Anal. Chem. 2005, 77, 5019-5027
Tertiary Amine Appended Derivatives of N-(3,5-Dinitrobenzoyl)leucine as Chiral Selectors for Enantiomer Assays by Electrospray Ionization Mass Spectrometry ChengLi Zu, Bobby N. Brewer, Beibei Wang, and Michael E. Koscho*
Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762
Derivatives of the chiral selector N-(3,5-dinitrobenzoyl)leucine were prepared and used as chiral selectors for enantiomer discrimination in single-stage electrospray ionization mass spectrometric experiments. The chiral selectors were designed to remove the ionization site from the sites required for effective chiral recognition. Addition of a chiral analyte to a solution of the two pseudoenantiomeric chiral selectors, which differ in absolute stereochemistry and the length of the tether connecting the tertiary amine site used for ionization via protonation and the rest of the chiral selector, affords selector-analyte complexes in the electrospray ionization mass spectrum where the ratio of these complexes is dependent on the enantiomeric composition of the analyte. The relationship between the ratio of the selector-analyte complexes in the electrospray ionization mass spectrum and the enantiomeric composition of the analyte can be used to relate the extent of enantioselectivity that is being observed and for quantitative enantiomeric composition determinations. Investigations into the scope and limitations of this method, plus a comparison to the enantioselectivities observed by chiral HPLC using a N-(3,5-dinitrobenzoyl)leucine-derived chiral stationary phase, is presented.
Interest in the development of methods that utilize only mass spectrometry for investigating chiral recognition systems, and for the determination of enantiomeric composition, has recently increased dramatically.1-4 Since Fales and Wright first reported on the observation of chiral recognition between protonated diastereomeric dialkyl tartrate dimers in the chemical ionization (CI) mass spectrum,5 a number of reports have illustrated mass spectrometric chiral recognition with a myriad of host-guest systems using a variety of ionization methods, including CI,5 fast* To whom correspondence should be addressed. Tel.: 662-325-9500. Fax: 662-325-1618. E-mail:
[email protected]. (1) Speranza, M. Int. J. Mass Spectrom. 2004, 232, 277-317. (2) Tao, W. A.; Cooks, R. G. Anal. Chem. 2003, 75, 25A-30A. (3) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, M. Int. J. Mass Spectrom. 2000, 198, 137-163. (4) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73-90. (5) Fales, H. M.; Wright, G. J. J. Am. Chem. Soc. 1977, 99, 2339-2340. 10.1021/ac050438n CCC: $30.25 Published on Web 06/28/2005
© 2005 American Chemical Society
atom bombardment,6-11 matrix-assisted laser desorption,12 and electrospray ionization (ESI).6,13-19 Though the focus of the majority of these reports was the observation of chiral recognition, recent progress has been made in the development of quantitative enantiomer assays via single-stage mass spectrometric experiments.18-22 By far, the greatest strides in developing methods for the determination of enantiomeric composition have been by tandem mass spectrometry (MS/MS). The tandem mass spectrometric measurements rely on isolating a specific ion and allowing this ion to react with another reagent or on observing the collisioninduced dissociation (CID) of the complex. The first type of tandem measurement has mainly been applied to cyclodextrin(6) Liang, Y.; Bradshaw, J. S.; Izatt, R. M.; Pope, R. M.; Dearden, D. V. Int. J. Mass Spectrom. 1999, 185, 977-988. (7) Swada, M.; Okumura, Y.; Shizuma, M.; YoshioTakai; Hidaka, Y.; Yamada, H.; Tanaka, T.; Kaneda, T.; Hirose, K.; Misumi, S.; Takahashi, S. J. Am. Chem. Soc. 1993, 115, 7381-7388. (8) Swada, M.; Takai, Y.; Yamada, H.; Hirayama, S.; Kaneda, T.; Tanaka, T.; Kamada, K.; Mizooku, T.; Takeuchi, S.; Ueno, K.; Hirose, K.; Tobe, Y.; Naemura, K. J. Am. Chem. Soc. 1995, 117, 7726-7736. (9) Dobo, A.; Liptak, M.; Huszthy, P.; Vekey, K. Rapid Commun. Mass Spectrom. 1997, 11, 889-896. (10) Pocsfalvi, G.; Liptak, M.; Huszthy, P.; Bradshaw, J. S.; Izatt, R. M.; Vekey, K. Anal. Chem. 1996, 68, 792-795. (11) Swada, M.; Takai, Y.; Yamada, H.; Nishida, J.; Kaneda, T.; Arakawa, R.; Okamato, M.; Hirose, K.; Tanaka, T.; Naemura, K. J. Chem. Soc., Perkin Trans. 2 1998, 701-710. (12) So, M. P.; Wan, T. S. M.; Chan, T.-W. D. Rapid Commun. Mass Spectrom. 2000, 14, 692-695. (13) Nierengarten, H.; Leize, E.; Garcia, C.; Jeminet, G.; Dorsselaer, A. V. Analusis 2000, 28, 259-263. (14) Czerwenka, C.; Maier, N. M.; Lindner, W. Anal. Bioanal. Chem. 2004, 379, 1039-1044. (15) Sawada, M.; Takai, Y.; Yamada, H.; Yoshikawa, M.; Arakawa, R.; Tabuchi, H.; Takada, M.; Tanaka, J.; Shizuma, M.; Yamaoka, H.; Hirose, K.; Fukuda, K.; Tobe, Y. Eur. J. Mass Spectrom. 2004, 10, 27-37. (16) Mehdizadeh, A.; Letzel, M. C.; Klaes, M.; Agena, C.; Mattay, J. Eur. J. Mass Spectrom. 2004, 10, 649-655. (17) Seymour, J. L.; Turecek, F.; Malkov, A. V.; Kocovsky, P. J. Mass Spectrom. 2004, 39, 1044-1052. (18) Koscho, M. E.; Zu, C.; Brewer, B. N. Tetrahedron: Asymmetry 2005, 16, 801-807. (19) Brewer, B. N.; Zu, C.; Koscho, M. E. Chirality. In press. (20) Shizuma, M.; Imamura, H.; Takai, Y.; Yamada, H.; Takeda, T.; Takahashi, S.; Sawada, M. Int. J. Mass Spectrom. 2001, 210/211, 585-590. (21) Sawada, M.; Yamaoka, H.; Takai, Y.; Kawai, Y.; Yamada, H.; Azuma, T.; Fujioka, T.; Tanaka, T. Int. J. Mass Spectrom. 1999, 193, 123-130. (22) Sawada, M.; Yamaoka, H.; Takai, Y.; Kawai, Y.; Yamada, H.; Azuma, T.; Fujioka, T.; Tanaka, T. Chem. Commun. 1998, 1569-1570.
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analyte complexes,23-27 and chiral crown ether-chiral ammonium cation complexes.28,29 The rate at which the analyte exchanges for an achiral reagent gas in the host-guest complex is used as a metric for determining the stereochemical composition of the analyte. For the other type of tandem experiments, higher order complexes are mass-selected and allowed to undergo CID, and the observed relative branching ratios are related to the enantiomeric composition by the kinetic method of Cooks.30-49 In addition to the methods above, where in each case diastereomeric noncovalent complex ions are produced in the experiment, it is also possible to measure enantiomeric composition using preformed covalent diastereomers.50-52 Derivatization of a chiral analyte with a mixture of mass-labeled, pseudoenantiomeric chiral reagents (where each pseudoenantiomer has the opposite stereochemistry, but a slightly different mass due to labeling of one enantiomer at a remote position) affords derivatives that can be discriminated by mass spectrometry. As long as kinetic resolution is observed in the derivatization step, the relative amounts of the derivatives can be related back to the enantiomeric composition of the analyte. Of course, one major drawback to this method is the requirement for the preparation of derivatives before the analysis. (23) Ramirez, J.; He, F.; Lebrilla, C. B. J. Am. Chem. Soc. 1998, 120, 73877388. (24) Ramirez, J.; Ahn, S.; Grigorean, G.; Lebrilla, C. B. J. Am. Chem. Soc. 2000, 122, 6884-6890. (25) Grigorean, G.; Ramirez, J.; Ahn, S. H.; Lebrilla, C. B. Anal. Chem. 2000, 72, 4275-4281. (26) Grigorean, G.; Lebrilla, C. B. Anal. Chem. 2001, 73, 1684-1691. (27) Grigorean, G.; Cong, X.; Lebrilla, C. B. Int. J. Mass Spectrom. 2004, 234, 71-77. (28) Dearden, D. V.; Dejsupa, C.; Liang, Y.; Bradshaw, J. S.; Izatt, R. M. J. Am. Chem. Soc. 1997, 119, 353-359. (29) Chu, I.-H.; Dearden, D. V.; Bradshaw, J. S.; Huszthy, P.; Izatt, R. M. J. Am. Chem. Soc. 1993, 115, 4318-4320. (30) Bagheri, H.; Chen, H.; Cooks, R. G. Chem. Commun. 2004, 2740-2741. (31) Wu, L.; Meurer, E. C.; Cooks, R. G. Anal. Chem. 2004, 76, 663-671. (32) Yu, C.-T.; Guo, Y.-L.; Chen, G.-Q.; Zhong, Y.-W. J. Am. Soc. Mass Spectrom. 2004, 15, 795-802. (33) Wu, L.; Clark, R. L.; Cooks, R. G. Chem. Commun. 2003, 136-137. (34) Wu, L.; Cooks, R. G. Anal. Chem. 2003, 75, 678-684. (35) Augusti, D. V.; Augusti, R.; Carazza, F.; Cooks, R. G. Chem. Commun. 2002, 2242-2243. (36) Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G. Anal. Chem. 2002, 74, 3458-3462. (37) Tao, W. A.; Clark, R. L.; Cooks, R. G. Anal. Chem. 2002, 74, 3783-3789. (38) Fago, G.; Filippi, A.; Giardini, A.; Lagana, A.; Paladini, A.; Speranza, M. Angew. Chem., Int. Ed. 2001, 40, 4051-4054. (39) Paladini, A.; Calcagni, C.; Palma, T. D.; Speranza, M.; Lagana, A.; Fago, G.; Filippi, A.; Satta, M.; Guidoni, A. G. Chirality 2001, 13, 707-711. (40) Tao, W. A.; Cooks, R. G. Angew. Chem., Int. Ed. 2001, 40, 757-760. (41) Tao, W. A.; Gozzo, F. C.; Cooks, R. G. Anal. Chem. 2001, 73, 1692-1698. (42) Tao, W. A.; Wu, L.; Cooks, R. G. J. Med. Chem. 2001, 44, 3541-3544. (43) Tao, W. A.; Wu, L.; Cooks, R. G. Chem. Commun. 2000, 2023-2024. (44) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609. (45) Yao, Z.-P.; Wan, T. S. M.; Kwong, K.-P.; Che, C.-T. Anal. Chem. 2000, 72, 5383-5393. (46) Yao, Z.-P.; Wan, T. S. M.; Kwong, K.-P.; Che, C.-T. Anal. Chem. 2000, 72, 5394-5401. (47) Yao, Z.-P.; Wan, T. S. M.; Kwong, K.-P.; Che, C.-T. Chem. Commun. 1999, 2119-2120. (48) Cooks, R. G.; Wong, P. S. H. Acc. Chem. Res. 1998, 31, 379-386. (49) Vekey, K.; Czira, G. Anal. Chem. 1997, 69, 1700-1705. (50) Yao, S.; Meng, J.-C.; Siuzdak, G.; Finn, M. G. J. Org. Chem. 2003, 68, 25402546. (51) Diaz, D. D.; Yao, S.; Finn, M. G. Tetrahedron Lett. 2001, 42, 2617-2619. (52) Guo, J.; Wu, J.; Finn, M. G. Angew. Chem., Int. Ed. Engl. 1999, 38, 17551758.
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One main impetus for developing mass spectrometric methods for enantiomer assays is the potential for rapid analysis. Such a method would definitely be a boon to the development of catalytic enantioselective reactions, particularly by combinatorial asymmetric catalysis, whereby libraries of potential asymmetric catalysts are produced in parallel and each catalyst is screened for its ability to produce a product of high enantiomeric purity.53-57 Typically, the size of combinatorial catalyst libraries has not been limited by the number of potential catalysts that one can prepare, but rather by the time needed to evaluate large libraries using contemporary methods for enantiomer analysis. Such a method could also be used for the discovery and optimization of chiral selectors by combinatorial methods.58-64 Instead of measuring the enantiomeric composition, one would instead be using mass spectrometry to directly measure the relative binding of analyte enantiomers to potential chiral selectors. In addition to the potential for rapid analysis, other attributes such as a high tolerance to impurities, broad analyte scope, and high sensitivity are beneficial for the determination of the enantiomeric composition of samples originating from a variety of sources, particularly samples of biological origin. Previously, we have reported the use of soluble analogues of Pirkle-type chiral stationary phases (CSPs) as chiral selectors for enantiomer assays by electrospray ionization mass spectrometry.18,19 In each case, a solution of pseudoenantiomeric chiral selectors (where each enantiomer is mass-labeled at a remote site), when mixed with a chiral analyte whose enantiomers are known to be resolved on the corresponding CSP, was shown to afford selector-analyte complexes in the mass spectrum where the relative peak intensities of the complexes depend on the enantiomeric composition of the analyte. For example, mass-labeled, soluble analogues of the N-(3,5dinitrobenzoyl)leucine CSP (DNB-leucine CSP, Chart 1), such as a 1:1 mixture of (S)-1 and (R)-2 which differ only by the length of the N-alkyl chain of the amide, when mixed with analyte 10 (Chart 2) and excess lithium chloride afforded peaks in the electrospray ionization mass spectrum that correspond to the following complexes: [1 + 10 + Li]+ and [2 + 10 + Li]+. It was found that a plot of the natural log of the relative peak intensities of the selector-analyte complexes in the mass spectrum versus the enantiomeric composition of the analyte is linear.18 Similar results were found using soluble analogues of the N-(3,5dinitrobenzoyl)phenylglycine CSP as chiral selectors (e.g., (R)-3 (53) Finn, M. G. Chirality 2002, 14, 534-540. (54) Reetz, M. T. Angew. Chem., Int. Ed. 2002, 41, 1335-1338. (55) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284-310. (56) Reetz, M. T.; Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew. Chem., Int. Ed. 2000, 39, 3891-3893. (57) Schrader, W.; Eipper, A.; Pugh, D. J.; Reetz, M. T. Can. J. Chem. 2002, 80, 626-632. (58) Wang, Y.; Blum, L. H.; Li, T. Anal. Chem. 2000, 72, 5459-5465. (59) Bluhm, L. H.; Wang, Y.; Li, T. Anal. Chem. 2000, 72, 5201-5205. (60) Brahmachary, E.; Ling, F. H.; Svec, F.; Frechet, J. M. J. J. Comb. Chem. 2003, 5, 441-450. (61) Welch, C. J.; Pollard, S. D.; Mathre, D. J.; Reider, P. J. Org. Lett. 2001, 3, 95-98. (62) Lewandowski, K.; Murer, P.; Svec, F.; Frechet, J. M. J. J. Comb. Chem. 1999, 1, 105-112. (63) Welch, C. J.; Bhat, G.; Protopopova, M. N. J. Comb. Chem. 1999, 1, 364367. (64) Weingarten, M. D.; Sekanina, K.; Still, W. C. J. Am. Chem. Soc. 1998, 120, 9112-9113.
Chart 1
Chart 2
and (S)-4). Overall, it was found that the selectivity was rather low, compared to the enantioselectivity obtained by HPLC using either of the N-(3,5-dinitrobenzoyl)amino acid CSPs, presumably due to interference of the requisite selector-analyte hydrogen bonds needed for effective chiral recognition by the lithium cations. Subsequently, mixtures of deprotonated N-(3,5-dinitrobenzoyl)amino acids were used as chiral selectors (e.g., binary combinations of 5, 6, and 7).19 As before, it was found that a plot of the natural log of the relative peak intensities of the selector-analyte complexes in the negative ion ESI-mass spectrum versus the enantiomeric composition of the analyte is linear. In these cases, it was found that the selectivity was indeed comparable to the selectivity obtained by chiral HPLC. In both instances, it was found that the reproducibility of enantiomeric composition determinations using the linear calibration plot was ∼(0.05 for the calculated mole fraction of either enantiomer. In the first instance, with the lithiated selectoranalyte complexes, the poor reproducibility was due primarily to a low selectivity where small differences in measured relative peak intensities will afford substantial differences in the calculated enantiomeric composition value. In the second instance, with the deprotonated chiral selectors, the low reproducibility was due to a small absolute value of the intensities of the selector-analyte complexes in the ESI-mass spectrum. In fact, the ion counts were ∼2 orders of magnitude less for the complexes derived from the
deprotonated selectors compared to the lithiated selectors. The variability was therefore a manifestation of a decreased signal with respect to experimental noise. In this paper, we report the use of tertiary amine appended N-(3,5-dinitrobenzoyl)leucine derivatives, 8 and 9, as pseudoenantiomeric chiral selectors for enantiomer analyses by electrospray ionization mass spectrometry. These chiral selectors were designed to overcome the problems delineated above, via a separation of the requisite chiral recognition sites from the ionization site. Addition of an acid to the solution to be assayed should protonate the tertiary amine, affording high ion counts for the selector in the electrospray ionization mass spectrum. Additionally, since the ionization site is removed from the sites needed for chiral recognition, its interference with the formation of selector-analyte complexes should be minimized. In order for this to be a viable analytical method, one would like to be able to measure the enantiomeric composition of the analyte at a variety of concentrations, even without necessarily knowing the concentration of the analyte. Additionally, one would like to be able to use a variety of solvent compositions to accommodate samples of many types. Herein is reported the scope and limitations of this method, including the effect of analyte concentration and a survey of solvent effects, using protonated pseudoenantiomeric chiral selectors, (S)-8 and (R)-9, for enantiomer discrimination and for the determination of enantiomeric composition by electrospray ionization mass spectrometry. Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
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Figure 1. Electrospray ionization mass spectrum of a solution of pseudoenantiomeric chiral selectors (S)-8 and (R)-9 (1.0 mM each), racemic analyte 10 (1.0 mM), and ammonium chloride (10 mM) in methanol/water (1:1).
EXPERIMENTAL SECTION Procedures for the preparation of chiral selectors (S)-8 and (R)-9 and characterization data for all new compounds are included in the Supporting Information. The chiral stationary phase DNB-leucine (4.6 × 250 mm column) was prepared by literature methods.65 All solvents used were HPLC grade and used without further purification. All mass spectra were obtained on a Micromass Quattro Micro (Beverly, MA) triple quadrupole mass spectrometer with electrospray ionization running in the positive ion mode. Solutions were flow injected into the electrospray ionization source through a 10µL injection loop with mobile phase running at a flow rate of 200 µL/min. The full positive ion spectrum was recorded every 0.7 s. All scans for which a significant total ion count was observed were averaged together to afford the final spectrum, typically requiring ∼10 s/injection. Spectrometer conditions are as follows: capillary voltage, 3.0 kV; cone voltage, 12 V; extractor voltage, 1.0 V; rf lens, 0.5 V; source temperature, 80 °C; desolvation temperature, 325 °C; cone gas flow, 62 L/h; desolvation gas flow, 608 L/h. For data collected by direct infusion with a syringe pump (entries 6-11 in Table 4), scans collected over a 2-min period were averaged together to afford the final spectrum. RESULTS AND DISCUSSION Chiral Recognition. The chiral selectors 8 and 9 were designed to (1) remove the ionization site from the sites required for chiral recognition and (2) act as enantiomers where both can be detected in a single mass spectrometric experiment (i.e., masslabeled pseudoenantiomers). As has been demonstrated through a number of studies,65-69 the primary interactions between the DNB-leucine chiral selectors and analyte that are necessary for effective chiral recognition are as follows: (1) a π-stacking interaction with the aromatic ring of the electron-deficient dinitrobenzamide, (2) a hydrogen bond with the electron-deficient benzamide proton, and (3) a hydrogen bond with the electron(65) Pirkle, W. H.; Welch, C. J. J. Org. Chem. 1984, 49, 138-140. (66) Pirkle, W. H.; Murray, P. G.; Wilson, S. R. J. Org. Chem. 1996, 61, 47754777. (67) Pirkle, W. H.; Pochapsky, T. C. J. Am. Chem. Soc. 1986, 108, 56275628. (68) Pirkle, W. H.; III, J. A. B.; Wilson, S. R. J. Am. Chem. Soc. 1989, 111, 92229223. (69) Pirkle, W. H.; Tsipouras, A. Tetrahedron Lett. 1985, 26, 2989-2992.
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rich C-terminal carbonyl group of leucine. Any chiral analyte that possesses complementary interaction sites, (1) an electron-rich aromatic ring, (2) a functional group that can accept a hydrogen bond, and (3) a hydrogen bond donor is a candidate for enantiodifferentiation by this type of chiral selector. Analytes 10-18 all fit within this rubric and were chosen for this study. The appended tertiary amine of chiral selectors 8 and 9 is superfluous for chiral recognition purposes, but has been added to allow ready ionization of the chiral selectors and any complexes derived thereof via protonation. Electrospray ionization mass spectrometry of solutions containing the chiral selectors, (S)-8 and (R)-9, the analyte, and ammonium chloride in methanol/water affords significant protonated selector-analyte complexes in the mass spectrum for each analyte. The samples were introduced into the electrospray unit via flow injection of a 10-µL sample loop at a flow rate of 200 µL/ min of water/methanol (1:1). The full positive ion spectrum was recorded every 0.7 s. All scans for which a significant total ion count was observed were averaged together to afford the final spectrum, typically requiring ∼10 s/injection. The full mass spectrum for the solution containing racemic analyte 10 is shown in Figure 1. The protonated analyte, [10 + H]+, and chiral selectors, [8 + H]+ and [9 + H]+, are observed at m/z 303, 424, and 438, respectively. Analyte dimers and selector dimers are observed at m/z 605 [102 + H]+, 622 [102 + NH4]+, 883 [(8 + H)2 + Cl]+, 897 [(8 + H) + (9 + H) + Cl]+, and 911 [(9 + H)2 + Cl]+. The selector-analyte complexes, which, in this instance, are the largest peaks in the spectrum, are observed at m/z 726 [8 + 10 + H]+ and 740 [9 + 10 + H]+. Figure 2 presents the portions of the mass spectra that contain the selector-analyte complexes obtained at three different enantiomeric compositions of analyte 10. The relative intensities of the selector-analyte complexes vary regularly with the enantiomeric composition of the analyte, clearly demonstrating chiral recognition. As the amount of (R)-10 is increased in the sample, the complex with the (R)-9 selector is increased relative to the complex with the (S)-8 selector; likewise, as the amount of (S)10 is increased in the sample, the complex with the (S)-8 selector is increased relative to the complex with the (R)-9 selector. The sense of chiral recognition is consistent with what is observed chromatographically, whereby the (S)-enantiomer of analyte 10 is more retained when using the (S)-DNB-leucine CSP. Addition-
assays but it is also a measure of the enantioselectivity of the chiral selectors toward the analyte enantiomers. The larger the slope of this plot, the greater the selectivity. Presumably, in this experiment, complexes that are formed in solution are being transferred to the gas phase such that the relative amount of each complex in solution is reported in the mass spectrum. This allows determination of the extent of enantioselectivity in solution based on the slope of the calibration plot derived from the mass spectrometric data. In general, given pseudoenantiomeric chiral selectors, ΨR and ΨS, and analyte enantiomers, AR and AS, the enantioselectivity in solution, R, will be given by any of the ratios of concentrations shown in eq 1. This, of course, assumes that the pseudoenantiomeric chiral selectors are acting as enantiomers, and [ΨR] ) [ΨS].
R) Figure 2. Partial electrospray ionization mass spectrum of solutions containing pseudoenantiomeric chiral selectors (S)-8 and (R)-9 (1.0 mM each), analyte 10 (1.0 mM), and ammonium chloride (10 mM) in methanol/water (1:1). Spectra: (a) 79.6% enantiomeric excess (R)10, (b) racemic 10, and (c) 81.8% enantiomeric excess (S)-10. Each spectrum is normalized to the intensity of the larger complex peak.
[ΨR‚AS]
)
[ΨS‚AS] [ΨS‚AR]
)
[ΨR‚AR] [ΨS‚AR]
)
[ΨS‚AS]
(1)
[ΨR‚AS]
In this instance, since the chiral selectors have a tertiary amine appended and a 5-fold excess of ammonium chloride has been added to the solution, the complexes are likely protonated. Electrospray ionization of the solution will transfer these protonated complexes to the gas phase with an efficiency given by the transfer coefficient: tX. Each of the four possible complexes will have its own transfer coefficient, though given the similarity of the complexes, and that the ionization site has been removed from the sites presumed to be involved in chiral recognition, it can be assumed that all four are equal. With this assumption, one can then relate the relative ion counts of the selector-analyte complexes observed in the mass spectrum with the relative concentrations of complexes formed in solution, and hence the enantioselectivity, as measured by mass spectrometry, RMS, since the ratios of the transfer coefficients will be unity.
RMS ) Figure 3. Complex intensity fraction in the electrospray ionization mass spectrum versus the mole fraction of analyte (R)-10 (200 µM) in the solution, using pseudoenantiomeric chiral selectors (S)-8 (1.0 mM) and (R)-9 (1.0 mM) with added ammonium chloride (10 mM) in methanol/water (1:1). Slope 0.260; intercept 0.302; r2 ) 0.999.
ally, chiral recognition was observed in the electrospray ionization mass spectrum for each of the remaining analytes, 11-18, and the sense of chiral recognition was consistent with the sense of chromatographic chiral recognition in each case. A plot of the complex intensity fraction (CIF; intensity of one selector-analyte complex divided by the sum of the intensities for both selector-analyte complexes) versus the enantiomeric composition of analyte 10 is presented in Figure 3. The plot is linear with a correlation coefficient of 0.999. This plot can be used for subsequent enantiomer assays. Simply measuring the CIF of the selector-analyte complexes in the mass spectrum and applying this value to the calibration line affords the enantiomeric composition. Enantioselectivity. The linear plot of the CIF versus enantiomeric composition is not only useful for subsequent enantiomer
[ΨR‚AR]
tRR[ΨR‚AR‚H]+ tRS[ΨR‚AS‚H]+
)
tSS[ΨS‚AS‚H]+ tSR[ΨS‚AR‚H]+
tRR[ΨR‚AR‚H]+ tSR[ΨS‚AR‚H]+
)
) tSS[ΨS‚AS‚H]+ tRS[ΨR‚AS‚H]+
(2)
From the mass spectrum, it is the CIF that is measured, which is given by sum of ion counts of both analyte enantiomers with one chiral selector divided by the sum of ion counts of all selector-analyte complexes.
CIF ) [ΨR‚AR‚H]+ + [ΨR‚AS‚H]+ [ΨR‚AR‚H]+ + [ΨR‚AS‚H]+ + [ΨS‚AR‚H]+ + [ΨS‚AS‚H]+ (3) The relation of each of the ion counts to solution concentrations, followed by substitutions, using the appropriate equilibrium expressions and eq 2, and rearrangement, affords eq 4. This equation predicts that the plot of the CIF versus the enantiomeric composition of the analyte (XR is mole fraction of Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
5023
the (R)-analyte) is linear.
R-1 1 CIF ) X + R+1 R R+1
(
)
(
)
(4)
Additionally, any differences in the transfer coefficients of the complexes, differences in enantioselectivity of the pseudoenantiomeric chiral selectors, or differences in the concentrations of chiral selectors, as long as the differences remain constant throughout, should manifest itself in the intercept of this plot. In fact, because of this, rarely do the RMS values determined from the slope and the intercept of this plot agree. For the pure enantiomers (XR ) 1, and XR ) 0), the CIF values will be RMS/ (RMS + 1) and 1/(RMS + 1). The ratio of these CIF values is RMS and affords an additional measure of the enantioselectivity. In practice, using the slope of the calibration plot is more convenient since one only needs to measure the CIF at two different enantiomeric compositions to construct the plot, whereas in order to determine the CIF values for both pure enantiomers, one needs access to both pure analyte enantiomers. All enantioselectivities reported herein were calculated from the slope of the calibration plots. In our previous reports,18,19 we plotted the natural log of the relative peak intensities of the selector-analyte complexes versus the enantiomeric composition, where the slope of this plot is then two times the natural log of the enantioselectivity (2 ln RMS). The RMS values calculated by this method agree, within experimental error, with the values determined as described above. In fact, the logarithmic function is an approximation of the hyperbolic function above. The logarithmic function will deviate from linearity as RMS increases, though eq 4 will still hold, and therefore, eq 4 has been used throughout for the determination of RMS. Enantioselectivity as a Function of Selector/Analyte Concentration. In order for this method to be practicable for quantitative enantiomer assays, the results of any assay should be independent of the absolute concentrations of the analyte as much as possible. One can readily control the concentrations of the selectors and additives and the solvent composition by preparing a stock solution, where this same solution is used for the construction of the calibration curve and for subsequent enantiomer assays. Ideally, one would like to add a sample of the analyte to a small aliquot of this stock solution and record the electrospray ionization mass spectrum, without measuring the amount of analyte. Table 1 presents the enantioselectivities obtained at a variety of different concentrations of analyte 10. In each case, the concentrations of the chiral selectors, ammonium chloride, and solvent composition were kept constant. The RMS values are relatively invariant, as long as the chiral selectors are in excess of the analyte. The RMS values for the entries at a selector/analyte ratio of 20:1, 10:1, 5:1, and 1:1 are all within 3% of the average RMS (1.66). As the concentration of the analyte is decreased, the intensity of the selector-analyte complexes in the mass spectrum also decreases, compared to the intensity of the monomeric ions. Since the measured enantioselectivity only depends on the relative amounts of the (pseudo)diastereomeric complexes formed in solution, the ratio of the complexes should not change in the limit of low analyte concentration. In essence, each pseudoenantiomer (via the pseudodiastereomeric selector-analyte complexes) is 5024 Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
Table 1. Observed Enantioselectivity as a Function of the Concentration of Analyte 10 at a Constant Concentration of Pseudoenantiomeric Chiral Selectors (S)-8 and (R)-9 selector:analytea,b
RMSc (σ)d
20:1 10:1 5:1 1:1 1:2 1:5 1:10
1.61 (0.02) 1.66 (0.03) 1.64 (0.02) 1.71 (0.04) 1.54 (0.02) 1.42 (0.02) 1.33 (0.02)
a Concentration of selectors (S)-8 and (R)-9 is 1.0 mM in all cases in methanol/water (1:1). b Concentration of NH4Cl is 10 mM in all cases. c Observed MS enantioselectivity. d σ is the standard deviation obtained from the calibration curve for three replicate measurements at three different enantiomeric compositions.
Table 2. Observed Enantioselectivity as a Function of the Concentration of Pseudoenantiomeric Chiral Selectors (S)-8 and (R)-9 and Analyte 10 at a Constant Selector/Analyte Ratioa,b [(S)-8]/[(R)-9], µM
[10], µM
RMSc (σ)d
4000 2000 1000 500 250 125 62.5 31.2
2000 1000 500 250 125 62.5 31.2 15.6
1.76 (0.03) 1.69 (0.04) 1.67 (0.02) 1.69 (0.02) 1.67 (0.02) 1.62 (0.03) 1.65 (0.02) 1.63 (0.02)
a Concentration of NH Cl is 10 times [10]. b Solvent is methanol/ 4 water (1:1) in all cases. c Observed MS enantioselectivity. d σ is the standard deviation obtained from the calibration curve for three replicate measurements at three different enantiomeric compositions.
acting as an internal standard for the other. Increasing the analyte concentration beyond the concentration of the chiral selectors results in saturation, where further increases in the analyte concentration will further reduce the observed enantioselectivity. The onset of this diminution of enantioselectivity was observed at a 2:1 analyte/selector ratio for analyte 10. Table 2 presents the observed enantioselectivities at a number of different absolute concentrations of selectors, analyte, and ammonium chloride. The relative concentrations of all the components were kept constant throughout. Relatively little variation of the RMS values was observed throughout this concentration range. All but one of the RMS values in Table 2 are within 3% of the average RMS (1.67) for this data set, and this value also agrees very well with the average RMS value for the data in Table 1. Again, since it is the ratio of (pseudo)diastereomeric selectoranalyte complexes that determines the selectivity, the relative amounts of these complexes should not be affected by dilution. At lower concentrations, the intensities of the selector-analyte complexes in the mass spectra are diminished, which limits the extent to which the solutions can be diluted by the signal-to-noise ratio that one can obtain experimentally. These two data sets have important implications for the use of this method for quantitative enantiomer assays: (1) as long as the concentrations of the selectors are in excess of the analyte, the calibration line and subsequent enantiomer assays can be done
Table 3. Determination of the Enantiomeric Composition of Five Different Samples of Analyte 10 by Mass Spectrometry at Three Different Concentrations Using a Single Calibration Linea,b mole fraction of (R)-10 [10], µM
HPLCc,d
MS (σ)e
500 500 500 500 500 100 100 100 100 100 50 50 50 50 50
0.976 0.774 0.497f 0.240 0.044 0.976 0.774 0.497f 0.240 0.044 0.976 0.774 0.497f 0.240 0.044
0.979 (0.009) 0.782 (0.009) 0.502 (0.008) 0.240 (0.009) 0.041 (0.009) 0.960 (0.009) 0.782 (0.009) 0.482 (0.008) 0.239 (0.009) 0.056 (0.009) 0.966 (0.009) 0.779 (0.009) 0.495 (0.008) 0.235 (0.009) 0.075 (0.009)
a [8] ) 1.0 mM, [9] ) 1.0 mM, and [NH Cl] ) 10 mM in methanol/ 4 water (1:1). b Calibration curve constructed at [10] ) 200 µM. c (S,S)Whelk-O1, hexanes/2-propanol/methanol (60:38:2) at 2 mL/min. d Repeated injections afforded standard deviations of
