Anal. Chem. 1996, 68, 1792-1797
Regulation of Multimer Formation in Electrospray Mass Spectrometry Morgan Stefansson,* Per J. R. Sjo 1 berg, and Karin E. Markides
Institute of Analytical Chemistry, Uppsala University, Box 531, 751 21 Uppsala, Sweden
Extensive multimer formation of small and uncharged analytes was observed in electrospray mass spectrometry. The concentration-dependent aggregation behavior, including species up to tetramers, resulted in highly nonlinear calibration curves. For some analytes, these effects were operating down into the nanomolar level. Varying the drift potential failed to selectively dissociate the multimer complexes in favor of the monomer, which severely obstructed quantitative work. However, by incorporating certain cationic additives into the electrosprayed solution, complete suppression of the multimers could be obtained. In addition, a gain in the signal intensities by a factor up to 7 was achieved. A variety of additives were investigated, and the structural requirements for optimum performance will be outlined. Electrospray is a new ionization technique which has literally revolutionized mass spectrometry. The analytical capabilities of electrospray mass spectrometry (ES-MS), as first demonstrated by Fenn and co-workers,1 have increased significantly during recent years, especially within the bioanalytical field in regard to the analysis of large biomolecules.2,3 The “soft” ionization methods of ES-MS, with its ability to desorb intact and multiply charged high molecular weight ions from aqueous solutions into the gas phase, supply data on molecular weights, and the possibility of direct assessment of sequence information has been suggested.4 One of the recent and exciting observations, with regard to ES-MS, is the presence of noncovalent complexes of large biomolecules and small molecules. Since the original report,5 a variety of noncovalent complexes have been reported, including protein-ligand, protein dimers, oligonucleotides, and antibody-antigen interactions.6-17 These remarkable findings (1) Mann, M.; Meng, C. K.; Fenn, J. B. Proceedings of the 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June 5-10, 1988; ASMS: East Lansing, MI, 1988; pp 1207-1208. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37. (3) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882. (4) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 201. (5) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 6294. (6) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 7818. (7) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1991, 113, 8534. (8) Ganguly, A. K.; Pramanik, B. N.;l Tsarbopoulos, A.; Covey, T. R.; Huang, E.; Fuhrman, S. A. J. Am. Chem. Soc. 1992, 114, 6559. (9) Jaquinod, M.; Leize, E.; Potier, N.; Albrecht, A. M.; Shanzer, A.; Van Dorsselaer, A. Tetrahedron Lett. 1993, 34, 2771. (10) Loo, J. A.; Holsworth, D.; Root-Bernstein, R. S. Biol. Mass Spectrom. 1994, 23, 6. (11) Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811. (12) Li, Y.-T.; Hsieh, Y. L.; Henion, L. D.; Senko, M. W.; McLafferty, F. W.; Ganem, B. J. Am. Chem. Soc. 1993, 115, 8409.
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have elicited keen interest in the possibility of applying ES-MS to probe or explore specific interactions in biochemical research as well as developing a methodology for rapid screening of new potential drugs. However, care must be taken in cases where mass spectral data indicate apparent association in order to differentiate molecular interactions in solution from processes occurring during droplet desolvation and/or in the gas phase as the species travel toward the mass spectrometer,18 i.e., artifacts due to gas-phase condensation reactions. The present limited understanding of the mechanisms involved in ion production and the processes taking place in the interface region strongly suggests a need for caution in data interpretation. Nonselective multimer formations of proteins,19 up to the pentamer of cytochrome c,20 as well as up to the 24-mer for arginine,18 have been demonstrated by manipulating sampling conditions in the ES interface. The individual multimer clusters seemed to exhibit a size dependence on the number of charges that could be accommodated.18 Multimer formation has been detected in both positive and negative modes, depending on the compounds studied. Although a vast amount of research has focused on the ESMS of large biopolymers, correspondingly, aggregation and multimer formation phenomena occurring with “common” small molecules, for example pharmaceuticals and other bioactive compounds, have received relatively little attention. Chargecarrying noncovalent complexes in the gas phase, such as protonor alkali metal ion-bound multimers, can be produced by ES and other ionization techniques. Regardless of the selective or nonselective processes responsible for these events taking place, the formation of dimers, trimers, tetramers, and so on will have a detrimental effect on the quantitative aspects of mass spectrometry. This is mainly due to the concentration-dependent formation behavior; hence, nonlinear calibration curves will be obtained. For some solutes, as will be demonstrated below, such effects might be operating down to the nanomolar level. Furthermore, a decrease in sensitivity usually results from the increased number of species created, along with a more complicated and and crowded mass spectrum. (13) Huang, E. C.; Pramanik, B. N.; Tsarbopoulos, A.; Reichert, P.; Trotta, A. K.; Nagabhushan, T. L.; Covey, T. R. J. Am. Soc. Mass Spectrom. 1993, 4, 624. (14) Ganem, B.; Li, Y.-T.; Henion, J. D. Tetrahedron Lett. 1993, 34, 1445. (15) Light-Wahl, K. J.; Springer, D. L.; Winger, B. E.; Edmonds, C. G.; Camp, D. G.; Thrall, B. D.; Smith, R. D. J. Am. Chem. Soc. 1993, 115, 803. (16) Ganem, B.; Li, Y.-T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 6294. (17) Baca, M.; Kent, S. B. H. J. Am. Chem. Soc. 1992, 114, 3992. (18) Meng, C. K.; Fenn, J. B. Org. Mass Spectrom. 1991, 26, 542. (19) Loo, J. A. J. Mass Spectrom. 1995, 30, 180. (20) Winger, B. E.; Light-Wahl, K. J.; Ogorzalek-Loo, R. R.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 536. S0003-2700(95)00980-2 CCC: $12.00
© 1995 American Chemical Society
Figure 1. Chemical structures of model analytes.
To dissociate complexes of this nature, thermal and collisional activation procedures21,22 or increases in curtain gas temperature20 have been performed. Measures of that kind require the multimer(s) to be less stable than the monomer. In our experience, this is rarely the case for nonselective complexes of small neutral and uncharged analytes. This is especially true when alkali metal cationization takes place. Additionally, a decrease in signal intensity of the monomer follows from a simultaneous dissociation of the monomer adduct. For such compounds, an activated dissociation by an increase in the drift potential cannot produce linear standard curves as, at maximum, one monomer from each multimer is formed, unless gas-phase reactions with available cations compensate for the charge deficiency experienced during multimer disintegration. The main purpose of this report is to demonstrate the possibility of regulating multimer formation of small and uncharged solutes through the use of organic ammonium com(21) Busman, M.; Knapp, D. R.; Schey, K. L. Rapid Commun. Mass Spectrom. 1994, 8, 211. (22) Loo, J. A.; Ogorzalek Loo, R. R.; Andrews, P. C. Org. Mass Spectrom. 1993, 28, 1640.
pounds as additives to the electrosprayed solution. Parameters influencing the complex stability and multimer formation will be discussed including hydrophobicity, concentration, and degree of substitution of the ammonium ion utilized. EXPERIMENTAL SECTION Mass spectra were recorded on a triple quadrupole atmospheric pressure ionization mass spectrometer (API III biomolecular mass analyzer; Sciex, Thornhill, ON, Canada) having an upper mass limit of 2400 m/z units. Data were acquired by scanning Q1 in increments of 0.1 amu with a dwell time of 1 ms and are presented as average values of 10-20 scans over the massto-charge ratio range of interest. The mass spectrometer was set to the following parameters: ion spray voltage (ISV), 3700 V (-2450 V); interface plate voltage (IN), 650 V (-650 V); orifice lens (OR), see below; AC entrance rod (RO), 30 V (-30 V); quadrupole rod offsets, R1 ) 28 V (-28 V), R2 ) -80 V (50 V), and R3 ) -50 V (50 V). The values in parentheses assign the negative mode. The potential difference between OR and RO is presented in the text as the drift potential. All of the spectra presented are raw data and have not been subject to smoothing, Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
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A
B
Figure 3. Influence of drift potential on multimer signal intensities. Conditions as in Figure 2A. 9, Monomer; 0, dimer; O trimer; 4, tetramer. Figure 2. (A) Positive electrospray mass spectra of artemisinin multimers at a drift voltage of 5 V. Sample at concentration of 70 µM dissolved in 50% acetonitrile, 1 mM formic acid, and 0.4 mM NaOH. (B) Collision-induced dissociation mass spectrum of the sodiated artemisinin trimer (m/z 869.6) into the corresponding dimer (m/z 587.4) and monomer (m/z 305.2) forms.
filtering, background correction, or other manipulative procedures. The sample solutions were electrosprayed from the tip of a fused silica capillary (Polymicro Technologies, Phoenix, AZ) of 50 µm i.d. and 187 µm o.d. connected to an air-tight glass syringe. The capillary was centered in a stainless-steel capillary auxiliary assembly delivering 1.8 L/min of synthetic air for pneumatically assisted ES-MS (hereafter refered to as electrospray mass spectrometry). A Harvard Apparatus (South Natick, MA) syringe infusion pump (Model 22) was utilized for a constant flow of 5 µL/min unless otherwise indicated. The volumetric flow of the countercurrent curtain gas (N2) over the orifice was 0.6 L/min at a temperature of 50 °C. A standard Galileo 4879 channeltron electron multiplier detector (Galileo Electro-Optics Corp., Sturbridge, MA) was used throughout the study and was operated at (3500 V, depending on the mode of operation. All chemicals were of analytical grade and were used without further purification. The chemical structures of the model substances used in this study are shown in Figure 1. Note that lonomycin A, lasalocid, benzyloxycarbonylglycyl-L-proline (ZGP), and (-)-2,3:4,6-di-Oisopropylidene-2-keto-L-gulonate (DIKG) are carboxylic acids. RESULTS AND DISCUSSION During initial electrospray mass spectrometric studies on some uncharged antimalaria drugs, extensive multimer formation was observed, and a typical mass spectrum of artemisinin is shown in Figure 2A. The aggregates were confirmed by tandem mass spectrometry using collision-induced dissociation; the CID spec1794 Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
trum of the sodium-cationized trimer is displayed in Figure 2B. The concentration of the drug was 70 µM in 50% acetonitrile containing 1 mM formic acid and 0.4 mM NaOH, pH 3.6. The presence of di-, tri-, and tetramers indicates a nonspecific kind of interaction. No pentamer or aggregates of higher order could be detected. The spectrum was, in essence, unaffected by variation of the acetonitrile concentration or by changing to methanol and tetrahydrofuran as organic modifiers. If formate only was used as the buffer component, a large number of peaks were obtained due to cationization of the analyte by ammonium, sodium, and potassium ions, in addition to the protons, present as trace impurities in the solvents and glassware. An increase in the drift potential revealed that sodium formed the strongest complexes; hence, addition of sodium resulted in one dominating species for the monomer and multimers, respectively. One question is where to draw a line between specific and nonspecific complex formation. In the case of sodium ions, it is suggested that the oxygen atoms in artemisinin can act as coordination centers much like a crown ether or a poly(alkylene oxide) structure and, possibly, such a complex could even involve two artemisinin molecules spatially arranged in a proper way. Higher aggregates would be less likely for steric reasons and might, therefore, involve a lower degree of specificity. The tri- and tetramers are the least stable of the multimers. However, the signal intensities decreased considerably at higher drift potentials, and the fraction of monomers, Θmono, never exceeded 0.8 (Figure 3). The increase in the monomer signal at lower drift potentials was due to activated dissociation of the monomer-sodium-acetonitrile and multimer complexes, the latter being more stable. Θmono is calculated as the intensity of the monomer peak divided by the sum of the monomer and multimer peaks. The intensities of the dimer, trimer, tetramer, etc. were corrected by factors of 2, 3, 4, etc., in order to compensate for the stoichiometry. Any degree of mass-to-charge
Figure 4. Concentration dependence of the multimer formation of artemisinin. Conditions as in Figure 2A. 9, Monomer; O, dimer; 4, trimer; 0, tetramer.
ratio discrimination, which is known to occur for quadrupole instruments,23 with increasing m/z values was not evaluated. A second factor contributing to a decrease in the signal intensity is due to an increase in isotope distribution of aggregates with increasing m/z values. Consequently, the real situation is actually worse than is apparent. For equilibria of this nature, a concentration dependence is to be expected and also is evident from the data presented in Figure 4. With increasing concentration of artemisinin, the relative abundance of monomers decreases rapidly in favor of the di-, tri-, and tetramers. As a consequence, the calibration curves become highly nonlinear, as shown for artemisinin, dihydroartemisinin, and artemether in Figure 5. Multimer formation could be observed, to varying degrees, for all of the test compounds investigated and for artemisinin down into the nanomolar range. At only 0.7 µM, the fraction of multimers ranged between 5 and 15%. This is evidence for multimer formation not being mainly a high-concentration phenomenon, which might severely limit the capacity to do quantitative measurements. Summation of the monomer and multimer peaks, in attempts to compensate for the monomer signal, proved unsuccessful. The spectra of lonomycin A, lasalocid, ZGP, and DIKG, i.e., carboxylic acid-containing compounds, are further complicated by the presence of sodiated alkali metal esters of the type [(M - H+ + Na+) + Na+]. The variation of Θmono was investigated in the flow rate range 1-100 µL/min using picrotin as the analyte. In accordance with an increasing number of solute molecules being transferred into the gas phase per time unit, i.e., apparent concentration, an increase in multimer formation was observed, leveling off, however, at even higher flow rates. This suggested a shift in the ionization and/or transmission mechanisms of the solute from solution to the gas-phase state inside the mass spectrometer. Furthermore, these results indicate that the multimers are formed as a consequence of gas-phase reactions, rather than originating from solution equilibria, as the concentration of the analyte was kept constant. Modulation of the curtain gas temperature between 50 and 150 °C had no effect on the monomer and multimer intensities. A general lack of detectable multimers was observed when working in the negative mode. This is most remarkable, in regard (23) Alexander, A. J.; Kebarle, P. Anal. Chem. 1986, 58, 471.
Figure 5. Calibration curves for artemisinin (0), dihydroartemisinin (O), and artemether (4) dissolved in 50% acetonitrile, 1 mM formic acid, and 0.4 mM NaOH. The drift voltages were 45, 30, and 30 V, respectively.
to the numerous publications on noncovalent complexes, where solution equilbria involving one or several different species are believed to be reflected as corresponding peaks in the mass spectrum. Studies on such species should, if truly characterized by the ES-MS methodology, be independent on the mode utilized, i.e., positive or negative. When the sodium salt of octyl sulfate in pure methanol was electrosprayed, a pronounced aggregation was registered covering the whole m/z range of the mass spectrometer. The complexes were of a [(M- + Na+)n + Na+] type with a degree of aggregation, n, between 1 and 10. The observation of these entities was highly dependent on the drift voltage employed and reached maximum signals around 170 V. This could be due to gas-phase reactions or dissociation or larger aggregates otherwise not discernible. Micelles are, however, not to be expected from such a solvent.24 When the analyte was run in the negative mode, only the monomer was observed. For the other test substances, only minute aggregation at very low drift potentials was registered, and the substances could easily be dissociated, except for picrotin, where an organic cationic additive had to be used (see below). The signal intensities were lower and suffered in terms of sensitivity compared to the positive mode, but they were acceptable as long as the chemical structure contained a proton-labile function group like carboxylate or alcohol. Hence, artemisinin, artemether, and related compounds were hardly detectable. In attempts to regulate the multimer formation and to obtain one dominating species, the monomer, different additives including neutral, anionic, and cationic modifiers were investigated. A main goal was to eliminate or, alternatively, to produce multimer complexes less stable than the monomer which then could be (24) Mukerjee, P.; Mysels, K. J. NSRDS-NBS, 1971.
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Figure 6. Positive electrospray mass spectra of artemisinin at a concentration of 70 µM in 50% acetonitrile, 1 mM formic acid, and 0.5 mM additive (Q): (A) ammonia, (B) propylamine, (C) hexylamine, and (D) dodecylamine. The drift voltages were optimized in terms of multimer suppression and signal intensity to 50, 65, 65, and 70 V, respectively.
selectively dissociated by suitable tuning of the drift potential. Due to the insignificant effects achieved in the negative mode (see above), the subsequent discussion is restricted to the positive mode only. After a brief survey, organic amines were found to have beneficial effects on complexation as well as signal intensity. Consequently, 13 amines differing in degree of substitution, carbon chain configuration, and hydrophobicity were investigated, and the results are presented in Table 1. The analyte was 70 µM artemisinin in 50% acetonitrile, 1 mM formate, and 0.5 mM ammonium additive at pH 3.8. The relatively high sample concentration was chosen as a “worst case” and to more easily assess the influence on multimer formation imposed by the modifier. The qualitative evaluation in Table 1 is based on changes in monomer peak intensity (additive complexation to the analyte, A), inhibition of multimer formation (B), and inhibition of sodium adduction (C) during variation of the drift potential. By suitable tuning of the drift potential, a minimum of multimers versus monomers could be obtained, probably caused by collisioninduced dissociation and/or gas-phase reactions between the analyte and the additive. Basically, two main physicochemical characteristics appeared crucial for an amine to exhibit a favorable and quantitative response: degree of substitution at the nitrogen center and hydrophobicity of the carbon chain(s). A complete suppression of multimer formation could be achieved only with primary amines, and the effect seemed to be correlated to the number of hydrogens present at the nitrogen, decreasing in the following order: primary . secondary > tertiary > quaternary. This indicates that hydrogen bonding to the oxygen atoms might be important for the complex stability, which is in agreement with the discussion above on sodium adduction to these kind of 1796 Analytical Chemistry, Vol. 68, No. 10, May 15, 1996
Table 1. Organic Ammonium Additivesa Ab
Bc
Cd
+ + + + + + +
+ + + + +
+ + + +
Secondary +
0
0
0 +
0
-
Quaternary tetraethylammonium-OH -
-
-
Primary ammonia guanidine ethylenediamine propylamine pentylamine hexylamine dodecylamine diethylamine dibutylamine
Tertiary triethanolamine triethylamine dimethyloctylamine
a Solution: 70 µM artemisinin in 50% acetonitrile, 1 mM formate, and 0.5 mM amine. A, B, and C are qualitative parameters assessed from scanning of the drift potential and ranked as follows: -, no or minor; 0, some; +, superior. b Complexation to analyte. c Inhibition of multimer formation. d Inhibition of sodium adduction.
structures. Primary amines are known to form strong complexes with crown ethers in solution25 and the gas phase.26 The complexation selectivities are, however, not the same.27 (25) Gokel, G. Crown Ethers and Cryptands; BlackBear Press: Cambridge, UK, 1991. (26) Cunniff, J. B.; Vouros, P. Rapid Commun. Mass Spectrom. 1994, 8, 715. (27) Zang, H.; Chu, I.; Leming, S.; Dearden, D. V. J. Am. Chem. Soc. 1991, 113, 7415.
Figure 7. Calibration curves for artemisinin (0), dihydroartemisinin (9), and artemether (O). Solutions: 50% acetonitrile in 0.2 mM formic acid and 0.1 mM dodecylamine, except for dihydroartemisinin (1 mM formic acid and 0.5 mM dodecylamine).
The carbon chains appeared to block further complexation, probably through steric hindrance, as indicated by the presence of dimers when ammonia was used (Figure 6A). The spectra in Figure 6B-D show the single peak obtained by primary amine additives and the shift to higher m/z values for the monomer peak with increasing size and chain length of the amine. The influence of hydrophobicity on cluster suppression was most apparent for the higher substituted amines, where an increase in the number of carbon atoms and/or the carbon chain length, compare diethylamine-dibutylamine and triethylamine-dimethyloctylamine, was advantageous. Hence, the opposite effect was brought about by introduction of hydrophilic functional groups (OH and NH2), i.e., triethylamine-triethanolamine and propylamine-ethylenediamine-guanidine, respectively. For ethylenediamine, only the monoprotonated form was detected. The analytes did not seem to experience any competition from the amines regarding transfer to the gas phase, a phenomenon which has been observed for compounds with the same type of charge.28 On the contrary, the signal intensities for artemisinin, dihydroartemisinin, and artemether increased by factors of 6.9, 6.8, and 1.4, respectively, after addition of dodecylamine (see Figure 7) to solutions containing 0.7 µM analyte. This might suggest that, during the evaporation process, transfer of an analyte (28) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654.
as complex with the amine is favored and, at the relatively high concentrations of additive (in the millimolar range) that may be used, the solute molecules experience negligible or minor competition. As a direct consequence of the multimer suppression, linear calibration curves (y ) (1.494 × 105) + (6.358 × 105)x, r2 ) 0.999; y ) -4560 + (2.441 × 105)x, r2 ) 1.000; y ) (3.174 × 104) + (1.934 × 105)x, r2 ) 1.000, respectively) were obtained, as displayed in Figure 7. Furthermore, of the carboxylic acids, lonomycin A and DIKG formed [(M- + NH4+) + NH4+] complexes that were more stable than the ammoniated complex of the neutral analyte (M + NH4+). The opposite was true when propylamine was used as additive, hence further simplifying the spectrum of acidic compounds. None of the ammonium additives exhibited any tendency to aggregate or form mixed multimers. Conversely, the additives octyl sulfate (see above) and octanoylN-methylgucamide (OMEGA), representing moderately hydrophobic anionic and neutral classes of compounds, displayed extensive aggregation. In the case of OMEGA, substantial amounts of mixed complexes with artemisinin (A) were found: A-OMEGA-Q+, A2-OMEGA-Q+, A-OMEGA2-Q+, A3-OMEGA-Q+, A2-OMEGA2-Q+, and A-OMEGA3-Q+, where Q+ is a proton or sodium ion. As a result, for these kinds of compounds, electrospray mass spectrometry in the positive mode seems to be limited to cationic additives. CONCLUSIONS Formation of noncovalent multimers of uncharged compounds could be regulated selectively through the addition of organic ammonium modifiers to the electrosprayed solution and finetuning of the drift potential. The largest effect was achieved with primary amines, followed by secondary, tertiary, and quaternary amines, indicating the importance of hydrogen bonding for the complex stability of the monomer. By increasing the carbon chain length and hydrophobicity, this effect could be optimized, and as a result of obtaining one dominating species, the signal intensities increased up to 7 times. Generally, the spectra were simplified, and the methodology seems highly suitable for quantitative work. ACKNOWLEDGMENT Financial support from the Swedish Natural Science Research Council, project K-KU 1439-314, is gratefully acknowledged. The authors also thank Dr. Y. Bergqvist (Clinical Chemistry Laboratory, Falu Hospital, Sweden) for providing artemisinin, dihydroartemisinin, and artemether, and Prof. D. Westerlund (Department of Analytical Pharmaceutical Chemistry, Uppsala University, BMC, Uppsala, Sweden) for supplying some of the organic ammonium compounds used in this study. Received for review September 29, 1995. Accepted March 4, 1996.X AC950980J X
Abstract published in Advance ACS Abstracts, April 1, 1996.
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