Characterization of Various Analytes Using Matrix-Assisted Laser

Certain limitations of DCTB are noted, for example, in the analysis of water-soluble compounds such as peptides, proteins, and oligonucleotides, and g...
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Anal. Chem. 2006, 78, 199-206

Characterization of Various Analytes Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2enylidene]malononitrile Matrix Mark F. Wyatt,* Bridget K. Stein, and A. Gareth Brenton

EPSRC National Mass Spectrometry Service Centre, Department of Chemistry, University of Wales Swansea, Swansea SA2 8PP, U.K.

Recently, our research has focused on the transfer of analyses from fast-atom bombardment (FAB) and liquid secondary ion mass spectrometry (LSIMS) to matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS). The driving forces for this work are the relative advantages and disadvantages associated with each technique. The soft ionization of thermally labile and ionic materials is possible with the techniques of FAB and LSIMS. The soft ionization of peptides and other biomolecules is also possible, as well as the analysis of organometallic and coordination compounds, though there may be significant fragmentation. The analyte ion beam can be maintained for tens of minutes, which is extremely useful for experiments with multiple analyzers. However, there are also many disadvantages. FAB and LSIMS both suffer significant

chemical background from the matrix, and alkali salts and buffers may suppress ionization. The analyte must be soluble in the liquid matrix, and this solubility is inversely proportional to molecular weight; hence sensitivity is limited as molecular weight increases. The observed ions only reflect the composition at the surface of the sample, and hydrophilic components, which can remain in the bulk of the sample, may be discriminated against. FAB and LSIMS are also generally not very successful for nonpolar molecules. They are slow to implement, so automation is not practical, and the ion source and optics quickly become very dirty, due to the spluttering nature of forming the ion beam. They are also not the most popular techniques with manufacturers, meaning the choice of instrumentation is limited and cost may be relatively high. Interestingly, MALDI-TOFMS has already superseded these techniques in the soft ionization of biomolecules and has been extended to analysis of synthetic polymers. The technique has high sensitivity, ∼5 fmol, and relatively high tolerance to salt and buffer impurities. The widest possible mass range is covered, and it can be coupled to chromatographic techniques, with the automated high throughput of chemically similar analytes also possible. However, no technique is without its disadvantages, and MALDI-TOFMS also suffers from significant chemical background from the matrix. Furthermore, sample preparation can often be slow and problematic when performed manually. Due to the explosion of research and development in the life sciences, the technique is popular with manufacturers, and therefore, instrumentation is becoming widespread. MALDI-TOFMS has already superseded FAB/LSIMS for a variety of samples, so extending this to coordination, organometallic, and nonpolar compounds is the next logical step. Little work has been done in this area so far, but interest has increased significantly in recent years. The first study of this kind was by Juhasz and Costello, who analyzed ferrocene and ruthenocene oligomers using 2-(4-hydroxyphenylazo)benzoic acid, quinizarin, dithranol, and 9-nitroanthracene matrixes.1 They noted that positive radical ions and not protonated ions were produced, even

* To whom correspondence should be addressed. E-mail: m.f.wyatt@ swansea.ac.uk. Tel: +44 (0) 1792 295653. Fax: +44 (0) 1792 295554.

(1) Juhasz, P.; Costello, C. E. Rapid Commun. Mass Spectrom. 1993, 7, 343351.

2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) is a nonpolar, aprotic matrix and was used in the analysis of a variety of compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The classes of compounds include coordination compounds, organometallics, conjugated organic compounds (including porphyrins and phthalocyanines), carbohydrates, calixarenes, and macrocycles. For some samples, comparisons are made with spectra acquired with the use of 1,8,9-trihydroxyanthracene (dithranol), 2,5-dihydroxybenzoic acid, and 2,4,6-trihydroxyacetophenone matrixes. Traditionally, the majority of these compounds would have been analyzed by fast-atom bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS), or electrospray techniques, but this work shows that MALDI-TOFMS using DCTB has advantages over these techniques, particularly FAB and LSIMS. Certain limitations of DCTB are noted, for example, in the analysis of water-soluble compounds such as peptides, proteins, and oligonucleotides, and good working practices for the use of the matrix are also outlined.

10.1021/ac050732f CCC: $33.50 Published on Web 11/18/2005

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with acidic polar matrixes. These observations are not so surprising given that similar observations are made when compounds of this type are analyzed by FAB/LSIMS. Other groups have analyzed neutral palladium metallodendrimers,2,3 metalloporphyrins and metallophthalocyanines,4,5 and metallosupramolecular assemblies,6,7 with acidic, basic, and neutral polar matrixes, and also observed radical cations. Polar matrixes have also been used to characterize ionic ruthenium metallodendrimers8 and ruthenium/ osmium/iridium bypyridine coordination complexes,9,10 though positive ions resulting from counterion loss(es) are observed in these instances. Positive radical ions have been observed for nonpolar compounds such as fullerenes11 and conjugated polymers.12 However, there was an element of fortune in these observations, as the polar matrixes were not specifically designed to promote radical ion formation. This was addressed by Limbach’s group, who experimented with nonpolar matrixes for promoting radical ion formation for various ferrocene derivatives and outlined the mechanism for charge transfer in the MALDI process.13,14 One such matrix is 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), which has been shown to be very effective for the characterization of fullerenes,15,16 rhodium coordination complexes,17 and giant metal oxide spheres.18 In this paper, we show initially an extended range of nonpolar, coordination, and organometallic compounds that may be characterized using DCTB. The relative softness of this matrix compared to other polar matrixes is highlighted for certain samples. For positive mode, dithranol and 2,5-dihydroxybenzoic acid (DHB) were chosen for comparison, as both are good, general purpose matrixes, while the latter is often used for smallmolecule work. 2,4,6-Trihydroxyacetophenone (THAP) was chosen for negative mode, due to its wide usage in the analysis of (2) Huck, W. T. S.; Prins, L. J.; Fokkens, R. H.; Nibbering, N. M. M.; van Veggel, F.; Reinhoudt, D. N. J. Am. Chem. Soc. 1998, 120, 6240-6246. (3) van Manen, H. J.; Fokkens, R. H.; Nibbering, N. M. M.; van Veggel, F.; Reinhoudt, D. N. J. Org. Chem. 2001, 66, 4643-4650. (4) Lidgard, R.; Duncan, M. W. Rapid Commun. Mass Spectrom. 1995, 9, 128132. (5) Srinivasan, N.; Haney, C. A.; Lindsey, J. S.; Zhang, W. Z.; Chait, B. T. J. Porphyrins Phthalocyanines 1999, 3, 283-291. (6) Schubert, U. S.; Eschbaumer, C. J. Inclusion Phenom. Macrocyclic Chem. 1999, 35, 101-109. (7) Stulz, E.; Mak, C. C.; Sanders, J. K. M. J. Chem. Soc., Dalton Trans. 2001, 604-613. (8) Bodige, S.; Torres, A. S.; Maloney, D. J.; Tate, D.; Kinsel, G. R.; Walker, A. K.; MacDonnell, F. M. J. Am. Chem. Soc. 1997, 119, 10364-10369. (9) Hunsucker, S. W.; Watson, R. C.; Tissue, B. M. Rapid Commun. Mass Spectrom. 2001, 15, 1334-1340. (10) Ham, J. E.; Durham, B.; Scott, J. R. J. Am. Soc. Mass Spectrom. 2003, 14, 393-400. (11) Sun, Y. P.; Ma, B.; Bunker, C. E.; Liu, B. J. Am. Chem. Soc. 1995, 117, 12705-12711. (12) Ra¨der, H. J.; Spickermann, J.; Kreyenschmidt, M.; Mu ¨ llen, K. Macromol. Chem. Phys. 1996, 197, 3285-3296. (13) McCarley, T. D.; McCarley, R. L.; Limbach, P. A. Anal. Chem. 1998, 70, 4376-4379. (14) Macha, S. F.; McCarley, T. D.; Limbach, P. A. Anal. Chim. Acta 1999, 397, 235-245. (15) Ulmer, L.; Mattay, J.; Torres-Garcia, H. G.; Luftmann, H. Eur. J. Mass Spectrom. 2000, 6, 49-52. (16) Brown, T.; Clipston, N. L.; Simjee, N.; Luftmann, H.; Hungerbu ¨ hler, H.; Drewello, T. Int. J. Mass Spectrom. 2001, 210, 249-263. (17) Go ¨ttker-Schnetmann, I.; Aumann, R.; Bergander, K. Organometallics 2001, 20, 3574-3581. (18) Mu ¨ ller, A.; Diemann, E.; Shah, S. Q. N.; Kuhlmann, C.; Letzel, M. C. Chem. Commun. 2002, 440-441.

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low molecular weight oligonucleotides. The advantages of MALDITOFMS, as compared to FAB/LSIMS and electrospray ionization (ESI), are given in terms of general clarity of spectra and unambiguous analyte identification. Finally, we examined the usefulness of DCTB for the characterization of polar analytes, and the disadvantages DCTB may have as a MALDI matrix.

EXPERIMENTAL SECTION Chemicals. All samples (1-17; see structures in Figure 1) were submitted for analysis to the EPSRC National Mass Spectrometry Service Centre (NMSSC),19 as part of the normal operation of the Centre. DCTB, dithranol, DHB, THAP, and 3-nitrobenzyl alcohol (3-NOBA) matrixes (highest purity available) were all purchased from Fluka (Dorset, U.K.). HPLC grade dichloromethane (DCM), acetonitrile (MeCN), and methanol (MeOH) solvents were purchased from Fischer Scientific (Loughborough, U.K.). Milli-Q water was used where appropriate. Sodium iodide, lithium chloride, potassium acetate (KOAc), and diammonium hydrogen citrate (DAC) salts were purchased from SigmaAldrich (Dorset, U,K,). MALDI Sample Preparation. DCTB and dithranol matrix solutions were made to a concentration of 10 mg mL-1 in DCM. DHB matrix solution was made to a concentration of 10 mg mL-1 in 1:1 (v/v) DCM/MeCN. In relation to 16, both DHB and THAP matrix solutions were made to a concentration of 10 mg mL-1 in 1:1 (v/v) H2O/MeCN. All salt solutions were made to a concentration of 10 mg mL-1 in MeOH, with the exception of DAC, which was made to a concentration of 50 mg mL-1 in H2O. Sample solutions were made to a concentration of 1 mg mL-1 in customerspecified solvent, generally DCM. However, it is often not possible to weigh an exact amount of sample due to the minuscule amounts submitted to the Centre. In these cases, 200 µL of solvent was added to the sample vial. A 1-µL aliquot of sample solution was vortex-mixed with 49 µL of matrix solution and with 0.5 µL of LiCl/NaI/KOAc salt solution as required. Where THAP was used, matrix solution was mixed 9:1 with the DAC solution, prior to mixing with the sample. A 0.5-µL sample of the final mixture was spotted onto the sample plate (gold-plated, deep-welled plates for organic solvent-based mixtures; stainless steel plates for aqueousbased mixtures) and allowed to dry. A stream of cool air was used to dry aqueous samples more quickly. Mass Spectrometry. MALDI-TOFMS spectra were acquired using an Applied Biosystems Voyager DE-STR spectrometer (Framingham, MA), which is equipped with a nitrogen laser (λ ) 337 nm). The instrument was operated in reflectron mode and in both positive and negative ion modes. The accelerating voltage was 20 kV, while the grid voltage was maintained at 66%. The delay time was optimized for each sample, as was the laser fluence, which was attenuated to just above the threshold of ionization for each sample. The laser was fired at a frequency of 3 Hz, and spectra were accumulated in multiples of 25 laser shots, with 100 shots in total, unless stated otherwise. Postacquisition processing of data was performed by utilizing Data Explorer V4.0 software supplied by Applied Biosystems. Theoretical isotope distributions were generated at a resolution of 15 000 fwhm. (19) http://www.swan.ac.uk/nmssc/.

Figure 1. Structural representations for compounds 1-17. aExact stereochemical structure of the saponin has yet to be determined fully.

ESI analyses were performed using a Waters ZQ4000 single quadrupole spectrometer (Manchester, U.K.). Samples were loop injected into a stream of water/methanol (1:1 v/v) using the customer-specified solvent for dissolution. The capillary (ionizing) voltage was either +3.6 kV for positive ion mode or -2.8 kV for negative ion mode. Postacquisition processing of data was

performed by utilizing MassLynx V4.00 software supplied by Waters. Theoretical isotope distributions were generated at a halfheight peak width of 0.1 Da. LSIMS analysis was performed using a MAT 95XP doublefocusing mass spectrometer (ThermoFinnigan, Bremen, Germany). Cesium ion bombardment was used at 20-22 kV energy Analytical Chemistry, Vol. 78, No. 1, January 1, 2006

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on to the sample dissolved in a matrix liquid (3-NOBA). Postacquisition processing of data was performed by utilizing Qual Browser V1.4 software supplied by ThermoFinnigan. Theoretical isotope distributions were generated at a resolution of 15 000 fwhm. RESULTS AND DISCUSSION Neutral, Nonpolar, Organic Compounds. Initial studies began with the choice of a class of conjugated compound, namely, phthalocyanines. Compound 120 was analyzed with DCTB, dithranol, and DHB matrixes (see Figure S-1 of Supporting Information). There is little difference observed on initial comparison of the three spectra, as all show the base peak to be the target compound. However, a closer look at the base peak of each spectrum reveals that in the DCTB spectrum it is at m/z ) 1171, corresponding to the positive radical ion species, but it is at m/z ) 1172 in the dithranol and DHB spectra, indicating the protonated species. There is also a peak at m/z ) 1171; so it is likely that a mixture of positive radical ion and protonated species is observed. Surrounding the base peak are several other ions due to impurities; the higher mass ions do not correspond to simple metal adducts, and the lower mass ions do not correspond to sensible fragments. The dithranol and DHB spectra were acquired at the same laser power, which is considerably greater than the power used to acquire the DCTB spectrum. The DHB spectrum has the lowest signal-to-noise ratio, and there are some weak matrix ions toward the lower mass range. While both dithranol and DCTB spectra have comparable signal-to-noise ratios, there are several, fairly significant, matrix ions visible in the latter. This phenomenon is illustrated better with 2,21 a porphyrin with a pendant triflate group. Figure 2 shows an expansion of the molecular ion region for 2, together with a theoretical isotope distribution for the radical ion (12C isotope at 100% relative intensity at m/z ) 762). The DCTB spectrum provides a very close match with the theoretical distribution and strong evidence for the correct empirical formula of the sample. With dithranol, the m/z ) 762 peak is only at 60% relative intensity, but it is fairly difficult to substitute an element or group to achieve this and not upset the remainder of the distribution. Therefore, one might start to think about a mixture of species, even when one is analyzing a complete unknown. Additionally, the m/z ) 762 peak in the DHB spectrum is only at 10% relative intensity, which may indicate that there is a boron atom in the sample or even that a lithium adduct is being observed. Hence, there is a clear advantage to using DCTB for this and similar compounds. We have also used DCTB to characterize conjugated oligomers based upon fluorene and thiophene and have observed positive radical ions. These compounds are synthesized stepwise and should be monodisperse, so in a fashion similar to dendrimers, the purity and extent of reaction can be checked with MALDI. Data have been obtained for oligomers up to roughly 4.5 kDa without any problems. Additionally, other techniques that commonly utilize quadrupole or sector mass analyzers, such as ESI and FAB/LSIMS, may begin to struggle at this mass. (20) Unpublished work. Sample submitted to the NMSSC by M. J. Cook and I. Chambrier, University of East Anglia, U.K. (21) Cammidge, A. N.; Scaife, P. J.; Berber, G.; Hughes, D. L. Org. Lett. 2005, 7, 3413-3416.

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Figure 2. (a) Expanded MALDI spectra of 2 comparing the theoretical isotope distribution for the positive radical ion, with data acquired with (b) DCTB, (c) dithranol, and (d) DHB.

Neutral Organometallic and Coordination Compounds. Having established DCTB to be an excellent matrix for conjugated compounds, attention turned to compounds containing metals, beginning with 3,21 a zinc-bound porphyrin with a pendant ferrocene group (see Figure S-2 of Supporting Information). The MALDI spectra for all three matrixes have the same base peak (m/z ) 910), corresponding to the positive radical ion species. The presence of multi-isotopic elements means the isotope distribution is very distinguished, almost like a fingerprint, and is shown to be an excellent match with the theoretical distribution. All spectra show low abundant ions at m/z ) 987/8, likely to be an impurity, but only in the DHB spectrum are fragment ions observed at lower m/z values. Meso-meso-linked, porphyrin dimers such as 422 can be analyzed in a similar fashion (see Figure S-3 of Supporting Information). The MALDI spectrum of 4 is easy to interpret, as the base peak (m/z ) 1495) corresponds to the positive radical ion species. This is the only signal observed; there are no fragmentation or matrix related ions lower than the base peak, and no signal corresponding to an adduct ion of sample and matrix at higher m/z values. The lack of other ions also gives an indication of the purity of the sample. An excellent isotope distribution match for the radical ion is observed, again providing strong evidence that the empirical formula is correct. Multi(22) (a) Osuka, A.; Shimidzu, H. Angew. Chem., Int. Ed. Engl. 1997, 36, 135137. (b) Sample submitted to the NMSSC by H. L. Anderson and M. J. Smith, University of Oxford, U.K.

Figure 3. (a) MALDI spectrum of 5 acquired with DCTB, with inset showing expanded region comparing (b) the theoretical isotope distribution for the positive radical ion, with (c) the data acquired; (d) LSIMS spectra of 5 acquired with 3-NOBA, with inset showing expanded region comparing (e) the theoretical isotope distribution for the positive radical ion, with (f) the data acquired.

porphyrin arrays are formed when several porphyrins are linked together and these can reach fairly high masses, where again other techniques may struggle. Arrays may also be formed by linking through the coordinating metal. We have successfully characterized such an array with DCTB, which contains four porphyrins at a mass of roughly 5.5 kDa. Compound 523 is a bimetallic platinum complex with S-donor bridging ligands, and a comparison is made between the MALDI and LSIMS spectra, acquired with DCTB and 3-NOBA matrixes, respectively (see Figure 3). Peaks relating to intact sample are observed in both spectra, but there are many more fragmentation peaks in the LSIMS spectrum. Ions at m/z ) 500 in the MALDI spectrum, and at m/z ) 460 and 613 in the LSIMS spectrum, are matrix related. The signal at m/z ) 1238 in the MALDI spectrum corresponds to an adduct ion of sample and matrix. However, while a high-intensity sample signal is observed in MALDI, only a very low intensity signal is observed in LSIMS and matrix ions dominate the spectrum. The tendency for LSIMS to produce a mixture of positive radical ion, protonated and [M - H]+ species can result in ambiguous isotope distribution comparisons. In this case, the pattern match with the theoretical isotopic distribution for the radical ion is above average quality for LSIMS. In contrast, (23) Unpublished work. Sample submitted to the NMSSC by J. A. Weinstein, University of Nottingham, U.K., and E. M. Budynina, Lomonosov Moscow State University, Russia.

Figure 4. (a) MALDI spectrum of 6 acquired with DCTB, with inset showing expanded region comparing (b) the theoretical isotope distribution for the positive radical ion, with (c) the data acquired; (d) positive ion ESI spectrum of 6 acquired at a cone voltage of +50 V, with inset showing expanded region comparing (e) the theoretical isotope distribution for the positive radical ion, with (f) the data acquired.

an excellent isotope distribution match for the radical ion is observed in the MALDI spectrum, again providing strong evidence that the empirical formula is correct. It is also possible to obtain ions for intact species for compounds with chloride bridging ligands. Sample characterization is not limited to just transition metal compounds. Compound 624 is a tin complex with two hydrotris(methimazolyl)borate ligands and is another example of a complex containing S-donor ligands. Initial analysis was performed by ESI, and both singly (m/z ) 822) and doubly charged (m/z ) 411) positive radical ions were observed. The result was unexpected, as radical ions are not normally formed by ESI, which may explain the low signal intensity of the species. Figure 4 gives a comparison between the ESI spectrum and the MALDI spectrum generated with DCTB. Only singly charged species are observed in the MALDI, and some ions, such as m/z ) 351 corresponding to the ligand, differ from those observed in ESI. This highlights the different fragmentation routes favored by each technique. Excellent matches for isotopic distributions are found with both techniques, though the MALDI comparison is clearer as a result of the stronger signal intensity and higher resolution for the observed species. Several other ions due to higher mass impurities are observed more clearly by MALDI. (24) Unpublished work. Sample submitted to the NMSSC by M. D. Spicer and J. Reglinski, University of Strathclyde, U.K.

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It is sometimes difficult to obtain a meaningful LSIMS spectrum for metal carbonyl complexes, as the signal for the intact species can be very weak and the spectrum may be dominated by ions corresponding to successive carbonyl losses. However, dendritic carbosilanes with dicobalt hexacarbonyl units have been successfully characterized as intact species by MALDI.25 Figure S-4 of Supporting Information shows the MALDI spectrum for 7,26 a rhenium carbonyl compound. Intact positive radical ions are observed for both 7 at m/z ) 616, and 7a, the tetracarbonyl, nonchlorinated analogue, at m/z ) 609, which appears to be present as an impurity. Some poorly resolved ions are visible between m/z ) 580 and 595 mass range and are likely the result of a single, postsource decay, carbonyl loss, but a series due to successive carbonyl loss is not observed. Additionally, the signal at m/z ) 831 is consistent with either the [7 - Cl + DCTB]+ or the [7a - CO + DCTB]+ species, while the signal at m/z ) 838 is consistent with the [7 - CO + DCTB]+ species. Further credence for these assignments is given by the isotope distribution comparison. These data form a stark contrast to that obtained using traditional acidic polar matrixes for various ruthenium carbonyl-porphyrin complexes, where significant carbonyl loss was observed.27 Further examples of MALDI spectra for neutral organometallic and coordination compounds include 828 and 9,29 which are a dirhodium-phosphate complex and 1,1,1-tris(ferrocenylselenomethyl)ethane, respectively, and these are given in Supporting Information (see Figures S-5 and S-6, respectively). The MALDI spectrum of 8 had a poor signal-to-noise ratio in relation to what was observed with other examples, but the base peak (m/z ) 3390) corresponds to the positive radical ion species. The signal observed at m/z ) 3505 does not correlate to an adduct ion of sample and matrix, so is presumed to relate to an impurity. There are other fragmentation or impurity related ions at lower m/z values. A good isotope distribution match for the radical ion is observed, indicating that the empirical formula is correct. The MALDI spectrum of 9 is easy to interpret, as the base peak (m/z ) 862) corresponds to the positive radical ion species. Overall, the spectrum is quite clean, but there are a few ions at low signalto-noise ratio, which could be either fragmentation or impurity related. An excellent isotope distribution match for the radical ion is observed, again providing strong evidence that the empirical formula is correct. Charged Coordination Compounds. Charged coordination compounds in the form of salts are analyzed commonly by ESI, but several different charge-state species and fragments complicate the spectra, so it may be advantageous to characterize them by MALDI. Compound 1030 is composed of a quadruply charged, ruthenium/osmium, terpyridine-based complex cation, balanced by hexafluorophosphate (PF6-) anions. The first feature to note about the MALDI spectrum is there are only a few number of peaks (see Figure S-7 of Supporting Information), and these are (25) Kim, C.; Jung, I. Inorg. Chem. Commun. 1998, 1, 427-430. (26) Unpublished work. Sample submitted to the NMSSC by J. A. G. Williams and K. J. Arm, University of Durham, U.K. (27) Frauenkron, M.; Berkessel, A.; Gross, J. H. Eur. Mass Spectrom. 1997, 3, 427-438. (28) Hodgson, D. M.; Selden, D. A.; Dossetter, A. G. Tetrahedron-Asymmetry 2003, 14, 3841-3849. (29) Unpublished work. Sample submitted to the NMSSC by S. Jing and C. P. Morley, University of Wales, Swansea, U.K.

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Figure 5. (a) MALDI spectrum of 11 acquired with DCTB, with inset showing expanded region comparing the (b) theoretical isotope distribution for the [M - NO3]+ ion, with (c) the data acquired; (d) positive ion ESI spectrum of 11 acquired at a cone voltage of +50 V, with inset showing expanded region comparing (e) the theoretical isotope distribution for the [M - NO3]+ ion, with (f) the data acquired.

consistent with the loss of a maximum of two PF6- ions, which is in contrast to the findings of other groups.8-10 Additionally, there does not appear to be any losses of hydrogen or transfer of fluorine, and the peak at m/z ) 1756 is of too low intensity to confirm either metal reduction or protonation, to obtain a singly charged species despite the loss of two PF6- ions; the expected doubly charged species is observed at m/z ) 878. There also appears to be a small amount of dimer present, observed at m/z ) 3949, and this is considered unlikely from a synthetic point of view. Therefore, it is possibly a dimer of aggregation, but the formation of such aggregates is supposed to be prevented by using a high matrix-to-sample ratio. This peak continued to be observed at successively higher matrix-to-sample ratios, and this phenomenon will be the subject of further investigation. Equally impressive results (given in Figure 5) may be obtained for complexes of heavy metals, such as 11,31 an erbium nitrate complex, containing bisphosphine dioxide ether ligands. The MALDI spectrum shows just a single signal at m/z ) 1432, which corresponds to a single nitrate loss; there is no fragmentation. In contrast, the ESI spectrum of 11 has a plethora of fragmentation peaks and multiply charged ions, and while this may provide extra structural information for a sample, it does take much longer to interpret and can be ambiguous. (30) Unpublished work. Sample submitted to the NMSSC by P. Li and A. C. Benniston, University of Newcastle, U.K. (31) Fawcett, J.; Platt, A. W. G.; Vickers, S.; Ward, M. D. Polyhedron 2004, 23, 2561-2567.

Figure 6. (a) MALDI spectrum of 13 acquired with DCTB and LiCl additive; (b) expanded region showing [M + Li]+, [M + Na]+, [M + K]+, and [M + Cu]+ species.

A further example of MALDI analysis is a charged coordination compound 12,24 the arsenic iodide analogue of 6 (see Figure S-8 of Supporting Information). The MALDI spectrum of 12 is not easy to interpret fully, but the base peak (m/z ) 777) does correspond to the [M - I]+ ion species. There are several other fragmentation or impurity related ions at lower m/z values, as well as impurity related ions at higher m/z values. An excellent isotope distribution match for the [M - I]+ ion is observed, and iodide was also observed in negative ion mode. Neutral, Polar, Organic Compounds. Having established DCTB to be an excellent matrix for organometallic, coordination, and nonpolar compounds, our attention turned to polar organic compounds, and whether DCTB had any advantages over traditional polar matrixes. Initial attempts at analyzing 13,32 an acetylated carbohydrate compound, with DCTB, failed to produce any sample ions. However, attempts at analyzing 13 with dithranol and DHB also failed, until a group 1 salt was added as a cationizing agent. Analysis was reattempted with DCTB and the addition of lithium salt (see Figure 6). The base peak (m/z ) 1184) corresponds to the lithiated species of 13, and there are ions at lower m/z values, indicating that the sample may contain impurities. There is then a difference of +16 from the base peak to the signal at m/z ) 1200, and again to the signal at m/z ) 1216, which could be considered oxidation products or other impurities were it not for the absence of ions at m/z ) 1199 and 1215, respectively, corresponding to the 6Li isotope. These ions, and that at m/z ) 1240, actually correspond to the sodiated, potassiated, and cuprated species for 13, which also indicates that the desired functionalization of the sugar moieties has progressed to completion. The source of these metals is likely to be the sample itself, particularly for copper, as these extra species were not observed for similar samples when the same sources of matrix, salt, and solvent were used. The presence of the 6Li isotope is the reason (32) Unpublished work. Sample submitted to the NMSSC by S. A. Nepogodiev and R. A. Field, University of East Anglia, U.K.

that lithium salts are a particularly useful additive, especially when analyzing samples about which very little is known. Further examples include 14,33 a macrotricycle, and 15,34 a calixarene. Analysis of 14 with DCTB and the addition of lithium and sodium salts failed to produce sample ions. However, the potassiated species was observed with the addition of potassium salt (see Figure S-9 of Supporting Information), and this observation may be explained by considering the rigid structure of the sample. The metal ion is most likely coordinated inside the cavity of the molecule, with the inward pointing carbonyl groups. The structure is possibly too rigid to conform in such a way to coordinate to smaller ions, such as lithium and sodium, but can adopt a suitable conformation for a larger ion like potassium. The spectrum is noisier than those shown previously, and there are significant matrix ions toward the lower mass range. Figure S-10 of Supporting Information shows the MALDI spectrum for 15, generated with the addition of sodium salt, which assists the ionization of calixarenes containing four rings. In addition to the sodiated species observed at m/z ) 1491, a peak consistent with the positive radical ion is observed at m/z ) 1468. The phenyl rings of calixarenes are not conjugated, but for this sample there may be enough conjugation between a phenyl ring and the substituents to stabilize the positive charge. There are other fragmentation or impurity related ions at lower m/z values, as well as impurity related ions at higher m/z values, while the signal at m/z ) 523 is likely to correspond to a sodiated matrix dimer. Negative Ion Mode. While the majority of samples were analyzed in positive ion mode, there are occasions where negative ion mode is more appropriate. Examples of samples where this is the case include oligonucleotides, acidic organic compounds, the anion component of salts, and fullerenes, which often give more intense negative radical ions, than positive radical ions. Compound 1635 is a saponin containing several unmodified sugars, a carboxylic acid moiety, and many alkyl substituents at chiral centers; the exact stereochemistry has yet to be determined fully. The sample was analyzed initially using DHB in positive ion mode, but despite containing several potential protonation sites, no ions were observed. Addition of lithium or sodium salts also failed to produce any ions for the sample. Therefore, polarity was switched to negative, and analysis was performed using both a typical oligonucleotide preparation with THAP matrix and DCTB; the results are given in Figure S-11 of Supporting Information. Upon comparison, both spectra appear to be similar, clearly showing the base peak (m/z ) 1512) to be the deprotonated molecular species. The spectra also indicate the sample to be a mixture, with the peak at m/z ) 1554 correlating to an analogue with a single acetylation modification. However, the signal-to-noise ratio is greater in the DCTB spectrum, so that ions at m/z ) 1380, 1675, and 1716 can be readily identified as being sample related. The species at m/z ) 1675 is the same as that at m/z ) 1674 in the THAP spectrum; the signal was so weak that the true isotope distribution was not observed. Additionally, there are no ions relating to the matrix in the lower mass region of the spectrum. (33) Unpublished work. Sample submitted to the NMSSC by L. Challinor and A. P. Davis, University of Bristol, U.K. (34) Unpublished work. Sample submitted to the NMSSC by, Y. H. Chan, H. Heaney, E. Moreno and P. C. B. Page, University of Loughborough, U.K. (35) Unpublished work. Sample submitted to the NMSSC by P. C. Stevenson and T. K. Jayasekara, University of Greenwich, U.K.

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Limitations of DCTB. The vast majority of researchers who use MALDI are analyzing samples that are water-soluble and probably biological in nature. DCTB is not water-soluble, and attempts to characterize common peptides, such as angiotensin, using a layered sample preparation technique, have failed. The majority of samples investigated in this study are soluble in DCM or a solvent miscible with DCM, for example, MeCN. However, it is not really solubility issues that prevents successful analysis but, more importantly, the mode of ionization of the matrix. Proteins and peptides are almost exclusively observed as protonated species, but with DCTB being an aprotic, charge-transfer matrix, protonation is highly unlikely. Similar attempts at analyzing oligonucleotides also failed, even in negative ion mode, where the matrix could act as a proton acceptor. DCTB should also be stored under nitrogen, and when failing to do this, we observed a gradual decrease in performance over roughly six months. Separating the matrix into small aliquots, of which all but the working aliquot is stored under nitrogen until required, appears to be good working practice. DCTB sublimes fairly quickly in the high-vacuum environment within the MALDI instrument. This property of DCTB has implications for automated analysis and sample spots are best analyzed within 0.5 h of introducing them into the instrument.

Figure 7. (a) Negative ion MALDI spectrum of 17 acquired with DCTB, with expanded region comparing (b) the theoretical isotope distribution for the complex anion, with (c) the data acquired.

Many organometallic and coordination compounds are air or moisture sensitive, which can make their analysis problematic. MALDI may often be disregarded for analyzing such compounds because of the relatively long sample preparation times. Compound 1736 is an air-sensitive salt, composed of a galliumphosphine complex anion, counterbalanced by a potassium diamine complex cation. The sample was analyzed initially by ESI (data not shown), but neither complex cation nor anion was observed; nor was the complex cation observed by MALDI. The negative ion MALDI spectrum generated with DCTB is shown in Figure 7. While the overall signal strength may be regarded as weak, and the signal-to-noise ratio very poor in relation to what was observed with other examples, it is still clear that the base peak (m/z ) 877) corresponds to the intact complex anion. Further evidence for this assignment is given by the isotope distribution comparison. The only other peak of significance is at m/z ) 893, a difference of +16 from the base peak, which is likely to correspond to an oxidation product of the sample, but the exact structure of the species is unknown. (36) Baker, R. J.; Jones, C. Coord. Chem. Rev. 2005, 249, 1857-1869.

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CONCLUSIONS It has been shown that DCTB is a very affective matrix in the characterization of a wide variety of compounds by MALDITOFMS. Clear, easily interpretable, spectra are obtained, generally offering unambiguous determination of empirical formulas. The technique is shown to have major advantages over, or at the very least comparable performance with, FAB/LSIMS in the case of air/moisture-sensitive compounds, and ESI in the case of salts. DCTB has also been shown to be comparable, if not better, in performance than traditional polar acidic matrixes, and much lower laser fluence is required to achieve desorption/ionization. However, DCTB is not well suited for the analysis of biologically derived samples. ACKNOWLEDGMENT The authors thank EPSRC for funding this work, and the users of the NMSSC who kindly gave us permission to use their mass spectrometry data for this publication. Contract/grant sponsor: Engineering and Physical Sciences Research Council (EPSRC); contract/grant GR/R70088/01. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 28, 2005. Accepted October 20, 2005. AC050732F