Quantitative Analysis of Recombinant N-terminal Pro-brain Natriuretic Peptide by Liquid Chromatography Coupled with Isotope Dilution Mass Spectrometry

N-terminal pro-brain natriuretic peptide (NT-proBNP) is an essential biomarker for diagnosis of heart failure. Currently, since reference materials of NT-proBNP are unavailable globally, value transfer activities are hard to implement from the higher level to the end-users, which makes terminal clinical testing results inconsistent and incomparable. To achieve standard detection of NT-proBNP, in this work, recombinant NT-proBNP was chosen as a target during traceable approach development. Firstly, the immune capacity and relative molecular weight were tested and varied through Western Blot assay and mass spectrometric methods. Next, two kinds of isotope dilution mass spectrometry were adopted for the absolute quantification of NT-proBNP. The results suggested that the protein hydrolysis reaction reached equilibrium in 48 h. NT-proBNP was calculated to be 79. 62 mu g/ g with an relative standard deviation (RSD) of 0. 89% by detection of valine, isoleucine and arginine. On the other hand, the isotope dilution mass spectrometry method was used to analyze signature peptides produced by enzymatic digestion. NT-proBNP was calculated to be 76. 04 mu g/ g with an RSD of 1. 85% by detection of the two peptides under the conditions of enzymatic digestion for 24 h and at mass ratio of 25:1 of NT-proBNP to protease. The quantitative analysis results from the two methods were consistent with each other. As the amino acids applied in quantifications were SI-traceable primary reference materials, all the results were also traceable. Such approaches were candidates for NT-proBNP reference material development.