Energy Resolved Tandem Mass Spectrometry Experiments for Resolution of Isobaric Compounds: A Case of Cis/Trans Isomerism

Abstract

A series of N-alkanol-N-cyclohexanol amine aryl esters cis/trans isomers that showed high efficacy to reverse the acquired resistance of cancer cells during chemotherapeutic therapy (MDR mechanism) was studied. These compounds were two 1,4 cyclohexane cis/trans derivatives (named ELF26A and ELF26B, respectively), and their positional isomers (named ELF34A and ELF34B, respectively) where the aryl-moieties were exchanged. In order to evaluate the behaviour of these compounds during biological tests, a method based on liquid chromatography coupled with mass spectrometry (LC-MS), operating in tandem mass spectrometry (MS/MS) mode, was developed. A unique chromatographic method suitable to separate the two pairs of cis/trans isomers was not achieved and the MS/MS experiments of the different compounds was not always able to characterise the different isomers. Therefore, a system of linear equations of deconvolution analysis (LEDA) tool was proposed to determine the relative proportions of individual cis/trans isomers in the sample. Considering the pharmaceutical interest of the compounds under investigation, the analytical method developed was tested to be effective at the active concentration levels, corresponding to a concentration of ng mL of compound in a processed sample. Precision and accuracy of the LEDA algorithm at three levels of relative concentrations of analytes were checked, i.e. low-level (about 25% in the mixture), mid-level (about 50% in the mixture) and high-level (about 70% in the mixture). Evaluation of performances of the algorithm proved that the accuracy (between 88.3% and 99.9%) and precision (between 2.0% and 3.7%) for simultaneous analysis of the mixtures of the four isomers is feasible. It is worth highlighting that the choice of characteristic product ions and optimal abundance ratios plays an important role in the application of the LEDA approach. Therefore, performing an investigation on the energetics of fragmentation pathway allowed the selection of the better product ions for each analyte in terms of both sensitivity of detection and specificity, i.e. the capability to distinguish between isomeric compounds. Finally, the developed approach was applied to determine the relative proportions of individual cis/trans isomers in spiked human plasma samples. The results obtained confirm the reliability of the proposed method in biological samples as well.

Differentiation of AB-FUBINACA and its five positional isomers using liquid chromatography–electrospray ionization-linear ion trap mass spectrometry and triple quadrupole mass spectrometry

“…Thus far, several research groups have differentiated positional isomers of other abused drugs using LIT-MS and/or QqQ-MS combined with gas chromatography (GC) or liquid chromatography (LC) [ 13 – 16 ]. Previously, we developed the method of distinguishing the 2-, 3-, and 4-fluorine substitution positions on the phenyl ring in SCs containing a fluorobenzyl group, such as AB-FUBINACA and its 2- and 3-fluorobenzyl isomers, based on the so-called energy-resolved mass spectrometry (ERMS) strategy [ 17 – 21 ] using GC–electron ionization (EI)-QqQ-MS [ 22 , 23 ]. We speculated that the methodology should be used in combination with other chromatographic and ionization techniques.…”

Resolution of co-eluting isomers of anti-inflammatory drugs conjugated to carbonic anhydrase inhibitors from plasma in liquid chromatography by energy-resolved tandem mass spectrometry

“…The Pi scan spectra were acquired in the m/z range from 50 to 650, scan time 600 ms; argon was used as collisional gas and the collision energy (CE) was increased stepwise in the range 5–40 V. A series of energy resolved tandem mass spectrometry (ERMS) experiments were performed to study the fragmentation of molecular species of each analyte and build its breakdown curves 26 , 27 . The ERMS experiments were carried out by introducing working solution 1 of each analyte, via syringe pump at 10 µL min −1 ; the protonated molecule was isolated and the abundance of Pis were monitored.…”

“…This algorithm (linear equations of deconvolution analysis [LEDA]) consists in the application of matrix of linear regression equations to different experimental data. In our case, the experimental data used were the abundance ratios of product vs. precursor ions selected during the MS/MS method set-up 26 , 27 . In this way, it was possible to resolve the MS/MS spectra assigning the correct signal to the isomer also for chromatographically unresolved peaks.…”

Synthesis and analytical characterization of new thiazol-2-(3H)-ones as human neutrophil elastase (HNE) inhibitors

“…Energy resolved tandem mass spectrometry (ERMS) experiments [28] were performed to evaluate the collision activated decomposition pathway of isomers 8 and 9 . The ERMS experiments involve a series of product ion scan spectra acquisitions, which were obtained by increasing the collision energy stepwise in the range from 0 to 20 V. The results obtained were used to study the fragmentation of molecular species from each analyte and build its breakdown curves.…”