Abstract
Objectives
Synthetic B-type natriuretic peptide (BNP) is employed in most clinical testing platforms as a raw material of calibrator. Characterization of impurities with structures similar (BNPstrimp compounds) to that of BNP is a reasonable way to decrease clinical measurement errors and improve drug safety.
Methods
A novel quantitative method targeted towards BNPstrimp compounds was developed. First, the peptide samples were separated and identified using ultra-performance liquid chromatography, coupled with high-resolution mass spectrometry (MS). To evaluate biological activity further, BNPstrimp immunoaffinity was investigated using western blot (WB) assays. Second, a quantitative label-free data-independent acquisition (DIA) MS approach was developed, and the internal standard peptide (ISP) was hydrolyzed. Absolute quantification was performed using an isotope dilution MS (ID-MS) approach. Third, method precision was investigated using the C-peptide reference material.
Results
Seventeen BNPstrimp compounds were identified in synthetic BNP, and 10 of them were successfully sequenced. The immunoassay results indicated that deaminated, oxidized, and isomerized BNPstrimp compounds exhibited weaker immunoaffinity than intact BNP1-32. The mass fraction of the synthetic solid ISP1-16, quantified by ID-MS, was 853.5 (±17.8) mg/g. Validation results indicated that the developed method was effective and accurate for the quantitation of the well-separated BNP impurities.
Conclusions
The developed approach was easy to perform, and it was suitable for the parallel quantification of low-abundance BNPstrimp compounds when they performed a good separation in liquid chromatography. The quantitative results were comparable and traceable. This approach is a promising tool for BNP product quality and safety assessment.
Funding source: National Natural Science Foundation of China
Award Identifier / Grant number: 21705144
Award Identifier / Grant number: 31800842
Funding source: National Institute of Metrology Fundamental Research Project
Award Identifier / Grant number: AKY1709
Award Identifier / Grant number: AKYZD1802-2
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant numbers 21705144, 31800842), and also by the National Institute of Metrology Fundamental Research Project (grant number AKY1709, AKYZD1802-2).
Research funding: The National Natural Science Foundation of China (grant numbers 21705144, 31800842), and the National Institute of Metrology Fundamental Research Project (grant number AKY1709, AKYZD1802-2).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Informed consent: Informed consent was obtained from all individuals included in this study.
Competing interests: Authors state no conflict of interest.
Ethical approval: The local Institutional Review Board deemed the study exempt from review.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-0012).
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