Development of a Reference Method for the Quantitation of by e295e75ae2526297


									   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
Mentor’s 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
Sigma Xi member: No
Category: Chemistry

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