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

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.