Supervisor: Koit Herodes
Opponent: Prof. Jonas Bergquist, Dr Med Sci, PhD, Uppsala University, Sweden
For sensitive amino acid analysis derivatization liquid chromatography mass spectrometry (LC/MS) with electrospray ionization (ESI) is used. In current work the compatibility of the method steps with LC-ESI-MS analysis was evaluated.
Method development was carried out for five derivatization reagents, Fmoc-Cl, DNS, Deemm, TAHS and in-house designed and synthesized FOSF. FOSF is a novel phosphazene based derivatization reagent that as designed and synthesized in this work keeping in mind the ionization efficiency scale. It was found out that novel reagents TAHS and FOSF provide sensitive analysis but not very good chromatographic separation. It was concluded that Deemm is an optimal choice for derivatization reagent since it provides sensitive analysis and better chromatographic separation. Moreover, it was found out that solid phase extraction is most suitable sample preparation technique.
In this work, derivatization procedures of the derivatization reagents were evaluated considering LC/ESI/MS compatibility. Results showed that derivatization buffer can have a significant effect on the stability and intensity of the signal. Main influences were related to the use of borate buffer. For all reagents, most suitable eluent components were determined and other signal enhancement techniques were sought for.
Experiments conducted in addition to the previously mentioned, such as the measurements of ionization efficiency in terms of logIE scale and a thorough MS optimization investigation, provided useful additional information for even more complete and systematic method development. Practical applications showed the high performance of the derivatization LC/ESI/MS for analysis of amino acid in honey and selenoamino acid in bloodserum and food (onion).
In conclusion, in this work, it is shown that for a sensitive derivatization LC/ESI/MS analysis, new aspects of method development are needed and therefore combines together most important aspects and proposes an advanced method development strategy for derivatization LC/ESI/MS.
Keywords: amino acids, LC/ESI/MS, derivatization, method development.