Development and Analytical Validation of a Multiplex LC-MRM-MS-Based Reference Measurement Procedure for...

Area of Science:

• Clinical Chemistry
• Mass Spectrometry
• Biomarker Standardization

Background:

• Accurate medical results from in vitro diagnostic devices are crucial.
• Previous reference measurement procedures for ApoA-I/ApoBtotal are no longer functional.
• A new reference measurement procedure is needed for standardization.

Purpose of the Study:

• To develop and validate a new reference measurement procedure (RMP) for apolipoprotein A-I (ApoA-I) and apolipoprotein B total (ApoBtotal).
• To ensure accurate quantification and standardization of ApoA-I and ApoBtotal in serum.
• To align with ISO 15193:2009 and ISO 17511:2020 calibration models.

Main Methods:

• Developed a multiplex liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) method.
• Serum samples were processed via reduction, alkylation, digestion, and peptide purification.
• Calibrators were indirectly traceable to WHO/IFCC standards with propagated measurement uncertainty.

Main Results:

• Demonstrated linearity and broad analytical suitability for ApoA-I and ApoBtotal quantification.
• Achieved low measurement uncertainty (2.3%) and high recovery (99.2%-100.3%).
• Established limits of blank, detection, and quantification, and showed data equivalence with existing assays.

Conclusions:

• The LC-MRM-MS based RMP is a next-generation method for ApoA-I and ApoBtotal standardization.
• Certification of commercial tests using this RMP will enable continued global standardization.
• Traceability to former WHO-IFCC reference materials is preserved within maximum allowable uncertainty.