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