Chiral Analysis by Electrospray Ionization Mass Spectrometry/Mass...
Anal. Chem. 2000, 72, 5383-5393
Chiral Analysis by Electrospray Ionization Mass Spectrometry/Mass Spectrometry. 1. Chiral Recognition of 19 Common Amino Acids Zhong-Ping Yao,† Terence S. M. Wan,*,‡ Ka-Ping Kwong,† and Chun-Tao Che†,§
Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, and School of Chinese Medicine, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong
Chiral recognition of 19 common amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to protonated dimers, and the intensity ratios of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be definitely differentiated. The method was demonstrated as rapid, sensitive, precise, robust, and requiring no reference standards and only minimal sample preparation. The chirality of all three amino acids in a mixture was determined without prior separation of the amino acids, consuming only 70 pmol of sample and requiring only ∼14 min of mass spectrometric measurements. A cyclodipeptide with unknown chirality was determined to be cyclo-(L-Pro-L-Leu) by acid hydrolysis followed by the present method, and the results were consistent with the physiochemical properties and NMR data of the compound. This study suggested that ESI-MS/MS can be a promising approach for the chiral recognition of other compounds. Chirality plays a crucial role in biochemical systems. The majority of organic substances from which all living creatures are built are chiral. Enantiomers often show very different physiological behaviors. For example, D-amino acids are generally sweet whereas the L-enantiomers are bitter or tasteless.1 The thalidomide tragedy is another example.2 (R)-Thalidomide has an antinausea * To whom correspondence should be addressed: (e-mail) terence.sm.wan@ hkjc.org.hk; (fax) +852 2601-6564. † The Hong Kong University of Science & Technology. ‡ The Hong Kong Jockey Club. § The Chinese University of Hong Kong. (1) Belitz, H. D.; Wieser, H. Food Rev. Int. 1985, 1, 271-354. (2) (a) Cayen, M. N. Chirality 1991, 3, 94-98. (b) Brown, J. M.; Davies, S. G. Nature 1989, 342, 631-636. 10.1021/ac000729q CCC: $19.00 Published on Web 09/28/2000
© 2000 American Chemical Society
effect, but the (S)-enantiomer is teratogenic and was thought to be responsible for the deformed babies born after their mothers had taken the racemic drug. Therefore, chiral recognition is an important topic in chemistry and biochemistry. A number of approaches3,4 have been used for the chiral recognition of organic compounds, including polarimetry, circular dichroism, nuclear magnetic resonance, chromatography, and capillary electrophoresis. In recent years, attention5-7 has also been paid to the use of mass spectrometry for chiral recognition due to its many advantages, e.g., high sensitivity, short analysis time, the ability to analyze mixtures by tandem mass spectrometry (MS/MS) or in combination with chromatography, and the ability to study intrinsic properties of the chiral effect by isolating the interacting molecules in the gaseous phase. Traditionally, mass spectrometry has been considered as insensitive to chirality because enantiomers have the same mass and show identical mass spectra. Enantiomers behave differently only in a chiral environment. Therefore, to differentiate a pair of enantiomers with mass spectrometry, the target enantiomers are ionized in the presence of a chiral selector (also called reference chiral compound, chiral reagent, resolving agent, etc.) to form diastereomeric complex ions. In most of the studies, the observation of chiral recognition was based on the following: (i) the difference in intensities of the diastereomeric complex ions;8-30 (3) Schreier, P.; Bernreuther, A.; Huffer, M. Analysis of Chiral Organic Molecules: Methodology and Application; Water de Gruyter: Berlin, 1995. (4) Juaristi, E. Introduction to Stereochemistry & Conformational Analysis; John Wiley: New York, 1991. (5) Winkler, F. J.; Splitter, J. S. In Application of Mass Spectrometry to Organic Stereochemistry; Splitter, J. S., Turecek, F., Eds.; VCH: New York, 1994; Chapter 16, pp 365-370. (6) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73-90. (7) Nibbering, N. M. M. In Advances in Mass Spectrometry; Karjalainen, E. J., Hesso, A. E., Jalonen, J. E., Karjalainen, U. P., Eds.; Elsevier: Amsterdam, 1998; Vol. 14, Chapter 2, pp 55-58. (8) Hua, S. M.; Chen, Y. Z.; Jiang, L. F.; Xue, S. M. Org. Mass Spectrom. 1986, 21, 7-10. (9) Chen, Y. Z.; Li, H.; Hua, S. M.; Qiu, W. K.; Deng, J. Z. Kexue Tongbao 1987, 32, 919-920. (10) Chen, Y. Z.; Li, H.; Yang, H. J.; Hua, S. M.; Li, H. Q.; Zhao, F. Z.; Chen, N. Y. Org. Mass Spectrom. 1988, 23, 821-824. (11) Martens, J.; Lubben, S.; Schwarting, W. Z. Naturforsch. 1991, 46B, 320325. (12) Sellier, N. M.; Bouillet, C. T.; Douay, D. L.; Tabet, J.-C. E. Rapid Commun. Mass Spectrom. 1994, 8, 891-894. (13) Okamura, K.; Sumida, Y.; Fujiwara, Y.; Terada, S.; Kim, H.; Hashimoto, K. J. Mass Spectrom. Soc. Jpn. 1995, 43 (1), 97-105.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000 5383
(ii) the difference in kinetic energy releases (KER) or daughter ion intensities in the MS/MS spectra of the diastereomeric complex ions;31-40 and (iii) the difference in equilibrium constants or rate constants between the chiral selector and the enantiomers as measured by Fourier transform ion cyclotron resonance mass spectrometry.41-44 In 1977, Fales and Wright45 reported the first observation of a chirality effect in mass spectrometry with optically active dialkyl tartrates, which was then studied46-56 in detail by Winkler and Nikolaev. Since then there have been more than 50 papers (14) Hashimoto, K.; Sumida, Y.; Terada, S.; Okamura, K. J. Mass Spectrom. Soc. Jpn. 1993, 41 (2), 95-100. (15) Williams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.; Taylor, L. C. E. J. Am. Chem. Soc. 1981, 103, 5700-5704. (16) Yang, H. J.; Chen, Y. Z. Org. Mass Spectrom. 1992, 27, 736-740. (17) Sawada, M.; Shizuma, M.; Takai, Y.; Yamada, H.; Kaneda, T.; Hanafusa, T. J. Am. Chem. Soc. 1992, 114, 4405-4406. (18) Sawada, M.; Okumura, Y.; Shizuma, M.; Takai, Y.; Hidaka, Y.; Yamada, H.; Tanaka, T.; Kaneda, T.; Hirose, K.; Misumi, S.; Takahashi, S. J. Am. Chem. Soc. 1993, 115, 7381-7388. (19) Sawada, M.; Okumura, Y.; Yamada, H.; Takai, Takahashi, S.; Kaneda, T.; Hirose, K.; Misumi, S. Org. Mass Spectrom. 1993, 28, 1525-1528. (20) Sawada, M. In Biological Mass Spectrometry: Present and Future; Matsuo, T., Caprioli, R. M., Gross, M. L., Seyama, Y., Eds.; John Wiley: Amsterdam, 1994; Chapter 3.19, pp 639-646. (21) Sawada, M.; Takai, Y.; Yamada, H.; Kaneda, T.; Kamada, K.; Mizooku, T.; Hirose, K.; Tobe, Y.; Naemura, K. J. Chem. Soc., Chem. Commun. 1994, 2497-2498. (22) Sawada, 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. (23) Pocsfalvi, G.; Liptak, M.; Huszthy, P.; Bradshaw, J. S.; Izatt, R. M.; Vekey, K. Anal. Chem. 1996, 68, 792-795. (24) Dobo, A.; Liptak, M.; Huszthy P.; Vekey, K. Rapid Commun. Mass Spectrom. 1997, 11, 889-896. (25) Drabowicz, J.; Dudzinski, B.; Mikolajczyk, M.; Sochacki, M. Pol. J. Chem. 1994, 68, 2265-2270. (26) Haskins, N. J.; Saunders, M. R.; Camilleri, P. Rapid Commun. Mass Spectrom. 1994, 8, 423-426. (27) Sawada, M., Takai, Y.; Kaneda, T.; Arakawa, R.; Okamoto, M.; Doe, H.; Matsuo, T.; Naemura, K.; Hirose, K.; Tobe, Y. Chem. Commun. 1996, 17351736. (28) Sawada, M.; Takai, Y.; Yamada, H.; Nishida, J.; Kaneda, T.; Arakawa, R.; Okamoto, M.; Hirose, K.; Tanaka, T.; Naemura, K. J. Chem. Soc., Perkin Trans. 2 1998, 701-710. (29) Sawada, M.; Shizuma, M.; Takai, Y.; Adachi, H.; Takeda, T.; Uchiyama, T. Chem. Commun. 1998, 1453-1454. (30) So, M. P.; Wan, T. S. M.; Chan, T.-W. D. Rapid Commun. Mass Spectrom. 2000, 14, 692-695. (31) Borges, C.; Ferreira, M. A. A.; Rauter, A. P. Biomed. Environ. Mass Spectrom. 1988, 18, 399-402. (32) Tabet, J. C. Spectrosc. Int. J. 1987, 5, 83-94. (33) Tabet, J. C. Tetrahedron 1987, 43, 3413-3420. (34) Tabet, J. C.; Hommet, G. L.; Royer, J.; Husson, H. P. Adv. Mass Spectrom. 1989, 11, 530-531. (35) Shen, W. Y.; Wong, P. S. H.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1997, 11, 71-74. (36) Hofmeister, G.; Leary, J. A.; Org. Mass Spectrom. 1991, 26, 811-812. (37) Dang, T. T.; Pederson, S. F.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1994, 5, 452-459. (38) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293-3294. (39) Vekey, K.; Czira, G. Anal. Chem. 1997, 69, 1700-1705. (40) Tao, W. A.; Zhang, D. X.; Wang, F.; Thomas, P. D.; Cooks, R. G. Anal. Chem. 1999, 71, 4427-4429. (41) Chu, I. H.; Dearden, D. V.; Bradshaw, J. S.; Huszthy P.; Izatt, R. M. J. Am. Chem. Soc. 1993, 115, 4318-4320. (42) Dearden, D. V.; Dejsupa, C.; Liang, Y. J.; Bradshaw, J. S.; Izatt, R. M. J. Am. Chem. Soc. 1997, 119, 353-359. (43) Camara, E.; Green, M. K.; Penn, S. G.; Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 8751-8752. (44) Gur, E. H.; Koning, L. J. D.; Nibbering, N. M. M. J. Mass Spectrom. 1996, 31, 325-327. (45) Fales, H. M.; Wright, G. J. J. Am. Chem. Soc. 1977, 99, 2339-2340. (46) Winkler, F. J.; Stahl, D.; Maquin, F. Tetrahedron Lett. 1986, 27, 335-338.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
published in this field. While chemical ionization (CI)8-14,32-35,45 was the main ionization mode used in the early development of chiral mass spectrometry, significant results have recently been reported using fast atom bombardment (FAB)15-25,28,29,36-39 and electrospray ionization (ESI).26,27,40,43 Because the differences in the spectra of diastereomers are usually very small and mass spectral signals often unstable, a number of data evaluation methods are available for the analysis. These include the relative peak intensity method (i.e., internal standard method),17,18 the deuterium-labeled method,15,21,22 the equilibrium constant method,23 and the two-internal-standards method.24 Despite these efforts, only limited success has been achieved; so far, not even a single class of chiral compounds can be systematically recognized by mass spectrometry, and to our knowledge there has been no example of a practical application of chirality determination by mass spectrometry. In addition to the poor reproducibility of mass spectra, an important obstacle to the application of chiral mass spectrometry is the need for consecutive measurements for comparison, which must be made carefully under identical conditions of sample compositions and instrumental settings. Amino acids are a class of compounds of great biochemical importance, especially the 20 common amino acids that are the fundamental units of proteins. All common amino acids except glycine are chiral. Since the introduction of chiral mass spectrometry, the chiral recognition of amino acids8-11,13,28,57 has been studied using this technique and (S)-2-methylbutanol, (R)- and (S)-mandelic acid, (R)- and (S)- 2-methylbutanoic acid, (R)- and (S)-R-phenylethylamine, (1R)- and (1S)-camphanic acid, (S)methylmandelate, cyclodextrins, or chiral crown ethers as the chiral selectors. However, only 10 common amino acids, namely, phenylalanine, methionine, alanine, threonine, tryptophan, leucine, isoleucine, proline, lysine, and valine, have been studied so far. The chiral discrimination between several pairs of common amino acids has also been observed.39,40 In general, the problems encountered in any attempt to systematically recognize the chirality of all 19 common amino acids would include the limited recognition ability of chiral selectors, the poor reproducibility of mass spectra, and the low volatility or solubility of most amino (47) Winkler, F. J.; Stahl, D.; Maquin, F. Adv. Mass Spectrom. 1986, 10, 829830. (48) Baldwin, M. A.; Howell, S. A.; Welham, K. J.; Winkler, F. J. Biomed. Envion. Mass Spectrom. 1988, 16, 357-360. (49) Winkler, F. J.; Winkler, J.; Krause, H. Adv. Mass Spectrom. 1989, 11, 620621. (50) Winkler, F. J.; Medina, R.; Winkler, J.; Krause, H. J. Chromatogr., A 1994, 666, 549-556. (51) Winkler, F. J.; Medina, R.; Winkler, J.; Krause, H. J. Mass Spectrom. 1997, 32, 1072-1079. (52) Nikolaev, E. N.; Goginashvili, G. T.; Tal’rose, V. L.; Kostyanovsky, R. G. Int. J. Mass Spectrom. Ion Processes 1988, 86, 249-252. (53) Honovich, J. P.; Karachevtsev, G. V.; Nikolaev, E. N. Rapid Commun. Mass Spectrom. 1992, 6, 429-433. (54) Denisov, E. V.; Shustryakov, V.; Nikolaev, E. N.; Winkler, F. J.; Medina, R. Int. J. Mass Spectrom. 1997, 167/168, 259-268. (55) Nikolaev, E. N.; Denisov, E. V.; Rakov, V. S.; Futrell, J. H. Int. J. Mass Spectrom. 1999, 182/183, 357-368. (56) Nikolaev, E. N.; Denisov, E. V.; Nikolaeva, M. I.; Futrell, J. H.; Rakov, V. S.; Winkler, F. J. In Advances in Mass Spectrometry; Karjalainen, E. J., Hesso, A. E., Jalonen, J. E., Karjalainen, U. P., Eds.; Elsevier: Amsterdam, 1998; Vol. 14, Chapter 12, pp 279-313. (57) Kwong, K. P.; Cheng, A. C. C.; Wan, T. S. M.; Wong, T.; Chan, D. T. W. Abs. 4th Chemistry Postgraduate Research Symposium in Hong Kong, Hong Kong Baptist University, 19 April 1997; p A-24.
acids, making them difficult to associate with chiral selectors to give abundant complex ions under conventional mass spectrometric conditions. In some cases, high concentrations or even saturated solutions of amino acids were required39 for chiral recognition. We report here the development of a simple and general method to recognize the chirality of all 19 common amino acids by mass spectrometry so as to promote the practical application of chiral mass spectrometry. MS/MS58,59 was used in our investigation. Although MS/MS has been used rather extensively31-40 in chiral recognition, some of its advantages (such as the ability to analyze mixtures and the relative indifference to changes in the sample composition) have not been explored. ESI60,61 was used to produce diastereomeric complex ions from an amino acid and a chiral selector. Commercially available modified amino acids were chosen as chiral selectors. The results62 are described below. EXPERIMENTAL SECTION Mass Spectrometry. Mass spectrometry was carried out on a Finnigan LCQ instrument (San Jose, CA) fitted with an ESI source. The conditions for measuring chiral recognition ratios were as follows: heated capillary 50 °C, sheath gas 60 psi, spray voltage 4 kV, and syringe pump 10 µL min-1. The instrument was operated with the automatic gain control (AGC) turned on. The AGC target values were as follows: full MS 5 × 107 and MSn 2 × 107. The default maximum injection time for MS/MS was 500 ms with five microscans. Helium was introduced and maintained at a pressure of 1 mTorr for improving the ion trapping efficiency and as the collision gas for collision-induced dissociation (CID). In the MS/MS mode, the isolation width was adjusted so that the maximum signal of precursor ions was obtained at 0% relative collision energy. The collision energy was then set to a value for which the product ions were produced in intermediate relative abundance, and this value was kept unchanged for all the four chiral combinations of the chiral selector and amino acid: LD, DL, LL, and DD. The measurements for each chiral combination were repeated three times. Unless otherwise stated, spectra were acquired for 7 min and the data obtained between 2 and 7 min after sample introduction were averaged. To ensure accurate measurement of ion intensities, CID spectra were all recorded using profile (rather than centroid) mode, where the sum of intensities of the seven highest submass peaks (corresponding to (0.2 u) in a profile was regarded as the intensity signal for the ion. Chemicals. All D- and L-amino acids and chiral selectors were purchased from Sigma Chemical Co. (Milwaukee, WI) and used without further purification. They were all assumed to be chemically and optically pure. For the measurement of chiral recognition ratios, 2 mM solutions of D- or L-amino acids and of D- or L-N-tertbutoxy-carbonylproline (BPro) were each prepared in 50% metha(58) Busch, K. L.; Glish, G. L.; Mcluckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH: New York, 1988. (59) Cooks, R. G.; Beynon, J. H.; Caprioli, R. M.; Lester, G. R. Metastable Ions; Elsevier: New York, 1973. (60) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (61) Gaskell, S. J. J. Mass Spectrom. 1997, 32, 677-688. (62) For the communication, see: Yao, Z. P.; Wan, T. S. M.; Kwong, K. P.; Che, C. T. Chem. Commun. 1999, 2119-2120.
Figure 1. Chemical structures of chiral selectors: (a) BPhe, (b) BPro, and (c) BBSer.
nol containing 1% acetic acid, and 2 mM solutions of L- or D-Ntert-butoxycarbonylphenylalanine (BPhe) and of L- or D-N-tertbutoxycarbonyl-O-benzylserine (BBSer) were each prepared in methanol. The solutions of an amino acid and a chiral selector were mixed in a 1:1 ratio prior to mass spectrometric analysis. Hydrolysis of the Cyclodipeptide.63 A solution of 0.1 mg of cyclodipeptide in 0.1 mL of 33% (v/v) aqueous sulfuric acid was heated overnight in an oven set at 105 °C. Saturated barium hydroxide solution was added to the reaction mixture until pH 2. The barium sulfate precipitate was then removed by centrifugation. Two portions of 25 µL of the supernatant solutions were each mixed with 100 µL of D- or L-BBSer (2 mM in methanol), respectively, prior to mass spectrometric analysis. RESULTS AND DISCUSSION Selection of Chiral Selectors and Measurement of Chiral Recognition Ratios. In choosing the chiral selectors for amino acids, we focused on the modified amino acids because they have physiochemical properties similar to the amino acids and are commercially available. Moreover, it has been reported39 that chiral discrimination exists between amino acids. The butoxycarbonyl (BOC) amino acids, often used in peptide synthesis,64 were of great interest because they bear a bulky tert-butyl group that can lead to a large steric hindrance with the amino acids and an extra carboxyl group that can result in increased hydrogen bonding with the amino acids. L- or D-N-t-BOC-phenylalanine (Figure 1, a) was investigated first because the rigid benzene ring could lead to steric hindrance and produce π-π interaction with the aromatic groups of some amino acids. Multisite interactions and steric hindrance have been thought to be the driving forces for chiral discrimination.46,65 A typical ESI mass spectrum of an amino acid/chiral selector mixture is depicted in Figure 2. Thus, amino acid phenylalanine (Phe) combines with BPhe to form protonated dimer (Phe)(BPhe)H+, protonated trimer (Phe)(BPhe)2H+, and protonated tetramer (Phe)(BPhe)3H+. Protonated pentamer (Phe)(BPhe)4H+ (63) Hydrolysis under conventional conditions (6 N HCl, 110 °C, 24 h) will cause appreciable recemization of the amino acids. For hydrolysis of peptides, see: (a) Johnson, J. L.; Jackson, W. G.; Eble, T. E. J. Am. Chem. Soc. 1951, 73, 2947-2948. (b) Hunt, S. In Chemistry and Biochemistry of the Amino Acids; Barrett, G. C., Ed.; Chapman and Hall: New York, 1985; Chapter 11, pp 376-398. (c) Bailey, P. D. An Introduction to Peptide Chemistry; John Wiley: Chichester, 1990; Chapter 4, pp 67-70. (64) Meienhofer, J. In Chemistry and Biochemistry of the Amino Acids; Barrett, G. C., Ed.; Chapman and Hall: New York, 1985; Chapter 9, pp 297-337. (65) Allenmark, S. Chromatographic Enantioseparation: Methods and Applications, 2nd ed.; Ellis Horwood: Chichester, 1991; pp 74-87.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
Figure 2. ESI spectrum of a mixture of L-Phe (1 mM) and L-BPhe (1 mM). Major complex ions are indicated. Conditions for ESI: heated capillary 50 °C, sheath gas 60 psi, spray voltage 4 kV, and syringe pump 10 µL min-1.
(namely, the intensity ratios of the product ion to the precursor ion, r) between the heterochiral and homochiral cases, i.e., between ([XYn-1H+]/[XYnH+])heterochiral and ([XYn-1H+]/ [XYnH+])homochiral, and a chiral recognition ratio CR can be defined according to eq 2. Since the LD and DL heterochiral complex ions,
CR ) ([XYn-1H+]/[XYnH+])heterochiral/ ([XYn-1H+]/[XYnH+])homochiral (2)
Figure 3. CID spectrum of protonated trimer from L-Phe and L-BPhe. Relative collision energy was 8%, isolation width was 18 u, and mass range was 350-750 u.
and the LL and DD homochiral complex ions, should in each case produce identical CID spectra, to rule out any measurement error CR is further defined according to eq 3, an equation similar to
CR ) is also observed but only at very low intensity. A trace amount of sodium ions in the instrument also combines with BPhe to form complex ions (BPhe)2Na+, (BPhe)3Na+, (BPhe)4Na+, etc. The collision-induced dissociations of the protonated dimer, protonated trimer, and protonated tetramer were studied. They all displayed the same dissociation pattern as showed in eq 1,
XYnH+ f XYn-1H+ + Y
where X is the amino acid, Y is the chiral selector, and n ) 1-3. A typical CID spectrum is showed in Figure 3. Protonated chiral selectors, YnH+, were not observed as product ions in all cases, probably because the proton affinity of amino acids bearing a BOC group is much lower than that of the free amino acids. For each complex ion, all four chiral combinations of the chiral selector and the amino acid, i.e., LD and DL (heterochiral) and LL and DD (homochiral), were studied. Chiral recognition can be measured by comparing the observed dissociation efficiencies 5386
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
([XYn-1H+]/[XYnH+])LD + ([XYn-1H+]/[XYnH+])DL ([XYn-1H+]/[XYnH+])LL + ([XYn-1H+]/[XYnH+])DD (3)
that used by Vekey and Czira.39 Thus, a CR value of 1 would mean there is no observable chiral discrimination. A CR value larger than 1 indicates that dissociation is more favorable in the heterochiral case, whereas a CR value smaller than 1 indicates that dissociation is more favorable in the homochiral case. The more deviation the CR value is from unity, the more significant is the chiral discrimination observed. As shown in Table 1, the protonated dimer from Phe and BPhe displayed very little chiral discrimination in their CID spectra, while a significant chiral discrimination could be observed for the protonated trimer and even more for the protonated tetramer. The larger CR with the protonated tetramer may be related to increased steric hindrance resulting from more aggregation in the complex ion. Because of the lower intensity of the protonated tetramer and a larger mass shift in the higher mass region, the
Table 1. CR Values According to Eq 3 and Based on the CID Spectra of Protonated Dimers, Trimers, and Tetramers Obtained from Phe and BPhe [XYn-1H+]/[XYnH+]a parent ion protonated
} } }
a n ) 1 for protonated dimers; n ) 2 for protonated trimers; n ) 3 for protonated tetramers. b Standard deviation of the mean value. c Standard deviation of the CR value. d Relative collision energy 10%, isolation width 10 u, and mass range 120-500 u. e Relative collision energy 8%, isolation width 18 u, and mass range 350-750 u. f Relative collision energy 0%, isolation width 50 u, and mass range 600-1050 u.
Table 2. CR Values According to Eq 3 and Obtained from the CID Spectra of the Protonated Trimers of Amino Acids with BPhe, BPro, or BBSer BPhe amino acid
Ala Val Leu Ile Pro Phe Tyr Trp His Met Cys Ser Thr Asp Glu Asn Gln Argd Lys
1.044 1.046 1.021 1.025 2.002 0.812 0.894 0.566 1.738 1.152 1.054 1.012 1.158 0.810 0.855 0.938 1.905 1.416 0.840
0.008 0.011 0.006 0.009 0.018 0.005 0.007 0.004 0.010 0.006 0.007 0.005 0.007 0.006 0.003 0.004 0.015 0.008 0.006
0.73 1.02 0.55 0.83 0.88 0.56 0.75 0.67 0.55 0.55 0.68 0.47 0.64 0.70 0.38 0.42 0.80 0.59 0.71
1.074 1.050 0.989 1.060 2.340 1.096 1.137 1.934 2.107 1.470 1.122 1.313 1.166 1.102 1.752 1.163 2.042 2.027 0.945
0.006 0.008 0.008 0.006 0.021 0.004 0.006 0.009 0.015 0.017 0.005 0.007 0.005 0.007 0.010 0.004 0.009 0.015 0.004
0.53 0.75 0.85 0.60 0.88 0.36 0.50 0.47 0.71 1.12 0.49 0.52 0.39 0.66 0.59 0.35 0.46 0.73 0.46
1.143 1.527 1.493 1.572 1.362b 0.928 0.963 0.494 4.316 1.277 1.262 0.992 1.078 1.061 1.626b 0.992 2.953 0.640 2.647b
0.012 0.014 0.012 0.012 0.012b 0.008 0.010 0.007 0.027 0.010 0.009 0.010 0.009 0.006 0.016b 0.011 0.039 0.007 0.028b
1.06 0.95 0.80 0.74 0.91 0.83 0.99 1.42 0.64 0.77 0.73 1.01 0.80 0.60 1.00 1.06 1.31 1.04 1.04
suitable chiral selectorsa
BPhe BPhe BPhe BPhe BPhe BPhe BPhe BPhe BPhe BPhe BPhe BPhe
BPro BProc BPro BPro BPro BPro BPro BPro BPro BPro BPro BPro BPro BPro
BBSer BBSer BBSer BBSer BBSer BBSer BBSer BBSer BBSer
BBSer BBSer BBSer BBSer
a CR > 1.1 or CR < 0.9. b The figures previously reported in ref 62 actually corresponded to 1/CR and were thus incorrect. c Should also be suitable. d Protonated trimers of low intensities; thus the CID spectra of protonated dimers were measured instead.
results obtained with the protonated tetramer had a larger error (Table 1). Therefore, the CID of protonated trimers was selected in general for our investigation of the chiral recognition of 19 common amino acids with different chiral selectors. Similar protonated dimer and trimer systems (formed instead from mixtures of amino acid pairs) were studied by Vekey and Czira;39 they observed no stereochemical difference in the dissociation of the homochiral and heterochiral protonated dimers, but using their protonated trimers and a kinetic method for studying very small energy differences in the product ions of competing reactions, they successfully discriminated between the homochiral and heterochiral amino acid pairs. The CR values for 19 common amino acids with BPhe as the chiral selector are shown in Table 2. BPhe provided large chiral
discriminations for amino acids having aromatic side chains, i.e., Phe, Tyr, Trp, and His, but little chiral discrimination for those having alkyl side chains, i.e., Ala, Val, Leu, and Ile. Significant chiral discriminations were also observed for the other amino acids except Asn, Cys, and Ser, the latter two have similar side chains, HSCH2- and HOCH2-. Among all the 19 common amino acids, the chiral discrimination of Pro with BPhe was the most pronounced. Apparently the rigid structure of Pro can play an important role. So we selected L- or D-N-t-BOC-proline (Figure 1, b) as another chiral selector for amino acids. BPro afforded insufficient improvement for the amino acids with alkyl side chains, but provided good discrimination for the other amino acids except Lys (Table 2). Apart from Leu and Lys, which showed a poor chiral discrimination with BPro, Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
Table 3. Chiral Discrimination of Leucine with L-BOC-amino Acids as Observed in the CID Spectra of Their Protonated Trimers chiral selector L-BOC-Asn L-BOC-Tyr L-BOC-Gln L-BOC-Leu L-BOC-Ile L-BOC-Val L-BOC-Met L-BOC-Ala L-BOC-N-methyl-Ala L-BOC-3-(2-naphthyl)-Ala L-BOC-O-benzyl-Ser L-BOC-O-benzyl-Thr a
1.019 0.892 0.984 1.061 1.073 1.114 1.050 1.064 1.049 0.926 1.413 1.226
0.014 0.009 0.015 0.012 0.008 0.011 0.011 0.011 0.021 0.017 0.011 0.011
According to eq 2, the chiral recognition ratio CR can be regarded as the equilibrium constant for a virtual equilibrium such as eq 4 or 5. The free energy for the virtual equilibrium, which indicates
XD(YL)2H+ + XLYLH+ T XL(YL)2H+ + XDYLH+
XL(YD)2H+ + XDYDH+ T XD(YD)2H+ + XLYDH+
the energy difference of the dissociation processes, as depicted in eq 1, between the heterochiral and homochiral complexes, can be calculated by eq 6, where R is the gas constant and Teff is the
-∆G ) RTeff ln(CR)
CR ) ([XYH+]/[XY2H+])DL/([XYH+]/[XY2H+])LL as in eq 2.
the same chiral discrimination trend was observed for all other amino acids; i.e., the dissociation of the heterochiral protonated trimers was more favorable than that of the homochiral protonated trimers with BPro as the chiral selector. To differentiate systematically, and with ease, the chirality of all 19 common amino acids, a suitable chiral selector for the amino acids with alkyl side chains was required. We thus investigated the chiral discrimination of Leu, as a representative amino acid with an alkyl side chain, with various L-BOC-amino acids (Table 3). Among all investigated, L-BOC-O-benzylserine (L-BBSer, Figure 1, c) provided the largest chiral discrimination for Leu. Therefore, L- or D-BBSer was investigated as another chiral selector for the 19 common amino acids. As expected, BBSer afforded significant chiral discriminations for the four amino acids with alkyl side chains (Table 2). It is interesting to note that both BBSer and BPhe afforded the same chiral discrimination trend for each of the amino acids with an aromatic side chain. This may be related to the phenyl group present in both selectors, and to their structural similarity, BBSer having just an additional OCH2 group in the side chain. The CR values for 19 common amino acids obtained with BPhe, BPro, and BBSer as the chiral selectors are summarized in Table 2, and a typical example of chiral discrimination, as observed in the CID spectra of all four chiral combinations of the protonated trimers, is shown in Figure 4. The highest relative standard deviation obtained in the determination of all the CR values in Table 2 was merely 1.4%. Therefore, at the 99% or better confidence level, any CR value obtained in a single measurement that falls outside the range of 1.00 ( 0.08 (i.e., 1.00 ( 9.925 × 1.4%/30.5)66 would indicate a significant chiral discrimination. In other words, chiral discrimination can be achieved with a high degree of certainty if CR is larger than 1.08 (or 1.1) or smaller than 0.92 (or 0.9). On this basis, our data clearly show that the chirality of any of the 19 common amino acids can be recognized by mass spectrometry with at least one of the three chiral selectors (BPhe, BPro, or BBSer). Energy Difference of the Dissociation Processes between the Heterochiral and the Homochiral Protonated Trimers. (66) For calculation of confidence intervals, see: Harris, D. C. Quantitative Chemical Analysis, 5th ed.; W. H. Freeman and Co.: New York, 1999; p 74.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
effective temperature of the system during the dissociation processes. In the present study, the effective temperature was estimated as 325 K according to the study by Lebrilla et al.67 on proton-bound systems in an ion trap instrument. Thus, the -∆G values for the complex ions formed from the 19 common amino acids and BPhe, BPro, or BBSer can be calculated from Table 2 and they are summarized in Table 4, where a -∆G value of less than zero would indicate a less favorable dissociation of the corresponding heterochiral complex and vice versa. In the case of Arg where the dissociation of the dimers rather than the trimers was studied, because the product monomer is degenerate, the -∆G value obtained reflects the stability difference between the homochiral and heterochiral protonated dimers. A -∆G value of less than zero, as for Arg(BBSer)H+, means the heterochiral complex is more stable. Effects of the Side Chains of Amino Acids on Chiral Discrimination. The data in Tables 2 and 4 provided some insights for the various effects of the amino acid side chains on the extent of chiral discrimination. The chiral discrimination observed for the protonated complexes should arise from the intrinsic gas-phase interaction between the protonated amino acid and the chiral selector in the absence of solvent. The group of four amino acids with alkyl side chains (Ala, Val, Leu, Ile) displayed similar chiral recognition effects in the presence of the three chiral selectors. Chiral differentiation for this group was poor with BPhe or BPro, but improved significantly with BBSer, which has a longer side chain for interaction and an extra oxygen for hydrogen bonding. The lack of steric or spacial interaction with a very short alkyl chain in alanine was probably the cause for its much-reduced discrimination even with BBSer. Changing the side chain to a more rigid arrangement could lead to a substantial improvement in chiral recognition, as was the case for Pro with BPhe or BPro as the chiral selectors. Asn, Gln, Asp, and Glu bear side chains that can be involved in hydrogen bonding, but the length of the side chains appeared to be crucial in chiral recognition. The amino acids with shorter side chains (Asp, Asn) generally showed much less chiral discrimination with the three chiral selectors. The additional methyl group in Thr, as compared to Ser, would increase steric interaction and enhance discrimination for the complexes involving BPhe or BBSer. However, such increased (67) Feng, W. Y.; Gronert, S.; Lebrilla, C. B. J. Am. Chem. Soc. 1999, 121, 13651371 and ref 43 therein.
Figure 4. Chiral discrimination of His with BBSer as observed in the CID spectra of protonated trimers. MS/MS conditions: relative collision energy 8% and isolation width 18 u: (a) D-His + D-BBSer; (b) L-His + D-BBSer; (c) L-His + L-BBSer; and (d) D-His + L-BBSer.
Table 4. Summary of the -∆G Values Estimated from the CR Values in Table 2 for the 19 Common Amino Acids with BPhe, BPro, or BBSer as Chiral Selectors BPhe
amino -∆G SD-∆Ga -∆G SD-∆G -∆G SD-∆G acid (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) Ala Val Leu Ile Pro Phe Tyr Trp His Met Cys Ser Thr Asp Glu Asn Gln Argb Lys
0.116 0.122 0.056 0.067 1.876 -0.563 -0.303 -1.538 1.494 0.382 0.142 0.032 0.396 -0.569 -0.423 -0.173 1.741 0.940 -0.471
0.021 0.028 0.016 0.024 0.024 0.017 0.021 0.019 0.016 0.014 0.018 0.013 0.016 0.020 0.009 0.012 0.021 0.015 0.019
0.193 0.132 -0.030 0.157 2.297 0.248 0.347 1.782 2.014 1.041 0.311 0.736 0.415 0.262 1.515 0.408 1.929 1.909 -0.153
0.015 0.021 0.022 0.015 0.024 0.010 0.014 0.013 0.019 0.031 0.012 0.014 0.012 0.017 0.015 0.009 0.012 0.020 0.011
0.361 1.144 1.083 1.222 0.835 -0.202 -0.102 -1.906 3.951 0.661 0.629 -0.022 0.203 0.160 1.314 -0.022 2.926 -1.206 2.630
0.028 0.025 0.022 0.021 0.024 0.023 0.028 0.038 0.017 0.021 0.019 0.027 0.023 0.015 0.027 0.030 0.036 0.030 0.029
a Standard deviation for -∆G. b The dissociation of the protonated dimer rather than the trimer was investigated.
interaction might not always be useful, as was the case with the more rigid chiral selector, BPro. For the group of aromatic amino acids (Phe, Tyr, Trp, His), the electron-donating nitrogen in Trp and His appeared to be important for substantial chiral discrimination. All three amino acids with a benzene ring (Phe, Tyr, Trp) showed the same chiral discrimination trend in the presence of any of the three chiral selectors; i.e., the dissociation of the homochiral protonated trimers was more favorable with chiral selectors bearing a benzene
Figure 5. Plot of the observed dissociation efficiency, r, versus scan time. Series 1-3 at the top are three repeat measurements for a mixture of L-Phe and D-BPro, while series 4-6 at the bottom are three repeat measurements for a mixture of L-Phe and L-BPro. The concentrations of L-Phe, D-BPro, and L-BPro were 1 mM each in 75% methanol containing 0.5% acetic acid.
ring (BPhe, BBSer), while that of the heterochiral protonated trimers was more favorable with BPro as the chiral selector. This might suggest an orientating role between benzene rings in chiral recognition. Molecular modeling has been investigated,68 but this proved to be difficult to achieve as there are too many variables in the protonated trimer systems used in the present study. Nevertheless, it can be concluded that steric hindrance, π-π interaction, and hydrogen bonding could play an important role in the chiral recognition of amino acids with BOC-amino acids as the chiral selectors. (68) We thank Mr. Zhitao Xu, Mr. Yilei Zhao, and Prof. Yundong Wu for their efforts in the molecular modeling of the protonated trimer systems used in this study.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
Figure 6. ESI spectrum of a mixture of Val (5 × 10-6 M), Ile (5 × 10-6 M), Trp (5 × 10-6 M), and D-BBSer (1 mM). Inset mass range: 690-810 u. ESI conditions: syringe pump 1 µL min-1, sheath gas 60 psi, spray voltage 4 kV, and heated capillary 50 °C.
Repeatability. The CR value for Phe with BPro as the chiral selector was 1.096, a little smaller than the defined limit of 1.1. To check the validity of this limit and the repeatability of our method, the observed dissociation efficiency r, where r ) [(Phe)(BPro)H+]/[(Phe)(BPro)2H+], for the dissociation of the protonated trimers obtained from mixtures of L-Phe and D-BPro, and L-Phe and L-BPro were plotted against scan time in Figure 5. The spectra were averaged for 1 min each during the whole scan time. The three replicate measurements for the mixture of L-Phe and D-BPro were clearly distinguishable from those of L-Phe and L-BPro, and the two groups never crossed over. Values obtained 2 min after sample introduction were more precise, and a definite chiral discrimination could be seen even for a CR value near our defined limit of 1.1. In the present study, repeatability was assured by a number of factors, including the following: (i) the use of ESI to produce complexes of very low internal energy; (ii) the MS/MS mode58,59 that is relatively insensitive to changes in the ESI conditions; (iii) the highly specific low-energy collision processes; (iv) the continuous introduction of samples via a syringe pump; (v) the AGC function that automatically adjusts the signal strength; and (vi) the averaging of profile data. Figure 5 also suggested that, in general, spectra averaged for 1 min and collected 2 min after sample introduction should be adequate for chiral recognition. Solvent and Concentration Effects. Previous studies22,27 revealed that chiral recognition by mass spectrometry was dependent on the solvent used and on the ratio between the sample and chiral selector, even for the enantiomer-labeled method. Water and methanol are common solvents for amino acids. As shown in Table 5 for Phe with L-BPro as the chiral selector, the CR values remained constant for different compositions of water and methanol and for a range of acidity. In addition, the CR values were insensitive to significant changes in the ratio of Phe to L-BPro, varying from 10:1 to 1:500 (Table 6). At the extreme amino acid/chiral selector ratio of 1:500, the signals for the protonated trimer and dimer were very weak. The above results indicate that our ESI-MS/MS method is rather insensitive 5390 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
Table 5. Solvent Effect on the CR Value of Phe with L-BPro as the Chiral Selectora solvent
water methanol water/methanol (50:50) water/methanol/acetic acid (50:50:1) water/methanol/acetic acid (50:50:10)
1.097 1.117 1.105 1.121 1.086
0.008 0.012 0.009 0.008 0.004
a The concentrations of D- or L-Phe and L-BPro were 1 mM each in the stated solvents. b CR ) ([(Phe)(BPro)H+]/[(Phe)(BPro)2H+])DL/ ([(Phe)(BPro)H+]/[(Phe)(BPro)2H+])LL.
Table 6. Concentration Effect on the CR Value of Phe with L-BPro as the Chiral Selectora D-
or L-Phe:L-BPro 10:1 1:1 1:10 1:50 1:200 1:500c
1.098 1.102 1.098 1.105 1.054 1.135
0.007 0.003 0.009 0.009 0.018 0.036
a Before mixing, the concentration of D- or L-Phe and L-BPro was 2 mM each in 50% methanol containing 1% acetic acid. b CR ) ([(Phe)(BPro)H+]/[(Phe)(BPro)2H+])DL/([(Phe)(BPro)H+]/[(Phe)(BPro)2H+])LL. c MS/MS signals were very weak.
to changes in the solvent or concentration conditions and suggest that chiral recognition using ESI-MS/MS is robust and requires minimal sample preparation. Chiral Determination of Amino Acids in a Mixture. Conventionally, the chirality of different components in a mixture cannot be determined3,4,65 by X-ray crystallography, polarimetry, circular dichroism, or nuclear magnetic resonance. It can be determined by chromatography or capillary electrophoresis, but suitable chiral stationary phases or additives as well as reference standards are required. In most cases, each component is required to be separated first from the mixture, and only then can its
Figure 7. CID spectra of protonated trimers formed from D- or L-BBSer and an amino acid component (Val, Ile, Trp) in a mixture. All products ion peaks are expanded 2 times. Spectra a, c, and e were obtained in a single 7-min sample introduction and spectra b, d, and f in another 7-min sample introduction. Spectra a and b were each averaged between 3 and 4 min after sample introduction, spectra c and d each between 4.5 and 5.5 min, and spectra e and f each between 6 and 7 min. MS/MS conditions: relative collision energy 8% and isolation width 18 u.
chirality be determined. It is quite laborious and time-consuming, and usually a relatively large sample size is required. A mixture containing Val, Ile, and Trp of “unknown” chirality, each at 0.01 mM in 50% methanol containing 1% acetic acid, was analyzed by the present method. The mixture solution was allowed to mix in a 1:1 ratio with respectively D- or L-BBSer (2 mM in methanol) prior to mass spectrometric measurements. The full ESI spectrum obtained with D-BBSer is shown in Figure 6. Ions m/z 708, 722, and 795, corresponding to the protonated trimers formed from BBSer and, respectively, Val, Ile, and Trp, were chosen for MS/MS analysis. The CID spectra were acquired for the three precursor ions consecutively during a single sample introduction for the mixture with D-BBSer and then another sample introduction for the mixture with L-BBSer (Figure 7). Dissociation was more favorable for all three ions with D-BBSer as the chiral selector. Therefore, with reference to Table 2, which shows the dissociation of the heterochiral BBSer complexes with Val or Ile is favored and that with Trp not favored, the three amino acids in the mixture were easily determined to be L-Val, L-Ile, and D-Trp.
The above chiral determination of three amino acid components in a mixture required only two sample introductions, ∼14 min of mass spectrometric measurements and consumed ∼70 pmol of each component. The results demonstrate that ESI-MS/ MS is a powerful and sensitive technique for chiral recognition, with distinct advantages over other methods. Chiral Determination of Amino Acids in a Cyclodipeptide. A compound isolated69 from sponge was identified as cyclo-(ProLeu) based on MS and NMR data. The hydrolysis product of the cyclodipeptide was mixed respectively with D- and L-BBSer and analyzed by mass spectrometry. The full ESI spectrum obtained with D-BBSer is shown in Figure 8. Ions m/z 706 and 722, corresponding to the protonated trimers formed from D- or L-BBSer and, respectively, Pro and Leu, were selected for MS/ MS analysis. Although the trace sulfuric acid70 present resulted in unstable and low signals for the protonated trimers, the CID (69) Zhang, W. H.; Che, T. C., unpublished results. (70) For interference to ESI spectra from sulfuric acid, see: Chowdhury, S. K.; Katta, V.; Beavis, R. C.; Chait, B. T. J. Am. Soc. Mass Spectrom. 1990, 1, 382-388.
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
Figure 8. ESI spectrum of a mixture of 25-µL digestion products of the cyclodipeptide and 100 µL of D-BBSer (2 mM in methanol). Inset mass range: 680-750 u. ESI conditions: syringe pump 3 µL min-1, sheath gas 60 psi, spray voltage 4 kV, and heated capillary 50 °C.
Figure 9. CID spectra of protonated trimers formed from D- or L-BBSer and an amino acid component (Pro, Leu) in the hydrolysis product of a cyclodipeptide. The product ion peaks at m/z 411 in (a) and (b) are expanded 3 times, while those at m/z 427 in (c) and (d) are expanded 1.5 times. Spectra a and c were obtained in a single 7-min sample introduction and spectra b and d obtained in another 7-min sample introduction. Spectra a and b were each averaged between 2 and 4 min after sample introduction and spectra c and d each between 4.5 and 5.5 min. MS/MS conditions: relatively collision energy 8.2% and isolation width 18 u.
spectra (Figure 9) clearly showed chiral discrimination. The dissociation of the protonated trimer formed with D-BBSer was more favorable for both Pro and Leu; thus, the two amino acids in the cyclodipeptide were determined to be L-Pro and L-Leu, and the peptide is cyclo-(L-Pro-L-Leu). This result is consistent with the physicochemical properties and NMR data of the compound in the literature.63a,69,71 Although the signal of m/z 706 was very low (Figure 8), the CID spectra gave satisfactory results for chiral recognition. This (71) Adamczeski, M.; Reed, A. R.; Crews, P. J. Nat. Prod. 1995, 58, 201-208.
5392 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000
demonstrated the high sensitivity and specificity of our method. Only 0.1 mg of the cyclodipeptide was consumed for the present chiral determination, compared to 3 g used previously.63a Moreover, the method we have developed is convenient and rapid, requiring no prior separation of the amino acids and taking only ∼11 min of mass spectrometric measurements.
CONCLUSION In summary, we have developed a mass spectrometric method to determine the chirality of all 19 common amino acids, using
BPhe, BPro, or BBSer as the chiral selector. Reference standards are not required. The method has been demonstrated to be simple, rapid, precise, sensitive, and robust. It can be used to identify the chirality of different compounds in a mixture and requires minimal sample preparation. The results indicate that ESI-MS/MS can be a good approach for chiral recognition. Electrospray ionization can provide complex ions of low internal energy even for large or labile components and also at low concentrations. The continuous introduction of samples via a syringe pump, the gentle collisional dissociation, and the time averaging of profile data of highly selective CID processes have ensured a good repeatability of the present results.
ACKNOWLEDGMENT Financial support from a RGC 1995 Competitive Earmarked Research Grant (HKUST 598/95P) and a Royal Society of Chemistry 1993 Research Fund (both to T.S.M.W.) is acknowledged. The authors are indebted to the Biotechnology Research Institute of HKUST for providing the LCQ instrument for this study. Thanks are also extended to Mr. Wei-Han Zhang for providing the cyclodipeptide sample for this study. Received for review June 26, 2000. Accepted August 10, 2000. AC000729Q
Analytical Chemistry, Vol. 72, No. 21, November 1, 2000