Quantification of coagulation factor VIII in human plasma with liquid chromatography tandem mass spectrometry using a selective sample purification with camelid nanobodies

Publication date: Available online 19 July 2019Source: Journal of Pharmaceutical and Biomedical AnalysisAuthor(s): Mohsin El Amrani, Anouk A.M. Donners, Gerard Graat, Eef G. Lentjes, Albert Huisman, Ruben E.A. Musson, Erik M. van MaarseveenAbstractPatients with hemophilia A are currently diagnosed and monitored by measuring the activity of coagulation factor VIII (FVIII) in plasma mostly with the one-stage clotting assay (OSA). Although the OSA is routinely available in many clinical laboratories, it has in some circumstances relatively low sensitivity and specificity. Therefore, the FVIII activity as a biomarker does not always correlate with the bleeding phenotype. Therefore, we have developed a liquid chromatography tandem mass spectrometry method to quantify the concentration of coagulation FVIII in plasma which would allow us to investigate the relation between FVIII plasma concentration, FVIII activity and bleeding tendency in future studies.LC-MS/MS method was set up by firstly dissociation Von Willebrand factor (VWF) from coagulation factor VIII by triggering the coagulation cascade to occur thus generating active factor VIII (FVIIIa). FVIIIa was then selectively extracted by means of immunoaffinity interaction using anti-FVIII camelid nanobody, after which FVIIIa was eluted, heat denatured and trypsin digested. Finally, a FVIII specific peptide was used as a surrogate for quantification by mass spectrometry. Critical method parameters such as antibody amount, incubat...
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research