MRM-based quantification of plasma Apolipoprotein A-I and B100 to help in identifying coronary-artery disease

Abstract

Background: Apolipoprotein A-I(ApoA-I) constitutes the major component of high-density lipoprotein(HDL) and Apolipoprotein B-100(ApoB-100) is the major protein constituent of low-density lipoprotein(LDL) and also constituents about 40 percent of the protein moiety of very low density lipoprotein(VLDL) and chylomicrons.The Apo B/ApoA-I ratio is a strong predictor of cardiovascular disease risk. Traditionally,ApoA-I and Apo B-100 are quantified using immunoassays,which may suffer from problems such as crossreactivity.A quantitative LC-MS/MS method for specific and simultaneous measurement of Apo A-I and B100 has been developed for assessment of cardiovascular risk. Methods: Apolipoprotein calibrators from Randox Laboratories were used for external calibration with Apo A-I and B100 concentrations traceable to WHO international reference materials.The tryptic digested peptides were analyzed by LC-MS/MS. The quantifying peptide ATEHLSTLSEK for ApoA-I and FPEVDVLTK for ApoB-100 was selected based on CPTAC assay portal. Synthetic Isotope labeled peptides (SIS) were used as internal standard. The LC-MS/MS results were compared to those from Nephelometric assay. Results: Our assay showed a linear range of 0.12-2.41g/L for ApoA-I and 0.12-2.31 g/L for ApoB-100 with Rsquare greater than 0.99.The LOQ is 0.12 g/L of ApoA-I and ApoB-100 in plasma.The Intra- and inter-assay coefficients of variation were less than 10%. The LC-MS/MS results of plasma ApoA-I and ApoB-100 correlated well with those from the existing Nephelometric method (Beckman IMMAGE). The Passing&Bablok(95%CI) slope is 1.03 for ApoA-I and 0.98 for ApoB-100 for 60 plasma samples.The Pearson correlation coefficient was r=0.97 and 0.98 for ApoA-I and ApoB-100.24 external quality control samples from CAP EQA program were analyzed ApoA-I and ApoB-100 level and the measured values were within +/-2.0 z-score from the peer mean. Conclusions: A LC-MS/MS method was developed for the accurate quantitation of ApoA-I and B100 aiding to identify cardiovascular disease.1.Irene VD Broek,Fred P.H.T.M. Romijn,and Christa M.Cobbaert, et al, Clin Chem,2016,62,188-197;2.Michel R. Langlois, et al, Clin Chem,2018,64,1006-1033;3.Michael E. Lassman, et al, Rapid Commun.Mass Spectrom.2012,26,101–108