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Development of an LC-MS/MS Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a) in Plasma



Santica Marcovina, Noemie Clouet-Foraison, Marlys Koschinsky, Mark Lowenthal, Michael Boffa, Andrew Hoofnagle, Tomas Vaisar


Background: Use of Lipoprotein(a) levels for identification of individuals at high risk for cardiovascular disease is hampered by the high size polymorphism of apolipoprotein(a) which strongly impacts immunochemical methods resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. Method: A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a primary reference material constituted by a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned at NIST using an amino acid analysis reference method, directly traceable to the SI units through an unbroken traceability chain. Digestion completeness, repeatability, intermediate precision and parallelism were assessed. Comparability to the designated gold standard method for Lp(a) quantification, a monoclonal antibody-based ELISA, was evaluated. Results: The LC-MS/MS method was highly optimized and digestion completeness and stability of the quantification peptides were verified. Method intermediate precision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Excellent agreement was found between the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA (Deming regression, y=0.98*ELISA+3.18, n=64). Conclusions: We have developed a method for the absolute quantification of Lipoprotein(a) in plasma and demonstrated that it has the required characteristics of a higher order reference method to be used for assay standardization.
Clinical Chemistry


lipoprotein(a), apolipoprotein(a), LC-MS/MS, standardization, isotope dilution, cardiovascular disease


Marcovina, S. , Clouet-Foraison, N. , Koschinsky, M. , Lowenthal, M. , Boffa, M. , Hoofnagle, A. and Vaisar, T. (2021), Development of an LC-MS/MS Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a) in Plasma, Clinical Chemistry (Accessed July 18, 2024)


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Created April 1, 2021, Updated February 27, 2023