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Extended Steroid Profiling in Human Serum and Plasma With Simultaneous Quantitative Determination Using One-Point

Mathieu Galmiche1,2,3, Oriane Strassel1,2, Marie-Anaïs Monat1,2

  • 1School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland.

Journal of Separation Science
|April 23, 2025
PubMed
Summary
This summary is machine-generated.

This study presents an advanced LC-MS/MS method for comprehensive steroid profiling in plasma, enabling accurate quantification of 171 compounds. The method overcomes challenges in analyzing low-concentration steroids in biological samples.

Keywords:
absolute quantificationendogenous compoundsliquid chromatographymass spectrometrysteroids

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Area of Science:

  • Endocrinology
  • Analytical Chemistry
  • Biochemistry

Background:

  • Steroids are vital endogenous compounds with complex analysis challenges in biofluids due to low concentrations and isobaric interference.
  • Absolute quantification of steroids in blood is difficult because obtaining a blank matrix for external calibration is impossible.
  • Existing methods struggle with comprehensive profiling and accurate quantification of a wide range of steroids in biological samples.

Purpose of the Study:

  • To develop and validate a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for extended steroid profiling in serum and plasma.
  • To enable the absolute quantification of four key endogenous steroids: cortisol, testosterone, progesterone, and androstenedione.
  • To establish a comprehensive method capable of identifying and quantifying up to 171 steroid compounds.

Main Methods:

  • Sample preparation involved protein precipitation using organic solvents followed by hydrophilic-lipophilic balance (HLB) cartridge filtration.
  • Liquid chromatography (LC) separation utilized a biphenyl stationary phase to resolve isobaric steroid species.
  • Mass spectrometry (MS) detection employed multiple reaction monitoring (MRM) with post-column ammonium fluoride addition for enhanced sensitivity; a one-point internal calibration strategy was used for absolute quantification.

Main Results:

  • The method successfully identified 69 distinct endogenous steroids in the NIST Plasma Reference Material (SRM 1950), representing the most comprehensive profiling to date for this matrix.
  • Quantitative performance was assessed using two certified materials, demonstrating satisfactory precision and trueness for steroid measurements.
  • The developed LC-MS/MS protocol effectively addresses challenges associated with low endogenous concentrations and isobaric compounds in steroid analysis.

Conclusions:

  • The presented LC-MS/MS strategy provides a powerful tool for extensive steroid profiling and accurate absolute quantification in human plasma and serum.
  • This method significantly advances the capability for analyzing steroid metabolomes in complex biological matrices.
  • The protocol offers a reliable approach for researchers investigating steroid-related biological processes and diseases.