Two-phase Matrix-assisted LaserDesorption/Ionization: Matrix Selection andSample Pretreatment for Complex AnionicAnalytes

Michael Dale, Richard Knochenmuss and Renato Zenobi*Department of Chemistry, ETH-Zentrum, CH-8092, Zuerich, Switzerland

Two-phase matrices, consisting of a binary mixture of solid particulates and a low vapour pressure liquid, areshown to be effective for the production of both positive-ion and negative-ion laser desorption/ionization massspectra (LDI-MS). It was found that matrix selection can be guided by criteria developed previously forfast-atom bombardment mass spectrometry. Two-phase matrix-assisted (MA)LDI-MS of a variety of complexanionic analytes is presented. Sample pre-treatment using ‘drop dialysis’, or exposure to an acid-activatedNafion® cation-exchange membrane, substantially enhances the quality of negative-ion mass spectra.© 1997 by John Wiley & Sons, Ltd.

Received 21 November 1996; Accepted 30 November 1996Rapid Commun. Mass Spectrom. 11, 136–142 (1997)No. of Figures: 7 No. of Tables: 2 No. of Refs: 13

It has recently been demonstrated that two-phasematrices, mixtures of solid particulates and liquids, arean attractive alternative to traditional solid matrices forthe laser desorption/ionization of intermediate molec-ular weight analytes.1–3 It is thought that laser radiationis absorbed by the solid particulates, causing desorp-tion/ionization via rapid thermal evaporation of theliquid matrix. The binary mixtures have a number ofadvantages over their solid counterparts for matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS). Most significantly, no co-crystallizationof the matrix and analyte is required, and analytediffusion through the liquid component can result inthorough mixing and homogenization of the sample. Inaddition, the liquid matrix is not required to have a UVchromophore. It has been shown previously3 that, intwo-phase matrices, the liquid provides protons andcations to the analyte, enhances analyte ion yields,improves resolution and increases signal stability anduniformity.

The separation of the energy absorption (solidparticulates) and solvent (liquid matrix) functions addsa new degree of flexibility which we use here to extendthe application of two-phase MALDI to include thenegative-ion mass spectrometry of involatile, inter-mediate-molecular-weight analytes. The choice of liquidmatrices and solid particulates is discussed in thiscontext. It is noted that liquid matrix selection can beguided by similar criteria to those previously deter-mined in fast-atom bombardment mass spectrometry(FAB-MS). It is demonstrated that a two-phase matrix,consisting of diethanolamine and silicon particulates,can be used to obtain the mass spectra of complexorganic phosphates and sulphonates, which are mostreadily analysed in the negative-ion mode. Sample pre-treatment by dialysis and cation exchange is demon-strated to significantly improve mass spectral qualityfor these analytes. The use of both membrane drop

dialysis and Nafion® cation exchange membranes isalso discussed.

EXPERIMENTAL

The experiments were performed on a home-built 2 mlinear time-of-flight mass spectrometer. Ions wereextracted from the source region using a static 25 kVacceleration potential. Desorption was performed usingthe 337 nm output from a nitrogen laser (Laser ScienceInc. VSL-337ND-T), incident at ca. 30° to the samplesurface. Laser attenuation was achieved using glassslides and an adjustable iris. The laser spot size wasestimated to be an ellipse of axes ca. 0.1 × 0.2 mm.Typical laser pulse energies were in the range 20 µJ to60 µJ. Pulse energies were measured using a pyro-electric detector. Typically, 50 consecutive laser shotswere summed in order to enhance the signal-to-noiseratio of the mass spectra.

MATRIX PREPARATION

Matrix–particulate mixtures were prepared by dilutingthe appropriate liquid in methanol, typically 30% byvolume of liquid matrix in methanol, and subsequentlyadding to this an approximately equal volume ofparticulates. The resulting mixture was then mechani-cally shaken for up to 1 h to ensure thorough mixing ofthe particulates and the liquid matrix. A small volume(ca. 0.5 µL) of the resulting slurry was then applieddirectly to the sample holder. Following evaporation ofthe methanol, a thin layer of the two-phase matrix wasleft on the sample probe.

It is important to note that the use of two-phasematrices can lead to particulate contamination of theMALDI source or damage to nearby turbo-molecularpumps. We have found that the use of low laserdesorption powers, along with careful matrix prepara-tion, minimizes the amount of particulate ejection fromthe sample. In addition, we have replaced the turbo-molecular pump in the desorption chamber with adiffusion pump. We would counsel caution in the use of

* Correspondence to: R. Zenobi.Contract grant sponsor: Kommision fur Technologie undInnovation; Contract grant number: 3165.1

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 11, 136–142 (1997)

CCC 0951–4198/97/010136–07 © 1997 by John Wiley & Sons, Ltd.

this methodology in instruments which cannot be easilycleaned if contamination occurs.

SAMPLE PREPARATION

Typically, a 0.5 µL volume of a 10–3M (aqueous)

solution of the analyte was placed on the surface of thetwo-phase matrix mixture.

Desalination of anionic analytes by membrane dialy-sis was performed using the Millipore ‘drop dialysismethod’.4,5 Typically, 10 µL of the sample solution wasplaced on a VS-membrane (0.025 µm, Millipore). Themembrane was allowed to float on a 200 mM solution ofdiammonium hydrogen citrate. After ca. 15–30 min,0.5 µL of the sample was removed from the membranefor analysis.

Pre-treatment of anionic analytes using an H-Na-fion®-117 membrane was carried out in a mannersimilar to that reported elsewhere.6 A disc of themembrane was regenerated overnight using 1M HCland then floated on the concentrated HCl solution.Approximately 5 µL of sample solution was depositedon the floating membrane for 15–30 min prior toanalysis. Some losses due to evaporation were observedduring this period. Approximately 0.5 µL of the resid-ual solution was placed on top of the two-phase matrixmixture. Acid (1M HCl) activated cation-exchangebeads (Dowex, 50W-X8, mesh size 100–200 µm) werealso used effectively in this work for pre-treatment ofthe anionic analytes.7 However, the Nafion-117 mem-brane proved to be simpler in practice and gave morereproducible results. Thus, the results described in thispaper are limited to those obtained using the Nafion-117 membrane.

MATERIALS

All the materials used in these experiments wereobtained from commercial sources. Substance P, grami-cidin D, adenosine-5-monophosphate, adenosine-5triphosphate disodium salt and the Dowex 50WH-exchange beads were obtained from Sigma (Buchs,Switzerland). The sulphonated dyes; methyl orange,direct yellow 50, direct red 80 and Ni (II) phthalocya-nine tetrasulphonic acid tetrasodium salt were obtainedfrom Aldrich (Buchs, Switzerland). Cibachrome Bluewas obtained from Fluka (Buchs, Switzerland). Theliquid matrices, glycerol (spectrophotometric grade)and diethanolamine, diammonium hydrogen citrate, theNafion 117 cation-exchange membrane, silicon (ca.40 µm particulates) and synthetic graphite (ca. 1–2 µmparticulates), were obtained from Aldrich.

RESULTS AND DISCUSSION

Choice of matrix components

Liquid matrix. Figure 1(a) and (b) shows the laserdesorption/ionization mass spectra of the peptideSubstance P obtained using a glycerol + silicon binarymatrix in positive- and negative-ion modes respectively.In the positive-ion mode the base peak of the spectrumcorresponds to the protonated molecule of Substance P(m/z 1348), while in the negative-ion mode no molec-ular ions are observed. Figure 2 shows the mass spectraobtained, in both positive- and negative-ion modes,using a diethanolamine + silicon binary matrix. Theselatter mass spectra are notably different from thoseobtained with the glycerol matrix. In the positive-ion

Figure 1. UV laser desorption/ionizationmass spectra of substance P obtained using aglycerol + silicon binary matrix in (a) pos-itive-ion and (b) negative-ion mode. A highintensity peak due to protonated moleculescan be observed in the positive-ion mode,whilst no molecular ion is observed in thenegative-ion mode. (X — derived from tracediethanolamine contamination in the spec-trometer source).

Figure 2. UV laser desorption/ionizationmass spectra of substance P obtained using adiethanolamine + silicon binary matrix in (a)positive-ion and (b) negative-ion mode. Anion corresponding to the addition of asodium ion to the molecule dominates in thepositive-ion mode, whereas an intense ioncorresponding to the molecule is observed inthe negative-ion mode.

TWO-PHASE MALDI FOR COMPLEX ANIONIC ANALYTES 137

mode only low intensity signals are observed in themolecular ion region of the mass spectrum; these beingdominated by a cationized molecule [M + Na]+ at m/z1370, rather than a protonated species. In the negative-ion mode, an intense ion signal corresponding todeprotonated Substance P is observed (m/z 1346).

Figures 1 and 2 demonstrate that protonation isfavoured by use of glycerol and deprotonation isfavoured by use of the diethanolamine-containingmatrix. This observation can be explained by thebasicities of the liquid matrices. At present, it is notclear whether ionization in two-phase MALDI is asolution-phase phenomenon or whether it occurs in thegas-phase during the desorption event. It is likely thatseveral mechanisms contribute to the final desorption/ionization yield. However, a previous study8 has shownthat gas-phase energetics dominate liquid-phase ener-getics in FAB-MS, suggesting that gas-phase protonaffinities can serve as a useful guide to the protonatingor deprotonating power of liquid matrices. Thus,glycerol, with a gas-phase proton affinity of 874 kJ/mol,9 is a more acidic matrix and a potential protondonor. Diethanolamine, on the other hand, with a gas-phase proton affinity of 954 kJ,9 is a proton-acceptingmatrix. In the light of these results, the negative-ionlaser desorption/ionization mass spectra of the complexanionic analytes discussed later were all obtained usingbinary matrices in which diethanolamine was the liquidphase.

It is important to note that, for analytes whichpreferentially form cationized molecules in the posi-tive-ion mode, considerations based on basicities areimportant only for anion formation. For example, usinga diethanolamine + silicon binary matrix for the laserdesorption/ionization mass spectrometry of GramicidinD (not shown), it is possible to obtain intense sodiatedmolecules in the positive-ion mode and deprotonatedmolecules in the negative-ion mode. Molecular ionformation in the positive-ion mode is not limited by thebasicity of the liquid component of the matrix, butrather by the concentration of cationizing agent (Na+ ),solubility of the analyte, fluidity of the matrix, etc.

Clearly, selection of the liquid component can becritical in optimizing the performance of a two-phasematrix. Physically, a successful matrix must have a lowvapour pressure to ensure vacuum compatibility and besufficiently fluid to enable sample flow over theparticulates. Chemically, it must be able to bothdissolve the target analyte and assist in its ionization. Inaddition, the chosen matrix must contribute minimalspectral interference in the mass range of interest.These properties are common to liquids used asmatrices in FAB-MS.

The results presented here, and elsewhere,3 suggestthat, for two-phase MALDI-MS, matrix selection maybe made using criteria similar to those developed forFAB-MS. The importance of this observation lies in thefact that one can draw on an extensive database of FABmatrix behaviour in order to assist selection of effectivetwo-phase MALDI matrices. The influence of variousproperties of liquid matrices on the FAB mass spec-trometry of involatile molecules has been reviewedpreviously.10–12 In addition, the physical and chemicalproperties of the more common liquid matrices havebeen tabulated by Cook et al.13 with the specificintention of assisting rational matrix selection in FAB-MS.

Solid particulates. In addition to the liquid componentof a binary matrix, the choice of solid particulates canaffect the quality of the resulting mass spectra. Figure3(a) and (b) shows the negative-ion mass spectra ofadenosine-5-monophosphate obtained using the two-phase matrices diethanolamine + silicon and diethano-lamine + graphite respectively. Both spectra containions, corresponding to deprotonated molecules, ofsimilar intensity at m/z 346, i.e. the choice of partic-ulates does not dramatically affect the absolute ionintensities observed. Furthermore, neither mass spec-trum exhibits significant interference from signalscorresponding to the diethanolamine. However, thespectrum obtained using graphite particulates is domi-nated in the low-mass region by intense carbon-clusteranions. These are absent in the mass spectrum obtainedusing silicon particulates. In the latter case, other thanthe ubiquitous signal at m/z 26 (C2H2

+ ), the low-massion signals can be attributed to fragments of the targetanalyte.

One of the goals in investigating two-phase matriceswas to develop a robust, simple and flexible, pre-parative methodology which could be used to obtainthe laser desorption/ionization mass spectra of a widerange of molecular analytes. Due to the presence ofcluster ions, graphite particulates are not optimal forthe production of low-molecular-weight negative ions.The combination of diethanolamine and silicon, whichcontributes minimal interference to the mass spectrumat low masses, can be considered a preferable combina-tion in this case.

Both binary matrices were prepared as outlined inthe experimental section. It was noted that the largersilicon particulates quickly settled out of the methanol

Figure 3. Negative-ion UV laser desorp-tion/ionization mass spectra of adenosine-5-monophosphate obtained using (a) die-thanolamine + silicon and (b) diethanola-mine + graphite binary matrices. Thespectra are plotted using equivalent inten-sity axes. The high intensity carbon clusteranion peaks which dominate the low-massregion on using a graphite-containingmatrix are notably absent in the case ofsilicon.

138 TWO-PHASE MALDI FOR COMPLEX ANIONIC ANALYTES

slurry whilst the graphite particulates remained sus-pended over extended periods of time. It provednecessary to agitate or stir the silicon matrix prior touse, in order to ensure adequate mixing of thecomponents. This problem may be overcome with theuse of smaller silicon particulates. Furthermore, it wasobserved that the mass accuracy and reproducibilitywas improved if a ‘flat’ surface was prepared prior toaddition of the sample. This could best be achievedusing a well-mixed slurry which spread evenly over theprobe prior to evaporation of the methanol.

Mass spectrometry of complex anionic analytes

The laser desorption/ionizaton mass spectrometry of anumber of complex anionic analytes — adenosine-5-tri-phosphate disodium salt, Cibachrome Blue, directyellow 50, nickel (II) phthalocyanine tetrasulfonic acidtetrasodium salt and direct red 80 — was studied usinga diethanolamine + silicon two-phase matrix. The struc-tures of these analytes are shown in Fig. 4. In general,the negative-ion mass spectrometry of these materialsproved much more successful than analysis in thepositive-ion mode. In all cases, enhanced performancewas obtained following desalination of the sample priorto analysis. Singly charged ions are observed, allowingfor simple spectral interpretation.

Figure 5(a), (b) and (c) shows the negative-ion massspectra obtained for adenosine-5-triphosphate (ATP)disodium salt (mol. wt. 551 Da) without desalination,following dialysis, and with Nafion cation exchange,respectively. Clearly, sample pre-treatment dramati-cally improves the information content of the massspectra. In Fig. 5(a), where no sample pre-treatmentwas used, only a low intensity molecular ion peak canbe observed at m/z 506 corresponding to [M–2Na + H]–

ions. The intense ion signals at m/z 79 and 181 can beassigned to the characteristic phosphate fragments[PO3]– and [P2O6Na]– respectively.

Following 15 min pre-treatment on a dialysis mem-brane the quality of the mass spectrum obtained (Fig.5(b)) is substantially improved. The base peak of themass spectrum, at m/z 191, can be assigned to thepresence of the diammonium hydrogen citrate. How-ever, strong analyte molecular ion signals are alsoobserved at m/z 506 and 528. It appears that thedisodium salt is proton exchanged during dialysis. Thepeak at m/z 506 can be now assigned to the deproto-nated free acid of ATP. The peak at m/z 528 can beattributed to singly sodiated ATP, i.e. a species in whichonly one of the sodium ions was replaced duringdialysis.

A similar spectrum (Fig. 5(c)) is obtained following30 min pre-treatment on a Nafion membrane. The base

Figure 4. Structures of the complex anionic analytes used in this study.

TWO-PHASE MALDI FOR COMPLEX ANIONIC ANALYTES 139

peak at m/z 506 corresponds to the deprotonated freeacid of ATP suggesting that complete H+ -exchange hasoccurred on the membrane. No signal is observedcorresponding to the singly, or doubly, sodiated molec-ular ion. A number of lower intensity ions are observedwhich correspond to various fragments of the molec-ular ion. Currently, it is not clear whether these aresolely the products of reactions in the mass spectrome-ter or are partially derived from hydrolysis occurring insolution during pre-treatment. More importantly,intense ion signals are observed at ca. m/z 859 and 881.Similar peaks can be observed in the spectrum obtainedafter dialysis, albeit with much lower intensities. Theirenhanced intensities following treatment on the cation-exchange membrane suggests that these are also theproducts of reactions occurring during pre-treatment,not the result of clustering in the ion source of thespectrometer.

The positive-ion mass spectra of disodium-ATP weregenerally of much lower quality than the negative-ionspectra. Sample pre-treatment did little to improve themolecular ion intensities, which were always small orabsent, and any low mass fragments were obscured byhigh intensity positive ions derived from the matrixitself.

Similar mass spectra could be obtained for a varietyof sulphonated dyestuffs. Mass spectra of lower

molecular-weight materials, e.g. methyl orange,(CH3)2NC6H4N = NC6H4SO3Na (mol. wt. 327 Da),could be readily obtained using direct laser desorption,i.e. no matrix was required. However, higher molecular-weight materials required matrix assistance to enhancethe ion yield. Figure 6(a), (b) and (c) shows thenegative-ion mass spectra obtained for the triplysulphonated dye, Cibachrome Blue (mol. wt. 840 Da),using no sample pre-treatment, following dialysis andwith Nafion cation exchange, respectively. While themass resolution of the instrument used in theseexperiments was insufficient to separate unit masses inthis mass range, it is assumed that molecular ions arethe result of proton loss, as observed in previousexamples.

With no sample pre-treatment (Fig. 6(a)) the spec-trum is dominated by a peak at m/z 26. The molecularion region contains only low intensity ion signalscorresponding to singly and doubly sodiated adductspecies. In addition, two intense fragment species canbe observed at m/z 197 and m/z 335. Following dialysis(Fig. 6(b)) stronger molecular ion signals are observedalong with the sodiated adduct ions. There is littleevidence, in this case, that significant cation exchangehas taken place during dialysis. The mass spectrumobtained using the Nafion cation-exchange membrane(Fig. 6(c)) is significantly different. In this case, almost

Figure 5. Negative-ion laser desorption/ionization mass spectra ofadenosine-5-triphosphate disodium salt obtained using a diethano-lamine + silicon binary matrix. Spectrum (a) was obtained withoutany sample pre-treatment, (b) following drop-dialysis and (c)following exposure of the sample solution to an acid-activatedNafion exchange membrane. In the spectra, M1 ≡ (ATP + 2Na),M2 ≡ (ATP + Na), M3 ≡ ATP (as free acid).

Figure 6. Negative-ion UV laser desorption/ionization mass spectraof Cibachrome Blue obtained using a diethanolamine + siliconbinary matrix. Spectrum (a) was obtained without any sample pre-treatment, (b) following drop-dialysis and (c) following exposure ofthe sample solution to an acid-activated Nafion exchange mem-brane. In the spectra, M ≡ trisodium salt, M1 ≡ free acid.

140 TWO-PHASE MALDI FOR COMPLEX ANIONIC ANALYTES

complete exchange of sodium ions for protons hasoccurred during sample pre-treatment. This results inthe generation of an intense, deprotonated, molecularion signal at m/z 773. A number of adduct peaks,including the triply sodiated molecular ion, can still beobserved above this mass but are now present with onlyrelatively low intensities. Furthermore, significant frag-mentation is observed in the mass spectrum; the basepeak corresponding to a fragment with m/z 197. Afurther intense peak is observed at m/z 512. Character-istic losses of — SO3 moieties can be observed fromboth the ions corresponding to deprotonated moleculesand the fragment ions of m/z 512.

It should be noted that the use of desalination pre-treatments also resulted in the successful production ofpositive-ion mass spectra for Cibachrome Blue. Thesetypically consisted of intense signals due to protonatedmolecules. (m/z 841), where the three sodium atomswere retained on the sulphonic acid groups. Followingextended exposure to the acid-activated Nafion cation-exchange membrane, substantial fragmentation, asso-ciated with successive losses of — SO3 groups, wasobserved. A similar phenomenon was observed in thecase if Ni (II) phthalocyanine tetrasulphonic acid,tetrasodium salt. However, as noted previously, in thepositive-ion mode, low-mass fragments are obscured byintense signals derived from the matrix.

The use of dialysis membranes, in comparison withthe use of acid-activated Nafion, typically results in thegeneration of simpler, more readily interpretable nega-tive-ion mass spectra. Figure 7(a), (b) and (c) shows thenegative-ion mass spectra of direct yellow 50 (mol. wt.957 Da), Ni (II) phthalocyanine tetrasulphonic acidtetrasodium salt (mol. wt. 979.5 Da) and direct red 80(mol. wt. 1373 Da), respectively. Simple mass, spectradominated by signals derived from the diammoniumhydrogen citrate (m/z 191 and 407), are observed. Ineach case, significant proton exchange has occurred.Direct yellow 50 and the sulphonated Ni(II) phthalocyanine undergo predominantly completeexchange of sodium ions for protons yielding intensesignals due to deprotonated molecules at m/z 868 andm/z 890.5, respectively. The mass spectra of both theseanalytes have ion signals which can be assigned tospecies that retain a singly sodiated sulphonate group,i.e. that have not undergone complete exchange duringdialysis. In the case of direct red 80, which has sixsodiated sulphonic acid groups, dialysis results in thecomplete exchange of all six sodium ions for protonsresulting in a peak due to deprotonated molecules atm/z 1235. However, the most intense signal in thisregion corresponds to a molecular ion at m/z 1257, thathas retained one of its sodiated sulphonate groups.

CONCLUSIONS

Two-phase liquid matrices can be used effectively togenerate both positive-ion and negative-ion mass spec-tra. A glycerol + graphite combination was previouslydemonstrated to be very generally useful for positive-ion formation.3 A diethanolamine + silicon combinationhas proved to be a very generally useful matrix fornegative-ion formation. The use of glycerol and dietha-nolamine liquid matrices favours the production of ionsdue to protonated and deprotonated analytes, respec-tively. This closely parallels their use in FAB-MS, whichsuggests that, in the development of optimal matrixcombinations for specific applications of two-phase

MALDI-MS, one could draw on the extensive databaseof published work already available for FAB-MS.

A complete mechanism for desorption/ionizationusing two-phase matrices has yet to emerge. However,in line with previous FAB investigations, it has beendetermined that the use of a more acidic matrix,glycerol, promotes protonation, whilst a more basicmatrix, diethanolamine, enhances the yield of neg-atively charged, deprotonated molecules. In addition,whereas graphite and silicon particulates appear to beequally effective as energy transfer media, the produc-tion of high intensity carbon cluster anions fromgraphite means that the use of silicon is optimal in thenegative-ion mode.

It has been demonstrated that a simple two-phasematrix of silicon and diethanolamine can be used toobtain the laser desorption/ionization mass spectra ofcomplex anionic analytes. The same preparation hasproved effective for a variety of molecular classes.Negative-ion mass spectra were typically of higherquality than the positive-ion mass spectra. In all cases,singly charged negative ions are observed, providing forsimple spectral interpretation. It was determined thatdesalination and cation exchange significantly improvethe spectral quality for complex anionic analytes. Bothdrop dialysis and exposure to a Nafion membrane can,

Figure 7. Negative-ion UV laser desorption/ionization massspectra of (a) direct yellow 50. In this spectrum, M ≡ (directyellow 50 as the free acid), M1 ≡ (direct yellow 50–1Na, 3 H), (b)Ni (II) phthalocyanine tetrasulphonic acid tetrasodium salt and(c) direct red 80, obtained using a diethanolamine + silicon matrix.In this spectrum, M ≡ (direct red 80 as the free acid), M1 ≡ (directred 80–1Na, 5 H) and M2 ≡ (direct red 80–2Na, 4 H). Each samplesolution was desalinated using drop-dialysis prior to analysis.

TWO-PHASE MALDI FOR COMPLEX ANIONIC ANALYTES 141

in some cases, result in complete exchange of sodiumions for protons to produce the free acid. The sub-sequent deprotonation of the free-acid groups producesintense, negatively charged ions. Furthermore, whereasthe use of dialysate with the dialysis membrane resultsin some spectral interference, the use of an acid-activated Nafion membrane appears to result in theproduction of extra fragment ions in the massspectrum.

Although this report focuses on the application oftwo-phase matrices to the mass spectrometry of com-plex anionic analytes, the methodology would appear tohave much broader potential. The obvious parallelsbetween two-phase MALDI and FAB-MS, e.g. the useof a liquid matrix component, suggest that MALDI-MS, hitherto predominantly associated with the massspectrometry of high molecular weight species, could beused to investigate the same variety of analytescurrently investigated using FAB. Such an extension ofthe capabilities of laser desorption/ionization massspectrometry, in tandem with the relatively inexpensivenature of the instrumentation, could result in thetechnique being increasingly adopted as a mainstreamanalytical mass spectrometric method.

Acknowledgements

MJD thanks the Royal Society of London for a European Exchange

Fellowship. Partial financial support by Ciba-Geigy (Basel, Switzer-land) and the Kommision für Technologie und Innovation (KTIGrant 3165.1) is gratefully acknowledged. The authors would like tothank Olaf Börnsen (Ciba-Geigy, Basel) for many fruitfuldiscussions.

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