Development of a Reference Method for the Quantitation of B-type Natriuretic Peptide in Serum by Liquid Chromatography-Mass Spectrometry

 

††††††††††† B-type natriuretic peptide (BNP) is a 32-amino acid cardiac biomarker utilized for the diagnosis, prognosis, and therapeutic guidance of heart failure.† BNP is a secreted, proteolytic cleavage product corresponding to amino acids 77-108 of the precursor peptide, proBNP.† Currently, there are several commercially-available in vitro diagnostic immunoassays for the determination of BNP levels in patient blood or plasma.† However, these assays lack standardization.† The calibration materials for these assays are not well characterized and inter-assay comparisons reveal differing values from patient samples.† We are developing a liquid chromatography-mass spectrometry (LC-MS) based reference method for quantitating BNP in human serum, which will allow for the traceability of routine, clinical BNP assays to a standardized method.† BNP is present in human plasma at very low levels (ng/L).† Therefore, removal of high-abundance proteins and concentration of serum samples are necessary to bring BNP levels into a range detectable by LC-MS.† To enable quantitation, an internal standard consisting of alkylated BNP that has been labeled with a deuterated reagent will be spiked into samples prior to processing.† The samples can then be treated with the undeuterated form of the same alkylating reagent to label endogenous BNP.† A variety of procedures, including acetonitrile precipitation, solid-phase extraction, and ultrafiltration can be employed to separate BNP from larger, high-abundance proteins prior to concentration and quantitative analysis by LC-MS or LC-MS-MS.

 

Name: Johanna Camara

Mentorís name: Michael J. Welch

Division: Analytical Chemistry Division

Laboratory: Chemical Science and Technology Laboratory

Room and Building address: 227/A153

Mail Stop: 8392

Telephone #: 301-975-4672

FAX #: 301-977-0685

Email: johanna.camara@nist.gov

Sigma Xi member: No

Category: Chemistry