62 Chapter 3 CONCLUSION Here we describe for the first time the use of LC-MS/MS for the quantification of FVIII in citrated plasma. Critical method parameters were optimized and the resulting method was subjected to validation according to EMA guidelines. All parameters were found to be well within predefined acceptance criteria. The method, which utilizes camelid anti FVIII nanobodies for sample purification and LC-MS/MS for measurement is highly selective. The removal of interfering plasma proteins lowered the detection threshold significantly, resulting in similar sensitivity as the one-stage activity assay. The lower limit of quantification of FVIII was 1 ng/mL which corresponds to 3.6 fmol/mL, which to the best of our knowledge has never been reported before in the analysis of biopharmaceuticals with LC-MS/MS. This was mainly achieved by using an easy and robust sample processing method which consisted of an efficient immunoaffinity purification in combination with a highly sensitive mass spectrometer. This method was developed for an ongoing study to investigate the pharmacokinetics of FVIII and its added value to existing activity-based diagnosing and monitoring. Furthermore, LC-MS/MS method enables ‘telemonitoring’ of patients by means sampling at home using dried blood spot sampling for instance. Finally, multiplexing capabilities of LC-MS/MS would allow for other coagulation factors to be included in the same assay thus providing a way to quantitate multiple coagulation proteins in patient plasma in one analysis. Author’s contribution AD helped the first author in analysis of data, drafted the first versions of the manuscript together with the first author, helped in implementation of significant contribution from co-authors up to the final publication.
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