Fragmentation Reactions of Rhodamine B and 6 G as Revealed by High Accuracy Orbitrap Tandem Mass Spectrometry

Correct interpretation of mass spectra is essential to our understanding of the ion chemistry occurring in the gas phase inside mass spectrometers and for constructing a solid knowledge from which mass spectrometry (MS) data of novel molecules will be interpreted. Assignments of product ions leading to incorrect dissociation mechanisms can also be dangerous in several disciplines such as in forensic chemistry and clinical diagnostics. Main fragmentation routes for rhodamines B and 6G were investigated by high accuracy Orbitrap tandem mass spectrometry (MS/MS). Isobars were properly resolved and molecular formulas were correctly attributed to all major product ions. This proper evaluation of ions composition and formula allowed us to propose a detailed mechanism for their dissociation. A comprehensive mechanistic fragmentation is proposed for rhodamine B and 6G using product ion assignments resulting from high resolution and accuracy tandem MS data, which should serve as a guide for MS and MS/MS data interpretation of homologues molecules.


Introduction
Mass spectrometry (MS) is a technique mainly based on the measurement of the masses of gaseous ions.If this measurement is done with sufficient accuracy, the ion molecular formula can be unequivocally attributed, but the connectivity of such ions cannot be determined from the molecular mass alone.To get information for the actual ion structure, MS investigations must rely on the dissociation chemistry of selected ions via tandem mass spectrometry (MS/MS).When this type of dissociation is investigated, it is therefore essential to use proper resolution and accuracy to be able to resolve isobars and to correctly assign masses for all fragment ions.Unfortunately, low resolution and accuracy tandem MS data had been often used to interpret MS and MS/MS data.Incorrect product ion composition and formulas are therefore sometimes obtained and used to construct dissociation mechanisms.These assignments can be particularly disastrous in some areas where false negatives or positives should not be tolerated such as in forensic chemistry and clinical analysis.An incorrect dissociation scheme can also be propagated leading to improper interpretations of the dissociation chemistry of similar or homologues molecules. 1nthene dyes represent a class of important organic compounds and are extensively employed. 2Rhodamines (Figure 1), such as rhodamine B and 6G and their derivatives, due to their unique photophysical 3 and photostability 4 properties, are among the oldest and most commonly used synthetic dyes. 5Rhodamine B is used widely as a colorant in textiles and in the plastic industries, and is also a well-known fluorescent dye applied in organic chemistry and biological studies. 6Rhodamine 6G is also widely used as a fluorescence tracer 7 and as a lasing 8 medium.These commercially available rhodamines are also used as a security feature in automatic teller machines (ATM's) to deter bank robberies. 9These dyes are also illegally used by sweet markets or bakers for coloring different confectionery. 10S is commonly used for rhodamine detection particularly due to its high sensitivity, selectivity and speed.Unfortunately, many contrasting interpretations for the ion chemistry of the cationic rhodamine B have been proposed. 11Lipson and co-workers 12 have studied the rhodamine 610's fragmentation using visible matrixassisted laser desorption ionization, suggesting that the degree of fragmentation is inversely proportional to the strength of the contact ion pair in the solid state.Similar results were also reported for salts of rhodamine 6G.In 1984, Brown et al. 13 reported the first investigation on the Vol.28, No. 1, 2017 fragmentation behavior of rhodamine B by means of fast atom bombardment (FAB) MS.Two years later, Ballard and Betowski 14 reported the MS/MS collision-induced dissociation (CID) of the cationic rhodamine B using thermospray triple quadrupole MS.The authors found a double loss of 44 Da as the main dissociation channel and attributed the first 44 Da loss to propane (C 3 H 8 ) and the second to CO 2 .However, it should be noted that the loss of 44 Da from the rhodamine B cation structure could also be attributed to either elimination of CO 2 or C 2 H 6 N molecules.A mechanism for rhodamine B dissociation was not discussed.In 2011, Peters and Grotemeyer 15

studied the fragmentation of xanthene dyes by means of Fourier transform ion cyclotron resonance MS (FT-ICR-MS).
Fragmentation of the electrosprayed cations of rhodamine B was induced by laser photodissociation (laser PD) or SORI CID (sustained off-resonance irradiation collision-induced dissociation).But now the two 44 Da losses were both attributed to propane (C 3 H 8 ) via either a concerted or a radicalar mechanism (Scheme 1).
Peters et al. 16 investigated the dissociation of rhodamine B using laser and collisional activation in an FT-ICR-MS.By D-labeling, they determined that the double 44 Da loss involves indeed the exocyclic NEt 2 group of rhodamine B. From high accuracy m/z measurements, they attributed the two sequential 44 Da losses to propane, favoring a concerted mechanism since no radical loss fragment ions are detected.Furthermore, using different isotopologues, they observed lack of fragment ions which incorporate both ethylated amine groups.Wang et al. 17  In view of these contrasting interpretations, we have therefore performed a comprehensive study to settle the dissociation chemistry for both cationic rhodamine B and its isomer protonated rhodamine 6G molecule using 140k of resolution and 1 ppm accuracy tandem mass spectrometry in an orbitrap mass analyzer.A revised MS/MS fragmentation mechanism for these dyes is therefore proposed.

Materials and samples
High performance liquid chromatography (HPLC)-grade methanol was purchased from Burdick & Jackson (Muskegon, MI).Rhodamine B and 6G were purchased from Sigma-Aldrich, Brazil Ltda, at purities of 95%.Rhodamine B and 6G were diluted in methanol and injected with 3.0 µL min -1 of flow rate on Q-Exactive TM (Thermo Scientific, Germany), mass spectrometer with orbitrap analyzer, via ESI, both experiments were performed in the positive mode.For collision was applied high collision dissociation (HCD) 45.00-55.00 of energy.

Results and Discussion
Figure 2 shows the MS/MS data for the precursor ion of rhodamine B cation of m/z 443.Note the most abundant ion of m/z 399 and a series of other characteristic fragment ions at medium abundances.
Although isobars have never been considered or detected, enlargements have shown that indeed two main fragment ions (Figure 3) of cationic rhodamine B are in fact composed of isobaric mixtures.
Table 1 shows the accurate m/z values and the correct assignments for the losses and fragment ions of the rhodamine B cation.Note that the first 44 Da loss is indeed found to correspond exclusively to propane.But for the second 44 Da loss leads to the product ion of nominal m/z 355, an isobaric mixture composed of two product ions at m/z 355.10684 and 355.17929 (Figure 3b).This second loss was attributed in fact to either loss of CO 2 and/or C 3 H 8 units.These two losses correspond to an m/z difference of only 0.07245 Da, and indeed MS data obtained with resolutions lower than 70k would not resolve these isobars, which explains the failure of some previous MS studies to resolve the mixture.Another isobaric mixture was found for the product ion having a nominal value m/z 371 and it was composed of product ion being formed by losses of a CH 2 N .radical (m/z 371.15054) or an ethylene (C 2 H 4 ) molecule (m/z 371.13821) (Figure 3a).
Figure 4 now shows the ESI(+)-HCD-MS/MS of protonated rhodamine 6G, whereas Table 2 summarizes the high accuracy data and interpretation of its major product ions.Please note that as the protonated rhodamine 6G in which the exocyclic N contains a single ethyl group, therefore it is not possible loss of propane, but that of ethane.Accordingly, the product ion with a nominal mass m/z 415 is an isobaric mixture containing two product ions formed by loss of either OEt and/or NEt groups, or both (Scheme 2).
Although of low abundance, a typical fragment ion for rhodamine 6G demonstrates the imperious need to   .or CH 2 N .radicals or a neutral CO 2 (Table 2 and Schemes 2 and 3).
In all, the high resolution and accuracy Orbitrap MS/MS data summarized in Tables 1 and 2 allowed us to propose

Conclusions
Using therefore proper high resolution and high accuracy MS/MS data, correct assignments of all fragment ions Scheme 2. Fragmentation routes eliminating radical species for (A) cationic rhodamine B; and (B) protonated rhodamine 6G.Scheme 2. Fragmentation routes eliminating radical species for (A) cationic rhodamine B; and (B) protonated rhodamine 6G.The color blue represented the first fragmentation, red second fragmentation and green third fragmentation sequential.from cationic rhodamine B and its isomeric protonated rhodamine 6G were made.Several fragmentation ions were found to be composed in fact by a mixture of isobaric ions, from which previous work based on low resolution or low accuracy MS/MS data failed to resolve and therefore to correctly interpret.But when the correct isobaric composition is determined and correct formula are assigned, previous misinterpretations could be corrected and a detailed fragmentation mechanism could be proposed for two important molecules widely used in forensic and food chemistry.
also investigated the fragmentation of the cationic rhodamine B by electrospray ionization-ion trap MS (ESI-MS) and also interpreted the double 44 Da loss to propane, whereas a third sequential 44 Da loss was attributed to CO 2 .

Figure 3 .
Figure 3. Expanded MS/MS data showing the isobaric composition of the rhodamine B fragment ions of nominal m/z 371 and 355.
have enough accuracy and resolution to properly interpret fragmentation routes in that of nominal m/z 371 (Figure5).Note that, in fact, it is composed of three isobars of m/z 371.13880, 371.15080 and 371.21131, which leads to different compositions: C 25 H 27 N 2 O, C 24 H 21 NO 3 , and C 23 H 19 N 2 O 3 , respectively.Therefore, three different mechanisms of fragmentation starting from the parent ion must be involved.These isobaric ions should therefore be formed by initial losses of either CH 3

Figure 5 .
Figure 5. Expanded MS/MS for the ion of m/z 317 from protonated rhodamine 6G.

Table 1 .
High accuracy data and interpretation for the fragment ions of cationic rhodamine B

Table 2 .
High accuracy data and interpretation for the fragment ions of cationic rhodamine 6G