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

Clinical Chemistry, vol. 72, pp. 776–790

Abstract

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.

Authors 24

1. Nina M. Diederiks Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

2. Fred P H T M Romijn Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

3. Nicolaas J.M. van Neer Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

4. Mervin Pieterse Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

5. Julia Dittrich

   University Hospital Leipzig · Leipzig University

   Affiliation as printed

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig Medical Center Hospital Leipzig , Leipzig ,

6. Uta Ceglarek

   University Hospital Leipzig · Leipzig University

   Affiliation as printed

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig Medical Center Hospital Leipzig , Leipzig ,

7. Christa M. Cobbaert Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

8. L. Renee Ruhaak Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

9. the IFCC Working Group for Standardization of Apolipoproteins by Mass Spectrometry (WG APO-MS)
10. Uta Ceglarek

    University Hospital Leipzig · Leipzig University

    Affiliation as printed

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig Medical Center Hospital Leipzig , Leipzig ,

11. V Delatour
12. L Dittrich
13. C Giangrande
14. Andrew N. Hoofnagle
15. Z Kuklenyik
16. A Lyle
17. H W Vesper
18. K Naumann
19. N Veprek
20. Christa M. Cobbaert Aachen Department of Clinical Chemistry Laboratory Medicine

    Leiden University Medical Center

    Affiliation as printed

    Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center , Leiden ,

21. E Angles-Cano
22. G M Kostner
23. L Deprez
24. L Noussis