An isotopologue-mixture-based internal standard strategy for LC-MS/MS quantification in plasma.

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Biomolecular Analysis

Background:

• Stable isotope-labeled internal standards (SIL-IS) are essential for precise LC-MS/MS quantification in complex biological samples.
• The high cost and limited commercial availability of SIL-IS hinder their widespread application for many bioactive analytes.

Purpose of the Study:

• To develop and validate a novel internal standard strategy using hydrogen-deuterium exchange (HDX)-derived deuterated isotopologue mixtures.
• To establish a framework for selecting and validating these mixtures for LC-MS/MS quantification.

Main Methods:

• Deuterated isotopologue mixtures (d₁-dₙ) were prepared for eight diverse bioactive compounds via HDX using Pd/C, Pt/C, and AlCl₃ catalysts in deuterated methanol.
• Isotopologues with Δm ≥ 3 were selected, and their co-elution and chromatographic isotope effects (CIE) were evaluated.
• LC-MS/MS assays were validated in plasma, assessing linearity, LOD, LOQ, trueness, precision, and matrix effects.

Main Results:

• HDX-derived d₃-dₙ isotopologues co-eluted with analytes (Δt < 0.01 min) with no significant CIE.
• Validated LC-MS/MS assays demonstrated excellent linearity (R² ≥ 0.995), low LOD/LOQ, acceptable trueness (85-115%), and good precision (RSD ≤ 12%).
• The HDX-derived internal standard performed comparably to commercial SIL-IS, reducing quantitative deviation by 15-25% compared to external calibration.

Conclusions:

• HDX-derived isotopologue mixtures offer a practical and effective alternative to commercial SIL-IS.
• This approach significantly improves quantitative accuracy in LC-MS/MS analysis, especially when commercial standards are unavailable.
• The established selection-and-validation framework supports the routine use of HDX-derived standards.