54 Chapter 3 1200 rpm on a plate shaker. The sample extracts were transferred to a 500 µL lobind eppendorf 96-well plate and heat denatured at 80 °C for 60 min. The samples were neutralized with 10 µL Tris (1M). Then, 5 µL trypsin (0.1 µg/µL) was added to each well and the sample plate was placed in a ThermoMixer set at 37 °C for 3 hour digestion at 800 RPM. Trypsin activity was stopped by adding 20 µL 10% formic acid dissolved in 100 % acetonitrile and finally, 50 µL was injected on a LC-MS/MS. Figure 1. Sample purification of FVIII in plasma. The light chain of active factor FVIII is captured by means of immunoaffinity interaction using anti FVIII camelid nanobody fixed onto a 96 well plate by means of biotin-streptavidin interaction. Camelid nanobody amount and signature peptide signal intensity This experiment was performed to determine the amount of biotinylated camelid nanobody required to retain 75 µL of the highest standard (500 ng/mL) Octocog alfa in FVIII deficient plasma. The plate was coated according to sample-preparation section with 10, 40, 100, 250 and 500 ng antibody per well each in triplicate. The highest standard was purified with a 4 hours incubation time and analyzed. Binding time and signature peptide signal intensity Citrate plasma sample from a volunteer was used to determine the time needed for the dissociation of VWF, conversion of FVIII to FVIIIa and for optimum sample binding during immunoaffinity interaction. Incubation times of 1, 2, 3, 4, 5 and 24h were evaluated in triplicate and the sample was run using the procedure described in sample-preparation section. Effect of sample volume on signal intensity Variable sample volumes were evaluated to determine the matrix effect on sample recovery. Citrated plasma from a volunteer was used for this test. The procedure in sample-preparation section was used with variable sample volumes (5, 10, 25 and 50 µL; each in duplicate).