Development and Analytical Validation of a Multiplex LC-MRM-MS–Based Reference Measurement Procedure for Apolipoprotein A-I and Btotal Quantification in Serum

BACKGROUND: Medical results from regulated in vitro diagnostic devices should be accurate. The previous reference measurement procedures (RMP) for apolipoprotein A-I (ApoA-I)/apolipoprotein B total (ApoBtotal) standardization are no longer functional. A new RMP aligned with ISO15193:2009 and calibration models of ISO17511:2020 was developed using multiplex liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) for quantifying ApoA-I and ApoBtotal in serum. METHODS: Serum samples underwent reduction, alkylation, lysyl endopeptidase/trypsin digestion, and proteotypic peptide purification. Optimal retention times, precursor/fragment ion m/z were selected using an Agilent-1290 ultra-high-performance liquid chromatography coupled to a 6495C triple-quadrupole mass spectrometer. Calibrators were indirectly traceable to WHO/IFCC SP1-01/SP3-08 (ApoA-I/ApoBtotal) via the RMP, with propagated measurement uncertainty (MU), supported by replicate SP1-01/SP3-08 measurements across independent batches. Validation followed ISO15193:2009 and included linearity, recovery, uncertainty, precision, stability, preanalytical conditions, carryover, limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), and comparability. RESULTS: Linearity was confirmed from 0.10 to 2.97 g/L and 0.27 to 3.83 g/L (ApoA-I/ApoBtotal), with <10% deviation, supporting broad analytical suitability. Duplicate MU was 2.3% for both analytes. Recovery ranged from 99.2% to 100.3%. Matrix effects were negligible except in citrate plasma (-14.1% to -15.7%). Carryover was <0.003 g/L (ApoA-I) and <0.004 g/L (ApoBtotal). LoB, LoD, and LoQ were <0.001/0.002 g/L, 0.0013/0.0019 g/L, and 0.01/0.02 g/L for ApoA-I/ApoBtotal, respectively. Compared with Roche immunoturbidimetric assays, data equivalence was demonstrated within allowable MU. CONCLUSIONS: This LC-MRM-MS-based RMP for ApoA-I and ApoBtotal provides a next-generation RMP succeeding the former ELISA-based RMP. Certification of commercially available ApoA-I/ApoBtotal tests will enable continued global standardization of ApoA-I/ApoBtotal measurements, while preserving traceability of test results to the former WHO-IFCC reference materials within maximum allowable uncertainty.