A-012 Novel Method for Determining the Concentration of Total N-terminal Pro-hormone BNP (NT-proBNP) in a Sample with Mass Spectrometric Detection
Authors: Jaquemar M , Knüttel T , Thi Tran L , Weikum J , Schmelck V , Nilsen T , David A 
Published: September 2023

Abstract

Background
NT-proBNP, the N-terminal fragment of the hormone B-type natriuretic peptide (BNP), is a useful clinical biomarker for diagnostic and prognostic evaluation of heart failure (HF). Recent studies show that commercially available NT-proBNP assays underestimate concentrations of NT-proBNP due to the usage of antibodies targeting the glycosylated central region of the NT-proBNP molecule. With this, a clinical need arises to establish a robust and reliable assay method which can measure the total amount of NT-proBNP, independent of the glycosylation status of the molecule.

Methods
To determine the concentration of NT-proBNP in patient samples, a known quantity of labelled NT-pro-BNP was added to each sample as standard. The capture of the analyte (native and standard) was performed by Dynabeads™ M-270 Epoxy beads coated with polyclonal goat antibodies, targeting non-glycosylated sites of the NT-proBNP molecule. Magnetic beads were added to the sample and incubated overnight. The isolated analytes were trypsinated directly on the beads after treatment for denaturation by unfolding and oxidation/alkylation. The reaction was stopped by addition of formic acid and the tryptic peptides were cleaned on C18 SPE columns, dried by SpeedVac and resuspended in acetonitrile for separation and analysis on LC-MS/MS (Agilent 1200 Series LC/MS system coupled to an Agilent G6490 triple quadrupole mass spectrometer).

Results
The Gentian LC-MS/MS validation was conducted including five studies such as Linearity, LoQ, Trueness, Precision and Carry-Over. All studies passed our defined acceptance criteria. To elucidate how glycosylation influences measurements of NT-proBNP, 81 plasma and serum samples covering a concentration range from 100 ng/L to 25 000 ng/L were measured by the Gentian LC-MS/MS method and the Roche Elecsys® proBNP I assay. Within the clinically relevant range of 100–2000 ng/L, the results between both methods exhibit a linear correlation of R2 = 0.97 and show that the commercial assay consistently underestimates the concentration of NT-proBNP by an average factor of 4.3 (3.5–5.8). Furthermore, results reveal that the concentration measurements between the commercial assay and the LC-MS/MS method converge at higher NT-proBNP concentrations. Method comparison was repeated using the Siemens ADVIA Centaur® and the Siemens IMMULITE Immunoassay systems, confirming the results of underestimation of NT-proBNP in clinically relevant areas. For method robustness investigations, the LC-MS/MS method was set up and used at two geographically different sites by different operators, using two different mass spectrometry systems (Agilent 1200 with triple quadrupole and the nano-LC-ESI-Orbitrap from Thermo Fisher Scientific), resulting in CVs from 13% to 2% for NT-proBNP concentrations ranging from 220 ng/L to 9700 ng/L.

Conclusion
Gentian AS presents a robust and reliable mass spectrometry method, capable of detecting the total amount of NT-proBNP in a sample, independent of its degree of glycosylation. This method is suitable for serving either as a reference method for calibrating and ensuring the accuracy of existing methods, harmonization of existing methods, or as a novel method in clinical chemistry for accurately determining the concentration of total amount of NT-proBNP.