Liquid chromatography/tandem mass spectrometry assay for the absolute quantification of the expected circulating...

Apelin peptides are of great interest owing to their involvement in physiological and pathological processes and they have been proposed as novel biomarkers for heart failure. The plasma concentrations of bioactive peptides of 12 (apelin-12), 13 (apelin-13) and pyroglutamyl apelin-13 (apelin-p13), 17 (apelin-17) and 36 (apelin-36) amino acids are reported to range from 20 to 4000 pg/mL in healthy subjects. As standard immunoassays cannot specifically quantify each apelin peptide, we have developed a sensitive and targeted multiplexed liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for each plasma apelin fragment. The approach was based on a cation-exchange extraction step of apelin forms present in human plasma. Apelin-12, -13, -p13, -17 and -36 were quantified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Stable isotope-labeled internal standards were used for quantification. Following assay validation, apelin peptide stability in plasma was investigated. Ten plasma samples from healthy donors were analyzed both with a standard immunoassay and with our LC/MS/MS method. The immunoassay results for the ten healthy donors showed immunoreactive plasma apelin concentrations ranging from 208 to 466 pg/mL. The lower limits of detection of our LC/MS/MS assay ranged from 10 to 50 pg/mL for apelin-12, -13, -p13, -17, and -36. Surprisingly, none of the five expected circulating forms of apelin was detected. These results question the nature and/or the concentration of circulating apelin peptides as well as the specificity of the immunoassays that have hitherto been used for clinical applications.