Quantitative charge-tags for sterol and oxysterol analysis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Metabolomics

Background:

• Global sterol analysis is complex due to sterol diversity, cholesterol dominance, and lack of strong chromophores.
• Existing methods face challenges in quantifying a wide range of sterols and oxysterols simultaneously.
• A novel approach is needed to overcome these analytical hurdles in sterol profiling.

Purpose of the Study:

• To develop and validate a quantitative method for analyzing a broad spectrum of sterols and oxysterols.
• To utilize isotope-labeled Girard P (GP) reagents as charge-tags for enhanced LC-MS analysis.
• To enable simultaneous profiling of sterols with different functional groups (3-hydroxy and 3-oxo).

Main Methods:

• Plasma sterols/oxysterols were extracted and internal standards were added.
• Separation was achieved using C18 solid-phase extraction.
• Derivatization with isotope-labeled Girard P (GP) reagents, with optional oxidation, followed by LC-MS analysis.

Main Results:

• A single LC-MS run could analyze both 3-oxo and 3β-hydroxy sterols/oxysterols.
• High accuracy and precision were achieved with intra- and interassay coefficients of variation <15% and recoveries of 85%-108%.
• The multiplex approach allowed analysis of up to 4 samples per run, demonstrating diagnostic capabilities for inborn errors of metabolism and oxysterol export.

Conclusions:

• The developed method provides the widest range of sterol/oxysterol profiling in a single analytical run.
• This technique is valuable for identifying inborn errors of cholesterol synthesis and metabolism.
• The method offers a significant advancement in quantitative sterol and oxysterol analysis.