Tandem mass spectrometry with ion trap in the isomers resolution

The analysis of isobaric molecules by tandem mass spectrometry, especially if they are isomers, is often complicated by the similarity of their fragmentation patterns. In fact, it is common that the same MS/MS product ions are present in the spectra of all isomers. In this case an adequate chromatographic separation between compounds should be developed in order to eliminate mutual interferences. The resolution of mixtures of isomers by liquid chromatography requires peculiar stationary phases and specific elution programs, which leads to lack of productivity, in terms of number of samples per time unit, and rise of analysis costs. The use of MS/MS experiments and the application of LEDA (Linear Equations Deconvolution Analysis) algorithm to the obtained data allows a remarkable reduction of these issues. Considering the energetic fragmentation profiles of pure isomers, the LEDA tool permits the deconvolution of isomers mixtures, assigning to each component the correct relative percentage. The aim of this work is the validation of a method to analyze five positionally isomers of multi drug resistance inhibitors (MDR) using an ion trap (IT) mass spectrometer. In fact, some issues were occur in the triple quadrupole (QqQ) LEDA application, that showed in some cases lack of precision on the obtained results. These problems were mainly due to the difficult evaluation of the abundance of the precursor ion before its decomposition and/or to the extensive number of formed product ions, which involves the subdivision of the ionic signal. To limit these occurrences, the MS/MS experiments in IT were carried out. The IT fragmentation mechanism allows a better management of the ions (precursor/products) ensuring their correct abundance rating. Indeed, the different energy transfer during collision process, lead to the formation of product ions directly by precursor ion. However, this characteristic often produce a poor number of product ions, which contrasts with necessary information for the LEDA application. The LEDA algorithm consists in the application of a matrix of linear regression equations to different experimental data. The LEDA tool was proposed to establish the relative proportions of individual isomers in the sample. This work is part of a wider project comprehending drug plasma stability and activity assays of these compounds. Considering the pharmaceutical interest of the compounds under investigation, the LC-MS/MS method developed was tested to be effective at the pharmacological active concentration levels of studied compounds, corresponding to a range between nM to µM (corresponding to ng mL-1 of processed sample). The performance evaluation of the proposed algorithm (LEDA) in IT application confirmed its effectiveness allowing an accurate and precise quantitative analysis of complex mixtures of isomers. Furthermore the product ion scan acquisition allows to measure the isotopic cluster of fragment ions and to determine the number of carbon atoms of these fragments. It is worth to observe that the LEDA tool is able not only to give the relative quantities of the mixture components but, overall, to distinguish immediately their combination (e.g. binary, ternary, quaternary, ....mixtures) or if the sample is represented by a pure compound. The LEDA approach has the advantage that isomers can be quantified without the need of LC separation or additional specialized ion mobility instrumentation.