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Purification and quantification of an isomeric compound in a mixture by collisional excitation in multistage mass spectrometry experiments. Dany Jeanne Dit Fouque, Alicia Maroto, and Antony Memboeuf Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03490 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016
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Analytical Chemistry
Purification and quantification of an isomeric compound in a mixture by collisional excitation in multistage mass spectrometry experiments. Dany Jeanne Dit Fouque, Alicia Maroto, Antony Memboeuf* UMR CNRS 6521, CEMCA, Université de Bretagne Occidentale, 6 Av. Le Gorgeu, 29238 Brest Cedex 3, France. ABSTRACT: The differentiation, characterization and quantification of isomers and/or isobars in mixtures is a recurrent problem in mass spectrometry and more generally in analytical chemistry. Here we present a new strategy to assess the purity of a compound that is susceptible to be contaminated with another isomeric side-product in trace levels. Providing one of the isomers is available as pure sample, this new strategy allows the detection of isomeric contamination. This is done thanks to a “gas-phase collisional purification” inside an ion trap mass spectrometer paving the way for an improved analysis of at least similar samples. This strategy consists in using Collision Induced Dissociation (CID) multistage mass spectrometry (MS2 and MS3) experiments and the Survival Yield (SY) technique. It has been successfully applied to mixtures of cyclic poly(L-lactide) (PLA) with increasing amounts of its linear topological isomer. Purification in gas phase of PLA mixtures was established based on SY curves obtained in MS3 mode: all samples gave rise to the same SY curve corresponding then to the pure cyclic component. This new strategy was sensitive enough to detect traces of linear PLA (˂3%) in a sample of cyclic PLA that was supposedly pure according to other characterization techniques (1H NMR, MALDI-HRMS and Size-Exclusion Chromatography). Moreover, in this case, the presence of linear isomer was undetectable according to MS/MS or MS/MS/MS analysis only as fragment ions are also of the same m/zs. This type of approach could easily be implemented in hyphenated mass spectrometric techniques to improve the structural and quantitative analysis of complex samples.
The structural and quantitative analysis of mixtures of isomeric and/or isobaric compounds is of paramount importance for various scientific fields ranging from e.g. pharmaceutics to environmental chemistry through biochemistry.1– 9 This, however, remains a challenging task by using mass spectrometric techniques. Those types of compounds may indeed not be separated effectively by single stage mass analysis due to their identical masses, in the case of isomers, or too close m/zs due to insufficient instrument resolving power and poor isolation efficiency in tandem mass spectrometry. A number of hyphenated mass spectrometric techniques have therefore been developed to overcome this issue using various chromatographic techniques10 such as HPLC,11,12 GC,13–15 SEC,16 electrophoresis17 and more recently ion mobility7,18,19 each with their own limitations; in particular increasing drastically the cost of analysis, its complexity and/or slowing down the analytical process that would be very fast, reliable, sensitive and very selective by using mass spectrometry only. Several tandem mass spectrometric techniques using Collision Induced Dissociation (CID) have thus been developed. They are based either on: (1) inspection of diagnostic fragment ions ratio,3,20 (2) Cook’s kinetic method21–23 and more recently (3) by using energy-resolved tandem mass spectra and the Survival Yield (SY) representation.24–29 In the first two tech-
niques, however, either in (1), fragmentation routes need to differ sufficiently so that parent ions lead to different fragment ions and differences in their MS2 or MS3 tandem mass spectra30,31 or in (2), the technique applies to low mass compounds. On contrary, in the latter technique (3), it was shown that mixtures of large mass compounds exhibiting identical tandem mass spectra or overlapping of diagnostic peaks may also be analyzed in terms of structures and quantitatively.26,27 In this communication, this method was extended to multistage mass spectrometry (MS3) to perform a gas-phase purification by collisional dissociation for a mixture of two topological isomers. Synthetic polymers such as cyclic poly(L-lactide) was obtained by CuAAC reaction from a linear isomeric precursor.27 Briefly, the proposed methodology consists in considering that the two isomeric forms of mass-selected parent ions will generate fragments, possibly with identical masses, on different ranges of excitation voltages in MS2 mode. This means that at a sufficiently large excitation voltage one of the two forms will not survive the CID process, while part of the ion population of the other form may remain intact. The remaining parent ions will then be of one isomeric form only, since parent ions of the other form have been fully fragmented. By performing an additional mass-selection on the same parent ions at such high excitation voltage, only one isomeric form can then be isolated: we have performed a “gas-phase
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Analytical Chemistry
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collisional purification” of an isomer in a mixture. To assess the above concept, we have proved the isomeric “gas-phase collisional purification” by performing CID experiments in both MS2 and MS3 modes on various mixtures of linear/cyclic PLA samples. In MS2 mode, we observed a larger fragmentation at low excitation energies when increasing the amount of linear compounds. In MS3 mode, after “gas-phase collisional purification” and a sufficiently high MS2 excitation voltage, no such tendency was observed: the degree of fragmentation remained unchanged whatever the quantity of linear PLA in the mixture. In the proposed strategy, energy-resolved tandem mass spectra were recorded and the SY technique used. The advantage of using SY curves is twofold: 1) it proves the fragmentation of linear PLA is not affected by using MS2 or MS3 modes (same curves are obtained); 2) it proves efficiency of purification at all amounts of linear PLA added (same curves obtained in MS3 mode whatever the mixture). Moreover, using the standard addition method and the SY, we have confirmed, without ambiguity, the presence of remaining small traces (
