Quantification of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS: a biomarker of drug-induced cardiac hypertrophy
Abstract
Brain natriuretic peptide (BNP) and N-terminal proBNP (NTproBNP) are well established in the clinic as biomarkers of heart failure. BNP hormone and the inactive NTproBNP are predominantly secreted in the ventricles of the heart in response to pressure overload and, consequently, are being investigated as markers of drug-induced cardiac hypertrophy in rat to support drug development. In the work presented here, an immunoaffinity-based LC/MS/MS assay was developed and validated to measure a selective tryptic fragment of NTproBNP in rat serum. The assay covers the range of 13-329 pg/mL of the tryptic fragment LLELIR, corresponding to 0.1-2.5 ng/mL intact NTproBNP. A stable isotope-labeled version of NTproBNP containing the tryptic fragment LLELI[13C615N1]R was prepared by solid-phase peptide synthesis and was used as an internal standard to minimize assay variability. Due to endogenous NTproBNP present in rat serum, human serum was used as the control matrix, and parallelism between rat and human serum was established by standard addition. Assay accuracy (% RE) and precision (% CV) were measured at three concentrations on each of 4 days and did not exceed 4.2 and 14.5%, respectively. Additionally, study data are presented from the...Continue Reading
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