Main author: Margrét Þorsteinsdóttir
Institution or Company: University of Iceland, ArcticMass
Co-Authors, Institution or Company:
Unnur Arna Þorsteinsdóttir, University of Iceland, ArcticMass.
Introduction: Method development can become much more efficient by utilizing design of experiments (DoE) approach. DoE offers many advantages including performing experiments in accordance with predefined plan, modelling by empirical functions and graphical visualization. Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) is an excellent analytical platform with ultimate selectivity and sensitivity needed for quantification of biomarkers in biological matrices. Examples will be given to illustrate the workflow of DoE for optimization of UPLC-MS/MS clinical diagnostic assays, allowing the effect of variables to be assessed with only a fraction of the experiments that would be required by changing one-separate-factor-at-time (COST) approach.
Methods: UPLC-MS/MS was utilized for simultaneous quantification of clinical biomarkers in biological matrices. A fractional factorial design was used for experimental screening to reveal the most influential factors. When multi-levels qualitative factors were included in the screening experiments D-optimal design was applied. Significant factors were studied via central composite design and related to sensitivity, resolution and retention time utilizing partial least square (PLS)-regression.
Results: DoE ensured that selected experiments contained maximum information and optimization of the UPLC-MS/MS clinical diagnosis assays was achieved. Results showed that many interaction factors were significant and therefore these variables could not be independently controlled to obtain optimal conditions. The optimized assays have been implemented for clinical diagnosis and therapeutic drug monitoring.
Conclusions: DoE is an excellent approach for optimization of UPLC-MS/MS assays especially when several experimental factors need to be simultaneously optimized to obtain maximum selectivity and sensitivity at minimum retention time.
