Validated multi-component CZE-UV procedure for the quantification of human hemorphin LVV-H7 in plasma stability studies |
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Authors: | Harald John Stefanie Schulz Wolf-Georg Forssmann |
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Institution: | (1) IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, 30627 Hannover, Germany;(2) Division of Experimental and Clinical Peptide Research, Center of Pharmacology and Toxicology, Hannover Medical School, Feodor-Lynen-Str. 31, 30627 Hannover, Germany;(3) Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany |
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Abstract: | The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin β, γ, ε or δ chain exhibiting potential pharmaceutical
relevance for blood pressure regulation, the treatment of Alzheimer’s disease or learning deficiencies. Here we present the
development of a multi-component capillary zone electrophoretic method (CZE-UV), allowing the simultaneous quantification
of LVV-H7 and four N-terminal degradation products generated in EDTA plasma. Hemorphins in the supernatant of precipitated
plasma samples are quantified by external calibration. Validation of the procedure oriented towards international pharmaceutical
guidelines and demonstrated excellent linearity ( r
2 ≥0.999), good precision (repeatability and reproducibility below 11%), accuracy (−8.4%–4%), ruggedness and an appropriate
lower limit of quantification (LLOQ 1.0 μg mL−1). This procedure was applied to stability studies of LVV-H7 in human EDTA plasma attended by profiling metabolites using
qualitative MALDI-TOF MS analysis. We detected the activity of a soluble plasma form of aminopeptidase M causing successive
N-terminal truncation. This is the first time that LVV-H7 degradation as well as its metabolite production have systematically
been monitored by a quantitative CZE-UV procedure, underlining the growing importance of such techniques in peptide analysis.
In addition, our results give useful hints for future drug development of LVV-H7. |
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Keywords: | Aminopeptidase M CZE-UV Hemorphins LVV-H7 Peptide quantification |
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