Supervisors info:
Γκίκας Ευάγγελος, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, Ε.Κ.Π.Α.
Ντότσικας Ιωάννης, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, Ε.Κ.Π.Α.
Summary:
Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization tend to be the method of choice for qualitative and quantitative determination of a wide range of compounds. However, the sensitivity and the response of each analyte in each analytical method is under the influence of both compound-dependent factors (chargeability, relative hydrophobicity, matrix characteristics etc.) and instrumental parameters. Despite the efforts there is no comprehensive model available to describe the interactions, the grade of interaction, as well as the mechanisms between the mentioned factors. Additionally, the demand of sensitive methods is very often mandatory, concerning the minimal volumes of biological samples and the effort to avoid or reduce matrix effects, that consist a major concern in bioanalytical methods.
A crucial step to this procedure is to distinguish the most important factors, that affect the MS signal, and the interactions between these factors. Instrument’s software perform usually an optimization but it is insufficient, considering that they study the influence of one factor at a time (OFAT) and they don’t distinguish the most important factors and the interactions between them. On the contrary, applying the theory of Design of Experiments (DOE) a more precise and efficient optimization can be achieved. DOE consists a tool of statistical science that is being applied in a wide range of scientific fields and in many areas of experimental work, as they solve problems and improve or optimize processes. According to the aim of the study, the number and the kind (numerical or categorical) of the experimental variables and the responses that are going to be measured the proper type of DOE can be chosen from a great variety of DOE types. Very often DOE and RSM (Response Surface Methodology) consist consecutively steps of a study, since they are basic components of a method optimization.
The aim of this work was to determine and compare the optimal experimental conditions through a stepwise optimization method of an UPLC-MS/MS method and the optimization being conducted from the instrument’s software. The model analytes chosen for this study are four macrolide molecules, erythromycin, clarithromycin, roxithromycin and azithromycin.
As a first step in the current study was to find the optimal experimental conditions according to instrument’ s software. After an experimental design was applied to define the UHPLC optimal conditions and a screening design was carried out aiming to distinguish the most important factors of the MS/MS analysis. The next step a CCI (Central Composite Inscribed) design was applied in order to optimize the important factors so as to maximize the MS signals of our analytes. A second ICC design was performed so as to optimize the liquid-chromatography conditions. Finally, a comparison was conducted between the optimized MS signals achieved from both the instrument’s software and DOE and an attempt to investigate these differences was made. The outcome of this current paper was a significant increase in MS signals, enabling the development of sensitive bioanalytical method for the quantification of these antibiotics in the future.
Keywords:
Optimization, Experimental design, UHPLC, mass spectrometry, macrolides
