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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Validated multi-component CZE-UV procedure for the quantification of human hemorphin LVV-H7 in plasma stability
Harald John1, Stefanie Schulz, Wolf-Georg Forssmann
1IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, 30627 Hannover, Germany. HaraldJohn@bundeswehr.org
The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin beta, gamma, epsilon or delta 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 (r2 > or = 0.999), good precision (repeatability and reproducibility below 11%), accuracy (-8.4%-4%), ruggedness and an appropriate lower limit of quantification (LLOQ 1.0 microg 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.
The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin beta, gamma, epsilon or delta 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 (r2 > or = 0.999), good precision (repeatability and reproducibility below 11%), accuracy (-8.4%-4%), ruggedness and an appropriate lower limit of quantification (LLOQ 1.0 microg 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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