Research Article

Received: 23 December 2010 Revised: 31 January 2011 Accepted: 31 January 2011 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2011, 25, 1169–1172

Fragmentation of xanthene dyes by laser activation andcollision‐induced dissociation on a high‐resolution Fouriertransform ion cyclotron resonance mass spectrometer

Jonathan Peters and Jürgen Grotemeyer*Institut für Physikalische Chemie, Christian Albrechts Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, Germany

Surprising fragmentation reactions in different xanthene dyes have been investigated by means of photodissociationand collision‐induced dissociation in a 9.4 T FT‐ICR mass spectrometer. It is shown that extensive rearrangementreactions lead to the formation of unexpected fragments which are identified for the first time by the use of highresolvingmass power. The observed reactions are an example of the fragmentation of a quinoidal even‐electron cation.Copyright © 2011 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.4972

Xanthene dyes, such as fluorescein, rhodamine and theirderivatives, have numerous applications throughout chem-istry, biochemistry and physics. Due to their high quantumyields they are used for fluorescence labeling experiments andas active media in dye lasers. Those experiments are restrictedby the laser power that can be applied to the dye. Investiga-tions showed that on raising the laser power, the fluorescencefirst increased, but at higher powers, this changed towards adecrease.[1,2] Probing of fluorescent molecules inside massspectrometers showed that this decrease in fluorescenceefficiency correlated with the appearance of photofragments,as demonstrated by Chingin et al.[2] who discussed the massspectrometric data of Rhodamine 6G and its isomers. Brownet al.[3] investigated the fragmentation behaviour of Rhoda-mine B after fast atom bombardment mass spectrometry. Thecollision‐induced dissociation (CID) of the Rhodamine Bcation was also investigated on a thermospray triple quadru-pole instrument by Ballard and Betowski.[4] In these papers aninteresting double loss of 44Da was observed. The authorsattributed the first loss of 44Da to the fragmentation of a C3H8

moiety. As shown in Fig. 1, this reaction can only take place ifa rearrangement occurs in the molecular cation. The authorsalso discuss the second loss, resulting in a product ion atM+–88, which they assign to the formation of a [M–(CO2+C3H8)]

+ ion. Since Rhodamine B has a highly symmetricalstructure (Fig. 1), this M+–88 product ion could also be due tothe loss of two C3H8 groups. The mechanism responsible forthe formation of the different observed product ions was not,however, discussed by these authors.For this reason, we have studied the fragmentation

behaviour of xanthene dyes by means of high‐resolutionmass spectra in a 9.4 T Fourier transform ion cyclotronresonance (FT‐ICR) instrument. Fragmentation of the electro-sprayed ions was achieved using laser photodissociation

* Correspondence to: J. Grotemeyer, Institut für PhysikalischeChemie, Christian Albrechts Universität zu Kiel, Olshausenstr.40, 24098 Kiel, Germany.E-mail: grote@phc.uni-kiel.de

Rapid Commun. Mass Spectrom. 2011, 25, 1169–1172

(laser PD) as well as SORI CID (sustained off‐resonanceirradiation collision‐induced dissociation). High‐resolutionmass spectra should allow us to distinguish between thedifferent mass signals thus leading to an understanding of thefragmentation mechanism. In the present study we have in-vestigated the following dyes, Rhodamine B, SulforhodamineB, Rhodamine B isothiocyanate and Oxazine‐1, as shown inFig. 1. All the compounds share the same xanthene dyebackbone and nitrogen side‐chain substitution and shouldallow a general insight into the fragmentation behaviour ofthese dyes.

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EXPERIMENTAL

All mass spectra have been recorded on a Bruker‐APEX QEFT‐ICR mass spectrometer equipped with a 9.4 Tesla magnet(Bruker Daltonics, Bremen, Germany).[5] For electrosprayionisation studies the samples were dissolved in dimethylsulfoxide and added to a standard solvent mixture (H2O/MeOH/formic acid; 50:50:0.2), yielding a final concentrationin the range of 1–100 pmol/μL. The instrument was calibratedon arginine clusters. For the laser photodissociation measure-ments the mass accuracy was better than 1mDa, taken fromseveral measurements of the Sulforhodamine B molecularcation at m/z 559, and 0.3mDa for the Oxazine‐1 molecularcation at m/z 324. For the SORI CID spectra, the accuracy wasbetter than 4mDa for Sulforhodamine B and 1mDa forOxazine‐1.

Fragmentation of the molecular cations was achieved byusing two different methods. For the photodissociation theions were isolated in the quadrupole section of the instru-ment and introduced into the ICR cell where the absorptionof photons from the laser (Innova 70 argon‐ion laser;Coherent Inc., Santa Clara, CA, USA) was performed. Typicallaser powers were in the range of 0.5 to 1W. Irradiance timesof 0.1 s were used in all cases. Depending on the experimentthe laser was used either in the multiline mode or at a fixedwavelength of 514.5 nm.

Copyright © 2011 John Wiley & Sons, Ltd.

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Figure 2. Laser PDspectra ofRhodamineB (a), SulforhodamineB (b), Rhodamine B isothiocyanate (c), and Oxazine‐1 (d).

Figure 1. Structures of rhodamine B (1), Sulforhodamine B(2), Rhodamine B isothiocyanate (3), and Oxazine‐1 (4).

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As an alternative method, collisional activation with argongas using the SORI CID techniques has been used. AfterSWIFT (stored waveform inverse Fourier transform) isolationof the ions of interest in the ICR cell, the CID process wasinduced. The SORI power was set between 3 and 4.5% andirradiation was applied for 0.1 to 0.5 s in all experiments.Rhodamine B and Sulforhodamine B were purchased from

Lambda‐Physik (Göttingen, Germany), Rhodamine B iso-thiocyanate was obtained from Sigma Aldrich (Schnelldorf,Germany) and Oxazine‐1 was bought from Thermo FisherScientific (Schwerte, Germany). All chemicals were usedwithout further purification.

RESULTS AND DISCUSSION

Figure 2 shows the laser‐induced photodissociation spectra ofthe molecular cations from the 4 different xanthene dyes. Ascan be seen from this figure, Rhodamine B (Fig. 2(a)) shows adouble loss of 44Da from the molecular cation in the uppermass range. The two product ions are the most intensesignals apart from the molecular cation. High mass resolutiongives exact masses of 399.170Da and 355.107Da for the twosignals. By using the exact measured mass of 443.232Da forthe molecular cation, it is obvious that the mass differencesfor the loss of the neutral fragment are 44.062Da and44.063Da, respectively.Loss of 44 mass units from the molecular cations of the

investigated xanthene dyes could be due to either CO2,C2H6N or C3H8 (in the case of the Rhodamine B isothiocya-nate also CS) molecules. However, the use of high massresolution should distinguish immediately between thesedifferent losses since their masses are CO2: 43.990Da, C2H6N:44.050Da and C3H8: 44.063Da. Comparing these values withthe measured mass differences observed in the PD spectrum,it is obvious that the two product ions in Rhodamine B areformed in a sequential double loss of C3H8 moieties.

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To test if this reaction is inherent to the structure ofRhodamine B, other xanthene dyes have been investigatedthat included (a) the same xanthene backbone and (b) thesame N(C2H5)2 substitution at the amine in the 3 and 6positions.

Figures 2(b) and 2(c) show the resulting PD mass spectra ofSulforhodamine B and Rhodamine B isothiocyanate (bothstructures are displayed in Fig. 1). Sulforhodamine B(M+= 559.157) shows two main fragmentations under tan-dem mass spectrometric (MS/MS) conditions that lead againto product ions at [M–44]+ and [M–88 or M–44–44]+, which isin very good agreement with the PD mass spectrum ofRhodamine B. High mass resolution shows that the exactmass for the neutral fragments is 44.063Da in both reactions.

Rhodamine B isothiocyanate (M+= 500.200, Fig. 2(c))shows the same fragmentation behaviour as the precedingcompounds. The most intense product ions in the upper massrange of the spectrum are again due to the losses of 44.063and 44.062Da. From these findings we can state that theremoval of propane residues is a common feature of theRhodamine B family.

To extend these statement to a more general rule weinvestigated Oxazine‐1 (M+= 324.207, Fig. 2(d)). This com-pound possesses the same xanthene backbone as themolecules mentioned before, but lacks the phenyl ring inthe 9 position (ring b). At this position in ring b the carbon isexchanged for a nitrogen atom. As can be seen from the PDmass spectrum (Fig. 2(d)), two major product ions are again

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Fragmentation of xanthene dyes by laser activation and CID

observed in the upper mass range. Both ions are due to theloss of propane molecules, as can easily be proved by usingthe high mass resolving power of the mass spectrometer. Thetwo losses of 44 mass units are measured exactly as 44.063and 44.062Da.It should be mentioned that if CO2 loss had occurred in

the molecules where a carboxyl group is present, a signal73mDa higher in mass than the described [M–C3H8]

+ and[M–(C3H8)2]

+ peaks would have been observed. A closerinspection of the spectra showed that this is not the case witheither of the Rhodamine species explored. Although we did

Figure 3. SORICIDspectraofRhodamineB (a), SulforhodamineB (b), Rhodamine B isothiocyanate (c), and Oxazine‐1 (d).

Figure 4. Possible fragmentation mechanisms: concerted (top) a

Copyright © 2011Rapid Commun. Mass Spectrom. 2011, 25, 1169–1172

observe a CO2 loss for Rhodamine B, it should be noted thatsuch a loss can only be found after the loss of two C3H8

fragments. Thus, formation of an [M–(C3H8)2–CO2]+ product

ion of noticeable intensity is detected at m/z 311.117. ForSulforhodamine B and Rhodamine B isothiocyanate, the CO2

losses were too weak to be detected.The results from the SORI CID experiments are displayed

in Fig. 3. This technique shows the same fragmentationproducts as the PD experiments. In all cases the formation ofthe [M–44]+ ion is observable. Due to the mass difference thision can be unambiguously attributed to the loss of a propanemolecule. Although the intensities of these product ions differsomewhat from those in the PD spectra, these ions are themost intense in the SORI CID spectrum. As discussed above,the second loss of 44Da can also be assigned to the loss of aC3H8 molecule. It should be noted that in case of RhodamineB isothiocyanate this second fragmentation is not observable.Even under higher collision gas pressures this reaction canonly be observed with low intensity.

The results mentioned above show that the structuralelement of resonantly stabilized ethyl‐substituted amine/imine groups is prone to lose C3H8 twice upon activation viamulti‐photon laser activation or by collisions with argon gasusing SORI CID. Losses of this group from other parts of thering system are very improbable. In fact, themechanism of thisloss is not directly evident from the structure of the molecule.

It is obvious that at least two different mechanisms for theloss of C3H8 can be responsible for the appearance of the prod-uct ions. As shown in Fig. 4 the reaction starts at the chargesite of the molecular cation or the first product ion. Clearlya concerted mechanism would lead to a methyl shift fromthe second ethyl group to form the propane moiety. Alter-natively, a sequential loss of an ethyl radical followedbymethylradical fragmentationwould lead to the same resulting iminumion. The loss of the second propane molecule is induced bythe shift of the charge center from the iminium group to theother amine function due to the resonance of aromatic ringsystem.

Regarding the results presented in this paper, we cannotdecide between the two mechanisms. It should be mentionedthat since the molecular cations of the investigated xanthenedyes are even‐electron ions, a radical mechanism would be aviolation of the so‐called ‘even‐electron’ rule.[6] This rulestates that an even‐electron cation will probably decay toanother cation and a neutral rather than perform a homolyticcleavage to yield a radical. We did not find any indication in

nd radical (bottom); adapted from Clemen.[7]

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J. Peters and J. Grotemeyer

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the mass spectra obtained by photodissociation or by CID forthe loss of an ethyl or methyl radical.However, a radical could be stabilised very well by the

delocalised electronic system, which would yield someprobability for its occurrence. Its absence in the spectrumwould not exclude the formation of the different intermedi-ates as the measurement takes place on a scale ofmilliseconds to seconds, which would give the radicalenough time to stabilise. Further experiments must beperformed to decide between the two mechanisms.

CONCLUSIONS

It has been shown that the [M–C3H8]+ and [M–(C3H8)2]

+ ionsin the photodissociation mass spectra, as well as in thecollisional activation experiments of Rhodamine B, are thetwo main dissociation products. This fragmentation has alsobeen found in the case of other dyes displaying the samexanthene backbone and the same substitution at the nitrogenatoms. However, the mechanism is not clearly understood

wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wil

and it will take further investigation to elucidate it. To gainfurther insight the different elementary reactions, we areinvestigating the effect of different substituents at the amineon the fragmentation pattern, which we will publish in afuture paper.

REFERENCES

[1] Q. Bian, M. W. Forbes, F. O. Talbot, R. A. Jockusch. Phys.Chem. Chem. Phys. 2010, 12, 2590.

[2] K. Chingin, H. Chen, G. Gamez, R. Zenobi. J. Am. Soc. MassSpectrom. 2009, 20, 1731.

[3] R. M. Brown, C. S. Creaser, H. J. Wright. Org. Mass Spectrom.1984, 19, 311.

[4] J. M. Ballard, L. D. Betowski. Org. Mass Spectrom. 1986,21, 575.

[5] C. Freudenhammer, J. Grotemeyer. Eur. J. Mass Spectrom.2010, 16, 489.

[6] F. W. McLafferty, F. Tureček. Interpretation of Mass Spectra,(4th edn.), University Science Books, Sausalito, CA, 1993.

[7] M. Clemen. Bachelor thesis, Universität Kiel, Germany, 2010.

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