Comprehensive targeted profiling of multiple steroid classes in rodent plasma using liquid chromatography-mass...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Endocrinology

Background:

• Simultaneous quantification of multiple steroids in biological fluids is analytically challenging due to issues like isomer crosstalk and matrix interference.
• Existing methods struggle to profile a wide range of steroids concurrently, limiting comprehensive analysis.

Purpose of the Study:

• To develop a derivatization-free, high-throughput, and cost-effective UHPLC-MS/MS method for simultaneous profiling of 38 steroids.
• To address analytical hurdles including isomer separation and matrix effects in steroid quantification.
• To validate the method according to ICH/EMA M10 guidelines using a novel surrogate matrix approach.

Main Methods:

• Developed a derivatization-free, achiral UHPLC-MS/MS approach for steroid analysis.
• Optimized protein precipitation (PP) and supported liquid extraction (SLE) methods using small sample volumes (40 μL).
• Implemented a novel surrogate matrix-background subtraction technique using adrenalectomized rat plasma.

Main Results:

• Successfully resolved 16 stereoisomers and 15 positional isomers within a 20-min run, profiling 38 steroids.
• Achieved low limits of quantification with PP and SLE, though some steroids were only qualitatively assessed.
• Validated the UHPLC-MS/MS workflow with the surrogate matrix approach, demonstrating its effectiveness in over 500 mouse plasma samples.

Conclusions:

• Accurate steroid quantification is often underestimated, compromising research and clinical applications.
• The comprehensive, validated UHPLC-MS/MS method mitigates steroid crosstalk and uses minimal sample volume.
• The novel surrogate matrix approach significantly advances steroid analysis for diverse biological applications.