52 Chapter 3 Therefore, in order to eliminate the drawbacks mentioned above, we have investigated the suitability of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of FVIII concentration in plasma. The method proposed is based on immunoaffinity purification in combination with tryptic digestion and LC-MS/MS analysis. Potentially, LC-MS/MS may be complementary to the current activity-based assay due to its ability to measure the absolute plasma concentration. Furthermore, pharmacokinetic and pharmacodynamics studies can be performed with FVIII plasma concentration which might be a better predictor for the bleeding phenotype compared to the current activity based assay. In addition, the stability issue of plasma samples, which has been proven to influence the activity results, could be circumvented by measuring FVIII concentration. This could bring home monitoring for haemophiliacs one step closer by allowing mail-in samples. Finally, other coagulation factors can also be added to the existing method at a later stage thus allowing for multiplexed LC-MS/MS analysis. MATERIALS AND METHODS Chemicals and reagents Octocog alfa (Advate®) was obtained from Baxter (Lessines, Belgium) as lyophilized powder and was reconstituted in LC-MS grade water to a final concentration of 500 IU/mL equivalent to 94 µg/mL FVIII; 40 µL aliquots of this solution were pipetted in Eppendorf LoBind Microcentrifuge tubes and stored at -80 °C. Stable isotope labeled peptide internal standard (IS) GELNEHLGLLGPYIR[13C 6, 15N 4] was synthesized by Pepscan (Lelystad, the Netherlands) as a 1 mg lyophilized powder and was dissolved in 1 mL elution solvent (0.5% trifluoroacetic acid (TFA) in 50 % methanol, 50% water). Biotinylated Anti FVIII conjugate, reference number 7102862100 was obtained from Thermoscientific (Waltham, MA, USA) as a 1 mg/mL solution and was stored in -20 °C. Streptavidin high binding capacity coated 96 well plates were obtained from Thermo Fisher (Waltham, MA, USA). MS grade modified trypsin was obtained from Promega (Madison, WI, USA) and was dissolved to 0.1 µg/µL in 50 mM acetic acid and aliquoted in Eppendorf LoBind microcentrifuge tubes. Aliquots were stored at -80 °C. FVIII deficient human plasma was obtained from Precision BioLogic Inc. (Dartmouth, NS, Canada). All other reagents and LC-MS grade mobile phase solvents were obtained from Sigma (Saint Louis, MO, USA). Preparation of standards, Internal standard and QCs The FVIII working solution (500 ng/mL) was prepared fresh from 94000 ng/mL stock solution by diluting in FVIII deficient human plasma. Standards at concentrations of 500, 200, 80, 40, 16, 4 and 1 ng/mL were prepared from the working solution by serial dilution in FVIII deficient human plasma. Before use, the IS solution (1 µg/µL) was diluted to 5 ng/mL in 0.1% formic acid (FA) and 0.005% zwittergent 3-16. Quality Control samples (QCs) where prepared at 4 levels namely; at lower limit of quantification (LLOQ) (1 ng/ mL), QC low (5 ng/mL), QC med (150 ng/mL) and QC high (300 ng/mL). Aliquots were stored at -80 °C.