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Comprehensive Quantitative Urinary Steroid Profiling of 29 Steroids Using Liquid Chromatography-Tandem Mass

Joshua T Bain1, Fozia Shaheen1, Alessandro Prete1,2

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Summary

This study developed a faster liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying urinary steroids, improving endocrine disorder diagnosis. The new method offers high throughput and comprehensive steroid metabolism analysis.

Keywords:
LC‐MS/MSandrogensglucocorticoidsmineralocorticoidssteroid precursorssteroid profilingurine

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Area of Science:

  • Endocrinology
  • Analytical Chemistry
  • Biochemistry

Background:

  • Steroid metabolism is crucial for physiological processes and endocrine health.
  • Disruptions in steroid metabolism are linked to various endocrine disorders.
  • Accurate steroid quantification is vital for understanding and diagnosing these conditions.

Purpose of the Study:

  • To translate a gas chromatography-mass spectrometry (GC-MS) urinary steroid profile to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform.
  • To establish a validated, high-throughput method for comprehensive steroid metabolism analysis.
  • To enhance the utility of steroid profiling for clinical and research applications.

Main Methods:

  • Urinary steroids were enzymatically hydrolyzed and extracted using C18 solid-phase extraction.
  • Quantification was performed using a triple quadrupole mass spectrometer with a 16-minute chromatographic separation.
  • A dual-column approach (C18-T3 and BEH-C18) was employed to resolve co-eluting analytes.

Main Results:

  • The LC-MS/MS method demonstrated good accuracy (bias -18.7% to 19.9%) and precision (CV 4.0% to 18.6%).
  • Most analytes were quantifiable in over 95% of samples from healthy volunteers.
  • Significant diurnal and sex-based variations in steroid excretion were observed.

Conclusions:

  • The developed LC-MS/MS method provides a robust and high-throughput solution for simultaneous quantification of multiple steroid classes.
  • This method offers improved sample preparation times and increased throughput compared to traditional GC-MS.
  • The enhanced steroid profiling facilitates advancements in clinical diagnostics and research within endocrine science.