Abstract 14919: A Novel and Validated Mass Spectrometry Assay for Characterizing in vitro Fragmentation of BNP and a Method to Inhibit Enzymatic Degradation of Intact BNP
Background: Elevated circulating levels of "BNP" as detected on commercial antibody-based assays in heart failure (HF) patients may reflect a mixture of degraded BNP fragments, rather than intact BNP1-32.
Methods: Blood samples from 20 HF patients were collected, immediately centrifuged with plasma promptly aliquoted and frozen at -80°C. In a single thaw, 50uL of 10N HCL was added to 200 uL of plasma, incubated for 10 minutes then centrifuged. 100 uL of supernatant was injected into liquid chromatography mass spectrometry system. On-line extraction and high performance liquid chromatography methods were optimized using Onyx C18 guard and analytical column. Purified synthetic BNP1-32 was then incubated in patient serum and BNP fragments identified. Lastly, a proprietary protocol to inhibit BNP degradation was developed.
Results: The estimated limit of detection of the mass spectrometry method was 0.78 ng/mL and the lower limit of quantitation was 1.5 ng/mL with a good linear relationship between the concentration of BNP and response. The analytically measurable range was 1.5-100 ng/mL (R2=0.996). After spiking the serum with purified BNP1-32 at room temperature there was rapid BNP degradation with most of BNP1-32 gone in 90 minutes, and concomitant accumulation of multiple ragged N-terminal peptides. Among the fragments identified, BNP9-32, BNP8-32, BNP7-32, BNP6-32, BNP5-32, BNP4-32, BNP3-32 and BNP2-32, BNP3-32 appeared to be the predominant products (Figure). Our proprietary protocol to inhibit degradation, entirely halted digestion of BNP1-32 at 90 minutes and at 3.5 hours. Further validation of this unique BNP mass spectrometry method is now underway with 40 age and sex matched patients with and without HF.
Conclusion: A novel mass spectrometry approach was developed for analyzing circulating BNP forms, including a novel method to arrest in vitro degradation. Expanded results examining whether circulating BNP is intact or degraded will be presented.
- © 2012 by American Heart Association, Inc.