensitivity, and the structural similarities among many steroids pose significant challenges for chromatographic separation. These issues make steroid analysis in lipid-rich matrices particularly challenging and underexplored.

OBJECTIVES:
This project aims to overcome matrix challenges in liver and plasma samples for steroid analysis by combining multiple derivatization chemistries with selective extraction strategies prior to LC-MS/MS detection.

METHODS:
Liver and plasma samples from mice, along with standard steroid solutions, were used in this study. Initial metabolite extraction was performed using solvent-based protein precipitation, followed by different derivatization reactions (using Girad P and isonicotinoyl chloride). Both underivatized and derivatized extracts were subjected to solid-phase extraction protocols focusing on the depletion of lipids prior to derivatization. Samples were analyzed using a UHPLC quadrupole time-of-flight system, using a C18 column with an optimized gradient. For liver samples, a step of beconjugation of phase II metabolites was also incorporated. The final method was then transferred to into targeted LC-MRM assay for a clinically relevant workflow.

RESULTS:
Preliminary results using steroid standard solutions demonstrated significant improvement in both separation and detectability for two derivatization protocols. The Girard P (GP) reagent, which specifically targets carbonyl and aldehyde-containing steroids, contains permanently charged quaternary amine groups. This results in a significant increase in signal in positive mode, facilitating the detection of poorly ionized steroids and enhancing their separation. An increased selectively is afforded by this permanent charge on GP derivatives, thus removing much of the background in complex samples. The second derivatization with isonicotinoyl chloride (INC), targeting hydroxyl groups, improved chromatographic retention and detectability.

CONCLUSION:
We have developed an optimized workflow that enables increased detectability of steroids in both serum and liver samples following the detailed comparison of different derivatization strategies and extraction methods, including a phospholipid depletion step. Significant improvement of ionizability and separation of steroids has been achieved. This is especially notable in liver samples where matrix effects from lipids was seen to cause deterioration of chromatographic behavior and sensitivity after only a few injections prior to using this optimized procedure. This workflow will be presented in the context of investigating variations in metabolic disorders.