UPLC-Orbitrap-HRMS application for analysis of plasma sterols

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Sterol biomarkers are crucial for diagnosing inherited metabolic disorders (IMDs).
• Current methods often lack the required selectivity and sensitivity for accurate sterol quantification.
• Developing precise assays is essential for improving IMD diagnosis.

Purpose of the Study:

• To develop and validate sensitive and selective assays for quantifying key sterol biomarkers.
• To establish robust analytical methods for cholesterol biosynthesis intermediates, phytosterols, and oxysterols.
• To demonstrate the diagnostic utility of these assays in patients with IMDs.

Main Methods:

• Quantification of six cholesterol biosynthesis intermediates, two phytosterols, and two oxysterols using UPLC-Orbitrap HRMS.
• Sterol derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) for specific cholesterol intermediates.
• Optimization of UPLC and MS parameters for enhanced sensitivity and selectivity, utilizing stable isotope-labeled analogs for accurate quantification.

Main Results:

• Developed two highly sensitive and selective assays for a comprehensive panel of sterol biomarkers.
• Achieved excellent analytical performance, including low limits of quantification (0.027 nM–50.5 nM) and high linearity (R² > 0.997).
• Demonstrated the diagnostic potential of the assays in a cohort of 31 patients with IMDs affecting cholesterol homeostasis.

Conclusions:

• The developed UPLC-HRMS assays provide accurate and reliable quantification of sterol biomarkers.
• These assays represent a significant advancement for the first-line diagnosis of IMDs related to cholesterol metabolism.
• The improved diagnostic capabilities can lead to earlier and more effective patient management.