Negative fast atom bombardment ionization ofaromatic sulfonic acids: unusual sample-matrixinteraction

Hung-Yu Lin†, George Gonyea, Susan Killeen and Swapan K. Chowdhury*‡Analytical Sciences Department, Sanofi Winthrop, Inc., 25 Great Valley Parkway, Malvern, PA 19355, USA

SPONSOR REFEREE: Dr. Cornelis E. C. A. Hop, Merck Research Laboratories, Rahway, NJ, USA

The most intense ion(s) in negative ion fast atom bombardment (FAB) mass spectra of 2- and 4-benzaldehyde sulfonic acid (BSA) in glycerol or 3-nitrobenzyl alcohol matrix corresponds to a covalentassociation of the analyte with one or two matrix molecules accompanied by the elimination of a molecule ofwater. The molecular ion [M ÿ H]ÿ, however, is of low abundance. The identity of the resulting ions[M � nA ÿ H2Oÿ H]ÿ (where M is the analyte and A is the matrix) was confirmed by exact massmeasurement using the peak matching technique. These covalent matrix-analyte complexes were notobserved when the sulfonic acid functionality in BSA was substituted with COOH, NO2, and OH or whenthe sulfonic acid was in salt form. These observations indicate that the free sulfonic acid group in BSA isresponsible for the covalent adduct formation. To our knowledge, analyte-matrix covalent association innegative ion FAB spectra of BSA has not been reported previously. Copyright# 2000 John Wiley & Sons,Ltd.

Received 24 January 2000; Accepted 25 January 2000

Aromatic sulfonic acids are well-known surfactants used assolubilizers for dyes and emulsifiers for liquid paraffins andfuels.1 The sulfonic acid derivatives of chalcones are used asfungicides.2 Benzaldehyde sulfonic acids (BSAs) arepivotal intermediates in the synthetic pathway for thepreparation of semicarbazones.3 Semicarbazones are com-mon antiviral drugs4 and were the first synthetic antiviraldrugs used in humans.5 Mass spectrometry with electronimpact ionization and chemical ionization has been usedextensively for the characterization of purified forms ofdifferent sulfonic acids.6 However, thermal decompositioncan be a major problem in generating the molecular ion.6

Fragmentation can also occur during ionization. Fragmenta-tion from either origin can be more problematic forinterpretation of spectra when the sample is not pure.

In this study we have examined the utility of fast atombombardment (FAB) and liquid secondary ionization massspectrometry (LSIMS)7 for the analysis of BSAs. In thenegative ion FAB analysis of 4-benzaldehyde sulfonic acid(4-BSA) in glycerol, we observed that the most intense peakin the spectrum is not the [Mÿ H]ÿ ion but corresponds to amuch higherm/z. The origin of this ion was not readilyunderstood. Additional unidentified ion peaks were alsoobserved. This observation was surprising because theliquid chromatographic analysis of this 4-BSA sampleshowed only one peak corresponding to 4-BSA. To establish

the purity of the sample it was necessary to identify theunknown peaks in the FAB mass spectrum. In an attempt tounderstand the origin and identity of these unknown peaksin the FAB spectrum, we investigated the 4-BSA sample ina number of matrices. Several other aromatic compoundswith sulfonic acid and aldehyde functional group(s) werealso investigated using FAB mass spectrometry.

EXPERIMENTAL

The FAB mass spectrometric analyses were conductedusing a Finnigan MAT (San Jose, CA, USA) SSQ-710single stage mass spectrometer equipped with an Ion Tech(Teddington, UK) FAB ionization gun. Data were acquiredin the centroided mode using Finnigan ICIS software on aDigital DEC5000/120 computer. The ionization conditionswere: ionizing atom beam, Xe; multiplier voltage,ÿ1000 V; conversion dynode voltage,ÿ15 kV; scan range,100–1000m/zunits; and scan time, 1 s.

The FAB data were acquired first on bare matrix andsubsequently sample-matrix mixture. Data for both the barematrix and the sample-matrix mixture were recorded in thesame data file. This procedure allows for the removal ofmatrix-related ions through subtraction of signals of thematrix ions from those of the sample-matrix mixture.

LSIMS experiments at low and high resolution innegative ion mode were performed on a double focusinghigh resolution mass spectrometer, Kratos Concept 1H,equipped with a Cs� gun that has been describedpreviously.8 For peak matching experiments, ions of interestwere bracketed using deprotonated polyethylene glycol ionsat a resolution of�5,000 (10% valley). Data were acquiredand processed using Kratos Mach3 software.

*Correspondence to: S. K. Chowdhury, Schering-Plough ResearchInstitute, K15-3-3700, 2015 Galloping Hill Road, Kenilworth, NJ07033, USA.E-mail: swapan.chowdhury@spcorp.com†Present address: Bristol-Myers Squibb Co., 6000 Thompson Road,East Syracuse, NY 13057, USA.‡Present address: Schering-Plough Research Institute, 2015 GallopingHill Road, Kenilworth, NJ 07059, USA.

Copyright# 2000 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.14, 520–522 (2000)

RESULTS AND DISCUSSION

A negative ion FAB mass spectrum of 4-benzaldehydesulfonic acidis shownin Fig. 1. Thespectrum wasacquiredin a glycerolmatrix. A numberof ion peaksareobservedinthe spectrum. Among the five most intenseions in Fig. 1,threewere readily identified to be [2A ÿ H]ÿ, [M ÿ H]ÿ,and [3A ÿ H]ÿ correspondingto m/z 183, 185, and 275;where A is a moleculeof glycerolandM is 4-benzaldehydesulfonic acid. The two other ions at m/z 259 and 351(designatedas?in Fig.1) arenotadductsof intactanalyteormatrix molecules.

In anattempt to identify theunknown peaksin Fig. 1, 4-benzaldehyde sulfonic acid was studied separately in 3-nitrobenzyl alcohol(3-NBA) andthioglycerolmatrices.Thespectrum of 4-BSA obtained in 3-NBA is shownin Fig. 2.Similar to the spectrum obtained in glycerol (Fig. 1), thespectrum in 3-nitrobenzyl alcohol also yielded (Fig. 2) alow-intensitypeakcorrespondingto the[M ÿ H]ÿ ion atm/z185with themostabundant ion at m/z473.Thespectrum of4-BSA obtainedin thioglycerol alsoproducedanunknownion at m/z383 (datanot shown).

Analysisof thespectragivenin Figs1 and2 revealssomecommon featuresto the origin of someof the unidentified

Figure 1. Negativeion FAB massspectrumof 4-benzaldehydesulfonicacidin glycerolmatrix. Ionsdesignated‘?’wereunexpected.Seetext for their identification.M is 4-BSA andA is glycerol.

Figure 2. Negativeion FAB massspectrumof 4-benzaldehydesulfonicacidobtainedfrom 3-nitrobenzylalcoholmatrix. M is 4-BSA andA is 3-nitrobenzylalcohol.

Copyright# 2000JohnWiley & Sons,Ltd. Rapid Commun.MassSpectrom.14, 520–522(2000)

UNUSUAL IONS IN FAB-MS OF AROMATIC SULFONICACIDS 521

ions. For example, m/z351 in Fig. 1, m/z473 in Fig. 2 andm/z 383 in the spectrum obtained with thioglycerol areconsistentwith formulae[M � 2Aÿ H2Oÿ H]ÿ, whereAis thematrix molecule andM is 4-BSA. Them/z259ion inFig. 1 is [M � A ÿ H2Oÿ H]ÿ. Thus, the commonfeatureof the FAB spectraof 4-benzaldehydesulfonic acid is theformation of analyte ions attached to one or two matrixmolecules with the loss of a molecule of water. Thisobservationis unusual becausetypical adductionsobservedin FAB spectraresult from the clusteringof analyteionswith intact matrix molecules, [M � nAÿ H]ÿ. To ourknowledgethese typesof analyte-matrix covalent associa-tion in negative ion FAB spectraof aromaticsulfonic acidshasnot beenpreviously reported.

The exactmassof m/z473wasdeterminedusing a peakmatching technique on a double focusing high resolutionmass spectrometer.8 This instrument was equippedwith aliquid secondaryionization source.The measured accuratem/z of the [M � 2Aÿ H2Oÿ H]ÿ ion obtained from the3-NBA matrix is 473.06582. The calculated m/z ofC21H17N2O9S is 473.06548. The excellent agreement of0.72ppm between the measured and the calculated m/zvalues confirms the identification of this unknown peakat m/z 473 to be [M � 2Aÿ H2Oÿ H]ÿ, where A is3-NBA.

To understandtheorigin of theseionsandthefactors thateffect their formation, we first changedthe sulfonic acidfunctional group to a -COOH, -NO2 or -OH group. TheFAB mass spectra of these materials obtained in 3-nitrobenzyl alcohol matrix did not produce [M � nAÿ H2Oÿ H]ÿ ions. Additionally, thesecovalent adductswerenot present in the FAB massspectraof 2-hydroxy-3-methoxybenzaldehyde, 4-hydroxy-3-methoxybenzalde-hyde, 3-formylbenzaldehyde, 2-carboxybenzaldehyde and4-carboxybenzaldehyde. These observations suggest thatthe sulfonic acid functionality is a requirement for theformation of [M � nAÿ H2Oÿ H]ÿ in the negative ionFAB spectra.

Next we investigatedthe FAB ionization of the sodiumsalt of 2-benzaldehydesulfonic acid (2-BSA). The FABspectrum of this sampleobtained in 3-nitrobenzylalcoholdid not yield [M � nAÿ H2Oÿ H]ÿ ions. However, whenthe sodium salt of 2-benzaldehyde sulfonic acid wasacidified with dilute nitric acid prior to FAB analysis, m/z473 ([M � 2Aÿ H2Oÿ H]ÿ) wasproduced.Theseresultsindicate that sulfonic acid in its acid form produces[M � nAÿ H2Oÿ H]ÿ in the FAB spectra. The proposedstructuresof m/z 259 and 351 in Fig. 1 are given below.Similar dehydration productscan be formed with 3-NBAandthioglycerol.

Recently, Boone et al.9 observed that phospolipidscontaining an amino group react in a triethanolamine(TEA) matrix to form abundantadductionsof m/z26and42

higher than the protonated (MH�) and deprotonated([M ÿ H]ÿ) analyteions.The authors9 proposedthat vinylgroups (�26) and acetyl groups (�42), generated underconditions of FAB from the TEA matrix, underwentassociation with thephospholipids to form theadductions.Additionally, Aubagnacet al.10 havesummarized severaltypesof reductionof analyte moleculesin FAB from theliterature, and also reportedthe observation of the samephenomenain Frit FAB mass spectrometry. However,analyte-matrix covalent association of benzaldehydesulfo-nic acidsin negativeion FAB analysishasnotbeenreportedpreviously, to our knowledge.

CONCLUSIONS

A free sulfonic acid group in 2- and 4-benzaldehydesulfonic acid (3-BSA was not investigated)appearsto beresponsible for the formation of [M � nAÿ H2Oÿ H]ÿ

ions in negative ion FAB spectra. The findings presentedhereindicate that the matrix moleculescanundergo beam-induced reaction with BSA in FAB/LSIMS ionization,producingotherwise unexpectedions andcomplicating theinterpretation of the spectra.

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Rapid Commun.MassSpectrom.14, 520–522(2000) Copyright# 2000JohnWiley & Sons,Ltd.

522 UNUSUAL IONS IN FAB-MS OF AROMATIC SULFONICACIDS