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Reduction of LC/MS In-Source Fragmentation of HFPO-DA (GenX) Through Mobile Phase Additive Selection: Experiments to Increase [M-H]- Formation
Citation:
Mulin, L., D. Katz, N. Riddell, R. Plumb, J. Burgess, AND I. Jogsten. Reduction of LC/MS In-Source Fragmentation of HFPO-DA (GenX) Through Mobile Phase Additive Selection: Experiments to Increase [M-H]- Formation. DioXin 2018: 38th International Symposium on Halogenated Persistent Organic Pollutants, Krakow, N/A, POLAND, August 26 - 31, 2018.
Impact/Purpose:
Tetrafluoro-2-(heptafluoropropoxy)propanoic acid (HFPO-DA) is a replacement compound used to manufacture products such as Teflon®, because it is considered less persistent and less toxic than previously used per- and polyfluorinated alkanoic substances (PFAS). While this compound has previously been detected in the environment downstream of certain manufacturing facilities, little is known about the persistence, fate and toxicity of HFPO-DA. Existing methods for PFAS can be used to detect HFPO-DA, however present methods are not specifically designed to detect this compound. As a result, our ability to detect this compound is poor in comparison to other PFAS compounds. This work was undertaken to investigate methods to improve our ability to detect HFPO-DA. Our results describe the best conditions found for detecting and analyzing for HFPO-DA in the laboratory.
Description:
Introduction: The compound 2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)propanoic acid (HFPO-DA) is a polyfluoroalkyl ether carboxylic acid which was introduced as replacement of perfluorinated alkanoic substances (PFAS) previously used in various industrial and consumer processes1. Commonly referred to by it’s ammonium salt derivative trade name GenX, environmental monitoring of this compound has increased notably in recent years due to prior high volume discharge by manufacturers, and continued occurrence in water ways. Analysis of HFPO-DA is performed most commonly by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using electrospray ionization in the negative polarity (ESI-). One challenge associated with the analysis of HFPO-DA resides in the moderate to intense fragmentation of the molecule upon ionization, as well as dimer formation. This fragmentation results in the diminishment of the deprotonated parent ion [M-H]- and potential loss in analyte specificity2. Here we describe method development characterizing the use of ammonium bicarbonate as a LC mobile phase additive to induce the controlled formation of the bicarbonate adduct [M+HCO3]- for HFPO-DA. This adduct formation was observed to reduce the degree of fragmentation contribution to overall molecular signal, as well as reduce dimer formation during ionization. Materials and Methods: HFPO-DA and other standards were purchased from Wellington Laboratories. Molecule fragmentation under various mobile phase additives at two concentrations each were assessed in aqueous (mobile phase A) and methanol (mobile phase B) compositions. These included ammonium acetate, ammonium formate and ammonium bicarbonate (2 and 10mM); and formic acid and ammonium hydroxide (0.1 and 0.5%). All additive experiments were performed on a Waters Acquity H-Class equipped with an isolator column in line with pre-injection solvent delivery and a BEH C18 2.1x100mm 1.7m analytical column. Mass spectrometry analysis and source condition optimizations were performed on a Waters Xevo TQ-D operating in both full scan and MRM modes. Adduct identity was confirmed using accurate mass measurement on a Waters Xevo G2-XS QTof. Method reproducibility was assessed by repetition of optimized conditions on additional systems and sites. Results: Assessment of all mobile phase additives found that the use of ammonium bicarbonate resulted in the regular formation of the [M+HCO3]- ion in addition to the [M-H]- ion. In monitoring the molecule stability across all mobile phases used, the previously characterized primary fragment generated from the loss of CO2 (-44Da) and additional fragments from breakage at either side of the ether linkage (-161 and -145Da) were assessed relative the parent molecule in full scan where minimal collision energy is applied. Formic acid mobile phase data showed the highest degree of molecule fragmentation, while ammonium bicarbonate resulted in the highest total parent molecule composition due to the combination of [M-H]- and [M+HCO3]- adduct formation. Optimization of the ammonium bicarbonate concentration were also performed, monitoring dimer ([2M-H]-) formation in addition to fragment molecule reduction. An assessment of mobile phase pH from both multiple pH adjustments of ammonium bicarbonate and the inherent pH of all mobile phase compositions showed no strong correlation to in-source fragmentation increase, decrease or relative proportion to parent molecule conservation. Additional data presented will compare and contrast the behavior of ADONA, another PFAS replacement, throughout the various mobile phase compositions, as well as the results for the 16 widely monitored PFASs using ammonium acetate versus ammonium bicarbonate.