Anal. Chem. 2004, 76, 663-671
Chiral Morphing and Enantiomeric Quantification in Mixtures by Mass Spectrometry Lianming Wu, Eduardo Cesar Meurer,† and R. Graham Cooks*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
A novel mass spectrometric method is introduced for rapid and accurate chiral quantification by examining a tetracoordinated transition metal complex into which a reference and a fixed ligand are incorporated simultaneously with the analyte. Chiral analysis is performed by measuring the dissociation kinetics of these trimeric cluster ions [(MII + Lfixed - H)(ref*)(An)]+ (MII ) a transition metal ion, Lfixed ) chiral peptide fixed ligand, ref* ) chiral reference ligand, and An ) chiral analyte) in an ion trap mass spectrometer. The ratio of the product ion branching ratios measured when a pair of pure chiral fixed ligands and chiral reference ligands (Lfixed D /ref*D fixed fixed and Lfixed /ref* ; or L /ref* and L /ref* ) are emL L D L D L ployed in separate experiments is related, via the kinetic method formalism, to the enantiomeric composition of the chiral mixture. This fixed-ligand quotient ratio (QRfixed) is logarithmically proportional to enantiomeric purity allowing construction of a calibration curve for chiral analysis when the analyte is only available in one form of known optical purity. There are reciprocal relationships when switching the chirality of the fixed/reference ligands. Improved quantification accuracy (due to simplified dissociation kinetics) and ready construction of two or more single-point calibration curves allow data to be crosschecked and represent an advantage of this approach. These features and the matrix tolerance of the kinetic method are demonstrated using the QRfixed method for determinations of enantiomeric excess of the drug DOPA in the presence of the co-drug compound L-carbidopa. The chiral selectivity of DOPA was found to vary from 0.0581 to 0.337 using this method, depending on the choices of fixed-ligand and reference chirality. The average relative errors are less than 1.2%. The potential of chiral morphing (changing chiral centers in the ligands) to further refine the chiral interactions and hence to maximize chiral recognition is shown. The accelerating trend toward the use of enantiomerically pure compounds as drugs has driven investigations of bioactivity of chiral drug molecules including their pharmacology and toxicology. Individual enantiomeric forms of drugs may produce different therapeutic effects.1 Quantitative determination of enantiomeric * To whom correspondence should be addressed. E-mail: cooks@ purdue.edu. Fax: (765) 494-9421. † On leave from State University of Campinas, Institute of Chemistry, CP6154 Campinas, SP, 13083-970 Brazil. 10.1021/ac0349072 CCC: $27.50 Published on Web 12/18/2003
© 2004 American Chemical Society
Chart 1
excess (ee) of chiral drugs and their metabolites sets new requirements in both pharmaceutical discovery and clinical pharmacy. Following the new FDA guidelines on chiral drugs, which recommend the study of enantioselective identity and stability, as well as assays for the contributions of individual enantiomers to pharmacological and toxicological activity, an increasing number of optically pure drugs have been approved and marketed.2,3 In addition, some drug formulations are composed of two or more different single-enantiomer compounds, which improve clinical efficiency. Sinemet, a Parkinson drug, is such a formulation consisting of levodopa (L-DOPA) and L-carbidopa (see Chart 1 for structures). When taken orally, L-carbidopa can prevent in-body breakdown of levodopa, a chiral drug used to increase production of dopamine, which reduces the symptoms of Parkinson’s disease.4 This combination was recently further developed by Novartis as approved Stalevo (carbidopa, levodopa, and entacapone) tablets. Kaletra is another example in which a combination of chiral compounds Lopinavir and Ritonavir is used in the treatment of HIV infection. Quantification of chiral drugs when only one pure optical analyte is available as a standard for calibration, especially in the presence of other co-drug compounds, is challenging. This issue is addressed in this study through introduction of a novel single-point calibration method that is applied twice using different chiral forms of the reference. Currently quantitative determinations of enantiomeric purity are mainly achieved using chromatographic methods such as highperformance liquid chromatography5,6 and, increasingly, capillary electrophoresis,7,8 as well as circular dichroism,9 nuclear magnetic (1) Aboul-Enein, H. Y.; Wainer, I. W. The Impact of Stereochemistry on Drug Development and Use; John Wiley & Sons: New York, 1997. (2) Stinson, S. C. Chem. Eng. News 1999, 77 (41), 101-120. (3) Stinson, S. C. Chem. Eng. News 2000, 78 (23), 55-79. (4) Bender, D. A.; Earl, C. J.; Lees, A. J. Clin. Sci. 1979, 56, 89-93. (5) Beesley, T. E.; Scott, R. P. W. Chiral Chromatography; John Wiley & Sons: New York, 1998. (6) Tang, Y.; Zukowski, J.; Armstrong, D. W. I. J. Chromatogr., A 1996, 743, 261-271. (7) Zhu, X.; Lin, B.; Jakob, A.; Wuerthner, S.; Koppenhoefer, B. Electrophoresis 1999, 20, 1878-1889.
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resonance,10 and enzymatic techniques.11 Internal standards are used and quantitation is achieved by regression (linear, quadratic, or curve fitting) using either peak height or peak area detector response ratios of the enantiomer to the internal standard plotted against relative enantiomer concentration. Selection of an appropriate approach, including the choice of chiral stationary phase and internal standard, is influenced by the nature of the chiral analyte of interest and the complexity of the biological extraction and derivatization processes necessary.12 In practice, such procedures are complicated and time-consuming. The capabilities of mass spectrometry for the rapid analysis of complex mixtures have encouraged its exploration for chiral recognition. As a result, especially in combination with soft ionization techniques such as electrospray (ESI)13 and matrixassisted laser desorption/ionization (MALDI),14 mass spectrometry has emerged as a powerful tool for direct measurements of enantiomeric excess.15,16 By creating a chiral environment, chiral analysis has been successfully achieved using mass spectrometric methods that fall into the following categories: (1) host-guest diastereomeric adduct formation with a chiral reference compound investigated by single-stage mass spectrometry.17 One of the enantiomeric guests (analytes) is isotopically labeled, and hence, the corresponding mixture of diastereomeric adducts can be massresolved. This method can be practiced easily using various kinds of ionization techniques including chemical ionization,18 fast atom bombardment,19 MALDI,20 and ESI.21 (2) Chiral recognition is achieved via guest-host type ion/molecule (equilibrium) reactions. Diastereomeric complex ions are generated from a chiral analyte and a chiral host molecule such as crown or cyclodextrin, then mass-selected, and allowed to react with a neutral reagent (either chiral or nonchiral).22-25 Chiral differentiation is achieved by investigating the difference in exchange rate over time due to the chirality change of the analyte, so the experiments are readily performed in traps including the quadrupole ion trap25 and Fourier (8) Blaschke, G.; Chankvetadze, B. J. Chromatogr., A 2000, 875, 3-25. (9) Hattori, T.; Minato, Y.; Yao, S.; Finn, M. G.; Miyano, S. Tetrahedron Lett. 2001, 42, 8015-8018. (10) Evans, M. A.; Morken, J. P. J. Am. Chem. Soc. 2002, 124, 9020-9021. (11) Abato, P.; Seto, C. T. J. Am. Chem. Soc. 2001, 123, 9206-9207. (12) Blomberg, L. G.; Wan, H. Electrophoresis 2000, 21, 1940-1952. (13) Fenn, J. B.; Mann, N.; Meng, C. K.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70. (14) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (15) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, M. Int. J. Mass Spectrom. 2000, 198, 137-163. (16) Finn, M. G. Chirality 2002, 14, 534-540. (17) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73-90. (18) Nikolaev, E. N.; Denisov, E. V.; Rakov, V. S.; Futrell, J. H. Int. J. Mass Spectrom. 1999, 183, 357-368. (19) 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. (20) So, M. P.; Wan, T. S. M.; Chan, T. W. D. Rapid Commun. Mass Spectrom. 2000, 14, 692. (21) 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, 3, 701-710. (22) Dearden, D. V.; Liang, Y.; Nicoll, J. B.; Kellersberger, K. A. J. Mass Spectrom. 2001, 36, 989-997. (23) Liang, Y. J.; Bradshaw, J. S.; Izatt, R. M.; Pope, R. M.; Dearden, D. V. Int. J. Mass Spectrom. 1999, 187, 977-988. (24) Grigorean, G.; Ramirez, J.; Ahn, S. H.; Lebrilla, C. B. Anal. Chem. 2000, 72, 4275-4281. (25) Grigorean, G.; Gronert, S.; Lebrilla, C. B. Int. J. Mass Spectrom. 2002, 219, 79-87.
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transform ion cyclotron resonance mass spectrometer.22-24 (3) Tandem mass spectrometric experiments provide the third way to perform chiral analysis. This is done by comparing differences in fragmentation patterns via collision-induced dissociation (CID) of diastereomeric adducts that are formed from an analyte and a chiral reference ligand.26,27 (4) The fourth approach employs cluster ion dissociation treated by the kinetic method28,29 to recognize and quantify chiral molecules. It is the subject of this study. Although this method is not limited to transition metalbound cluster ions examined in tandem mass spectrometry, this does represent a particularly favorable situation that gives rise to large chiral recognition due to enhanced stereochemical effects resulting from d-electronic orbitals of transition metals.30-34 So far the kinetic method has been successfully applied to chiral discrimination of amino acids,30,35 R-hydroxy acids,31 some important drugs such as an antiviral nucleoside36 and β-blockers,37 chiral peptides,38,39 and sugars.40 Chiral distinction is indicated by different relative abundances of product ions caused by differences in the dissociation energy required for their formation, as dictated by the kinetic method.28,29 This procedure has many attractive features (it is fast, simple, and accurate, there is no requirement for isotopic labeling or wet chemical processes, it is tolerant to impurities, and a commercial instrument can be used) and it allows rapid determination of very low enantiomeric excess. Significantly, the kinetic method procedure has also been extended to quantify ternary mixtures of optical isomers41 as well as binary and ternary mixtures of different amino acids.42 Recently, a novel fixed-ligand version of the kinetic method was introduced to improve the accuracy of chiral quantitation by increasing chiral selectivity and simplifying the dissociation kinetics. A fixed ligand is defined as a ligand in a cluster ion that is not lost upon collisional activation.43 Previous kinetic methods of chiral analysis were all single ratio (SR) methods since they allow enantiomeric mixtures to be analyzed by simply recording a ratio of two fragment ion abundances in a single (tandem) mass spectrum. The recently introduced fixed-ligand method43 falls into this category. A SR method allows the analysis of chiral mixtures within a few minutes, (26) Dang, T. T.; Pedersen, S. F.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1994, 5, 452-459. (27) Tabet, J. C. Tetrahedron 1987, 43, 3413-3420. (28) Cooks, R. G.; Patrick, J. S.; Kotiaho, T.; McLuckey, S. A. Mass Spectrom. Rev. 1994, 13, 287-339. (29) Cooks, R. G.; Wong, P. S. H. Acc. Chem. Res. 1998, 31, 379-386. (30) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609. (31) Wu, L.; Tao, W. A.; Cooks, R. G. Anal. Bioanal. Chem. 2002, 373, 618627. (32) Vekey, K.; Czira, G. Anal. Chem. 1997, 69, 1700-1705. (33) Paladini, A.; Calcagni, C.; Di Palma, T.; Speranza, M.; Lagana, A.; Fago, G.; Filippi, A.; Satta, M.; Guidoni, A. G. Chirality 2001, 13, 707-711. (34) Yao, Z.-P.; Wan, T. S. M.; Kwong, K.-P.; Che, C.-T. Anal. Chem. 2000, 72, 5383-5393. (35) Zhang, D.; Tao, W. A.; Cooks, R. G. Int. J. Mass Spectrom. 2001, 204, 159169. (36) Tao, W. A.; Wu, L.; Cooks, R. G. J. Med. Chem. 2001, 44, 3541-3544. (37) Tao, W. A.; Gozzo, F. C.; Cooks, R. G. Anal. Chem. 2001, 73, 16921698. (38) Tao, W. A.; Cooks, R. G. Angew. Chem., Int. Ed. 2001, 40, 757-760. (39) Chen, J.; Zhu, C.-J.; Chen, Y.; Zhao, Y.-F. Rapid Commun. Mass Spectrom. 2002, 16, 1251-1253. (40) Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G. Anal. Chem. 2002, 74, 3458-3462. (41) Wu, L.; Clark, R. L.; Cooks, R. G. Chem. Commun. 2003, 137, 136-137. (42) Wu, L.; Tao, W. A.; Cooks, R. G. J. Mass Spectrom. 2003, 38, 386-393. (43) Wu, L.; Cooks, R. G. Anal. Chem. 2003, 75, 678-684.
by constructing a two-point calibration line using a racemic sample and a sample of known ee or by using pure D- and L-enantiomers or any other two samples of known optical purity. Mixtures of unknown enantiomeric composition are analyzed by measuring the ratio of two fragment ions in a single spectrum. Despite the advantages of the SR method, it is not be applicable if only one pure isomer or only one sample of known optical composition is available. This is the often the case for drugs developed from natural products when even racemic analytes are not easily obtained. To overcome this drawback, we recently introduced the quotient ratio (QR) method.44 This version of the kinetic method allows one sample of analyte with known enantiomeric purity to be used for the construction of the calibration curve. Practically, however, there are risks in quantification based on calibration curves defined by the origin and a single point. So we here introduce a novel fixed-ligand43 quotient ratio (QRfixed) method for multiple single-point calibrations, which we believe is an important extension of the previously published fixed-ligand method.43 The QRfixed method has several novel features: (i) ready construction of a calibration curve by changing the chirality of both the fixed and reference ligands; (ii) facile cross-checks on the results by switching the chirality of the fixed ligand and the reference ligand; (iii) incorporation of the chiral morphing technique to refine the chiral interactions and so to maximize chiral selectivity; and (iv) chiral qualification of drugs in mixtures without separation.
EXPERIMENTAL SECTION All experiments were performed using a commercial LCQ ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA), equipped with an ESI source and operated in the positive ion mode under the following conditions: spray voltage, 5.00 kV; capillary voltage, 20 V; heated capillary temperature, 150 °C; tube lens offset voltage, 20 V; sheath gas (N2) flow rate, 30 units (roughly 0.45 L/min). For the ion trap mass analyzer, the automatic gain control settings were 5 × 107 counts for a full-scan mass spectrum and 2 × 107 counts for a full product ion mass spectrum with a maximum ion injection time of 200 ms. In the full-scan MS/MS mode, the parent ion of interest was isolated by using multiple waveforms to remove undesired ions through broadband excitation. The isolated ions were then subjected to a supplementary ac potential to resonantly excite them and so cause CID. The Mathieu qz values chosen for resonance excitation and the resonance ejection mass scan were 0.25 and 0.83, respectively. The excitation time used was 30 ms. The excitation amplitude could be varied from 0 to 100% relative collision energy corresponding to 0-2.5 V zero-to-peak resonant excitation potential; the value was optimized in each experiment but kept constant for the measurement of the D- and L- enantiomers. Spectra shown represent the average of ∼50 scans where each scan is an average of 5 individual microscans. Mass/charge ratios (m/z) are reported using the thomson unit (1 Th ) 1 atomic mass unit per unit charge).45 Gas-phase transition metal ion complexes were generated simply by electrospraying 50/50 water/methanol solutions containing a mixture of an analyte, a peptide fixed ligand, and a chiral (44) Tao, W. A.; Clark, R. L.; Cooks, R. G. Anal. Chem. 2002, 74, 3783-3789.
reference ligand, at a concentration of 100 µM each, with the metal salt present at 25 µM transition metal ion. The dipeptides, along with DOPA and L-carbidopa, chiral amino acid reference ligands, and copper chloride, were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Methanol (HPLC grade) was obtained from Fisher Co. (Pittsburgh, PA). Molecular modeling results were obtained using PM3 semiempirical calculations and the Spartan program (Wavefunction, Inc., Irvine, CA). RESULTS AND DISCUSSION Fixed-Ligand Quotient Ratio Method for Chiral Quantification. The methodology introduced here for quantitative enantiomeric analysis based on the QRfixed method is illustrated in Scheme 1, which also includes the previous QR method for purposes of comparison. Singly charged trimeric cluster ions [(MII + Lfixed - H)(ref*)(An)]+, instead of the [MII(ref*)(An)2 - H]+ clusters used in the QR method, are generated in the gas phase by electrospraying an aqueous methanol solution containing the analyte (An), a chiral reference (ref*), a chiral peptide fixed ligand, and a divalent transition metal ion (MII). Deprotonation is expected to occur at the peptide fixed ligand.43,46 Two pairs of enantiomerically pure fixed ligands and reference compounds (Lfixed D /ref*D fixed fixed fixed and LL /ref*L, or LD /ref*L and LL /ref*D) are employed separately in two consecutive experiments. The cluster ions are mass-selected and collisionally activated in the quadrupole ion trap; they dissociate competitively to form the dimeric complexes [(MII + Lfixed - H)(An)]+ and (MII + Lfixed - H)(ref*)]+, by loss of the neutral reference compound, ref*, and the analyte, An, respectively. The ratio of branching ratios, (RR)fixed (eq 1), depends on the enantiomeric composition of the analyte, An:
(RR)fixed ) fixed + II + [(MII + Lfixed DorL - H)(An)] /[(M + LDorL - H)(ref*DorL)] fixed + II + [(MII + Lfixed LorD - H)(An)] /[(M + LLorD - H)(ref*LorD)]
(1)
When the analyte is enantiomerically pure, (RR)fixed is or (RR)fixed . The chiral selectivity, the ratio of given as (RR)fixed D L fixed fixed fixed (RR)D to (RR)L , a triple ratio, defined as (RR)chiral , is a measure of the degree of chiral distinction achievable in a particular experiment:
(RR)fixed chiral )
( (
(RR)fixed D (RR)fixed L
)
) )
fixed + II + [(MII + Lfixed DorL - H)(AnD)] /[(M + LDorL - H)(ref*DorL)]
fixed + II + [(MII + Lfixed LorD - H)(AnD)] /[(M + LLorD - H)(ref*LorD)] fixed + II + [(MII + Lfixed DorL - H)(AnL)] /[(M + LDorL - H)(ref*DorL)] + II + fixed [(MII + Lfixed LLorD - H)(ref*LorD)]+ LorD - H)(AnL)] /[(M
(2) (45) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 93. (46) Gatlin, C. L.; Turecek, F. J. Mass Spectrom. 2000, 35, 172-177.
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Scheme 1. Quantitative Determination of ee by (a) QRfixed and (b) QR Method
As discussed in the previous study43 on the use of the fixed fixed ligands for chiral recognition, the more different the (RR)chiral value is from unity, the higher the degree of chiral recognition. fixed Thus, (RR)chiral ) 1 indicates a lack of chiral discrimination, which means that the particular combination of MII ion, fixed ligand, and reference ligand fails to create stereochemically distinctive interactions with the enantiomers under the conditions employed. The relationship between (RR)fixed and the ee of the analyte sample is derived from the fixed-ligand procedure43 using the kinetic method for chiral analysis. Ln((RR)fixed) is proportional to the differences between the free energy changes for the competitive dissociations to yield the two dimeric products as shown in Scheme 1, i.e.
ln((RR)fixed) ) (∆(∆G))fixed/RTeff
(3)
trimers,47 and (∆(∆G))fixed is defined as the difference in free energy change in reaction 4. fixed + II + [(MII + Lfixed DorL - H)(An)] + [(M + LLorD - H)(ref*LorD)] f fixed + II + [(MII + Lfixed (4) LorD - H)(An)] + [(M + LDorL - H)(ref*DorL)]
When a mixture of the two enantiomers of An (AnD and AnL) is examined, (∆(∆G))fixed is the sum of free energy changes (∆(∆G))fixed and (∆(∆G))fixed of the two independent reactions, D L 5 and 6: fixed + II + [(MII + Lfixed DorL - H)(AnD)] + [(M + LLorD - H)(ref*LorD)] f fixed + II + (5) [(MII + Lfixed LorD - H)(AnD)] + [(M + LDorL - H)(ref*DorL)] fixed + II + [(MII + Lfixed DorL - H)(AnL)] + [(M + LLorD - H)(ref*LorD)] f
where R in the denominator is the gas constant, Teff, the effective temperature, is the average temperature of the two activated complexes for the two competitive reactions of the activated 666
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fixed + II + [(MII + Lfixed (6) LorD - H)(AnL)] + [(M + LDorL - H)(ref*DorL)]
(47) Laskin, J.; Futrell, J. H. J. Phys. Chem. A 2000, 104, 8829-8837.
The reactants and products in reaction 5 are enantiomeric pairs of the products and reactants in reaction 6, respectively, and therefore (∆(∆G))fixed and (∆(∆G))fixed have the same value but D L opposite signs:
(∆(∆G))fixed ) -(∆(∆G))fixed D L
(7)
For an enantiomeric mixture with ee as the enantiomeric excess of the D-enantiomer, one can write
(∆(∆G))fixed ) (∆(∆G))fixed D )
1 + ee 1 - ee + (∆(∆G))fixed L 2 2
+ (∆(∆G))fixed ] [(∆(∆G))fixed D L + 2 - (∆(∆G))fixed ] [(∆(∆G))fixed D L ee (8) 2
Therefore, the relationship between (RR)fixed and ee can be expressed by combining eq 3 and eq 8 to obtain
ln(RR)fixed )
(∆(∆G))fixed + (∆(∆G))fixed D L + 2RTeff - (∆(∆G))fixed (∆(∆G))fixed D L ee (9) 2RTeff
When the sample is a racemic mixture (ee ) 0%), from eq 9 ln(RR)fixed will always be 0: fixed
ln(RR)ee ) 0%
(∆(∆G))fixed + (∆(∆G))fixed D L ) ) 0 (10) 2RTeff
Equation 9 predicts a linear relationship for ln (RR)fixed versus ee, a value of zero at zero ee, and a slope that depends on the magnitude of (∆(∆G))fixed or (∆(∆G))fixed . (Note that (∆ D L fixed fixed (∆G))D and (∆(∆G))L have opposite signs.) A large chiral or (∆(∆G))fixed ) will lead distinction (large value of (∆(∆G))fixed D L to a large slope. In the fixed-ligand method,43 at least two points are required to construct a calibration curve before quantitative analysis can be performed. However, since a racemic mixture always results in a zero value for ln(RR)fixed, according to eq 10, only one sample with known ee is needed to construct this calibration curve. This is clearly a great advantage when only one pure enantiomer is available. Unknown enantiomeric mixtures are analyzed by measuring (RR)fixed values obtained from two consecutive spectra. Because there are at least two combinations of each pair of fixed ligands and references (Lfixed D /ref*D and fixed fixed Lfixed /ref* or L /ref* and L /ref* ), two independent L L D L D L single-point calibration curves can be constructed. Note that if the fixed ligands have additional chiral centers, there are more than two optical isomers, and hence, more than two calibration curves could be constructed. For example, there are four possible combinations for a dipeptide such as Ala-Ala as the fixed ligand in this study, and so on for a tripeptide (8 combinations) and a
tetrapeptide (16 combinations). Furthermore, with the use of reference ligands with two chiral centers, the numbers of combinations will be doubled. Chiral Recognition of Amino Acids by Switching the Chirality of Fixed Liagnds and Reference Ligands. There are two known advantages in chiral analysis using the fixed-ligand form of the kinetic method:43 (i) improved chiral quantification accuracy due to simplifying the dissociation kinetics; (ii) improved chiral recognition by optimizing the properties (size and functionality) of the fixed ligands. In this part of the investigation, our main objective was to explore an additional feature of the singlepoint calibration method with the ability to obtain multiple calibration curves by chirality-switching the fixed/reference ligands. The value of this option is that it allows one to crosscheck data and avoids adventurous errors. The choice of the analyte and the reference ligand is based on their ready participation in the formation of metal-bound cluster ions and also dissociation rates that are comparable to each other to allow accurate measurement of relative abundance ratioss otherwise dissociation tends to proceed overwhelmingly to form the more stable product. One successful choice is CuII, D/Ltyrosine, and D/L-phenylalanine as the central metal ion, the analyte, and the reference ligand, respectively. To facilitate experiments, the small dipeptide Ala-Ala was selected as the fixed ligand with two different combinations of enantiomeric pairs D-AlaD-Ala/L-Ala-L-Ala and D-Ala-L-Ala/L-Ala-D-Ala. Table 1 shows exfixed perimental chiral selectivity (RR)chiral values as defined by eq 2. It is important to note that the high-degree of consistency of the experimental results in Table 1 confirms the inverse effects on the chiral selectivity by switching the chirality of the fixed ligands and the reference ligands, as described by eq 7. This forms the basis of the single-point calibration method for chiral quantification. Although it is not our intention to investigate in detail how the properties of the fixed ligand (such as the size and functionality) affect chiral recognition, it was found that the particular combination of chirality of the fixed ligand and the reference ligand in entries 3 and 4 in Table 1 gave obviously increased chiral selectivity. To understand what kinds of interactions promote large differences in chiral selectivity when changing the chirality of the fixed and reference ligands, molecular modeling using semiempirical PM3 calculations was employed. Although we realize that this low-level calculation cannot give accurate binding energy information, it is still possible to obtain correct qualitative results that are consistent with experiment by searching many conformations to get these two structures with lowest energies. The results show that cation-π interactions are promoted when L-DOPA, rather than D-DOPA, is bound to a Cu(II)-bound dipeptide, creating a large chiral effect and hence leading to the observed high chiral selectivity (Figure 1). In addition, there might also be agostic bonding involving the methyl group in the dipeptide AlaAla when a heterochiral dipeptide such as D-Ala-L-Ala or L-Ala-DAla is used as the fixed ligand and coordinated to the central metal ion CuII, in comparison with homochiral dipeptides D-Ala-D-Ala or L-Ala-L-Ala. If this agostic bonding exists, it is likely that it could make additional contributions to chiral recognition; a similar effect Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
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Table 1. Chiral Recognition of Amino Acids Measured with the Fixed and Reference Ligand of Different Chiralitya-c [CuII(Lfixed-H)(An)]+/ [CuII(Lfixed-H)(ref*)]+ entry
Lfixed
ref*
D-Tyr
L-Tyr
(RR)fixed
1/(RR)fixed
1
D-Ala-D-Ala
D-Phe
L-Ala-L-Ala
L-Phe
D-Ala-D-Ala
L-Phe
L-Ala-L-Ala
D-Phe
D-Ala-L-Ala
D-Phe
L-Ala-D-Ala
L-Phe
D-Ala-L-Ala
L-Phe
L-Ala-D-Ala
D-Phe
5.76 9.72 2.83 4.58 3.80 15.9 1.29 4.46
9.67 5.82 4.52 2.91 16.1 3.78 4.48 1.26
0.596 1.67 0.626 1.57 0.236 4.21 0.288 3.54
1.68 0.599 1.60 0.635 4.24 0.238 3.47 0.283
2 3 4
fixed (RR)chiral
0.357 0.398 0.0561 0.0813
a CuII as the central metal ion. b (RR)fixed is defined in eq 2. c CID activation level is optimized and kept constant for all measurements of chiral enantiomers.
Figure 1. PM3 semiemperical molecular calculation of (a) [(CuII + D-Ala-L-Ala - H)(L-DOPA)]+ and (b) [(CuII + D-Ala-L-Ala - H)(D-DOPA)]+ by the Spartan program.
was observed in isomeric tripeptides.48 The effects on chiral recognition due to changes in the chirality of auxiliary ligands indicates the potential value of chiral morphing techniques.49 These techniques allow utilization of the spatial diversity of multiple chiral centers to produce drug candidates with improved efficiency, stability, membrane permeability, and oral availability, as well as decreased toxicity and side effects. Chiral Quantification of DOPA by Chirality Switch of Fixed and Reference Ligands. For chiral quantification of DOPA, the same fixed ligands were employed as in the previous section in combination with D-Tyr or L-Tyr as the reference ligand. Figure 2 shows product ion mass spectra of the singly charged trimeric cluster ion [(CuII + Lfixed - H)(ref*)(An)]+ (Lfixed ) D-Ala-L-Ala and L-Ala-D-Ala; ref* ) D- and L-Tyr; An ) DOPA). The QRfixed method has been tested using both the D- and the L-forms of the analyte DOPA (panels a and c of Figure 2 are used to analyze D-DOPA, and panels b and d to analyze L-DOPA). The complex ions [(CuII + D-Ala-L-Ala - H)(L-Tyr)]+ (Figure 2a) and [(CuII + L-Ala-D-Ala - H)(D-Tyr)]+ (Figure 2d) are enantiomers, and (48) Wu, L.; Lemr, K.; Aggerholm, T.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2003, 14, 152-160. (49) Fromme, J. C.; Verdine, G. L. Nat. Struct. Biol. 2002, 9, 544-552.
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therefore, the two systems should display identical [(CuII + Lfixed - H)(A)]+/[(CuII + Lfixed - H)(ref*)]+ ratios. This is indeed the case within ∼1% error; the abundance ratios from consecutive measurements of the same system were also reproducible to ∼1%. The complex ion [(CuII + D-Ala-L-Ala - H)(D-Tyr)]+ (Figure 2b) likewise shows CID behavior identical to that of its enantiomer [(CuII + L-Ala-D-Ala - H)(L-Tyr)]+ (Figure 2c). The identical behavior of the clusters generated from the two homochiral enantiomers and the two heterochiral enantiomers justifies use of the present method and reflects its reproducibility. A large chiral fixed selectivity, (RR)chiral (eq 2), ranging from 0.463 to 0.0326 is measured for DOPA when using four possible combination sets of the fixed ligand and the reference ligand at the activation conditions described in the Experimental Section. As shown in Table 2, the predicted reciprocal relationships were observed when switching the chirality of the fixed/reference ligands. This further confirms that reversing the chirality of both fixed ligand and reference ligand reverses the sign of the energy difference between the two dimeric cluster ions (eq 7). Note that when the combinations of the fixed ligands and the reference ligands are not enantiomeric pairs, viz. when switching the chirality of either the fixed ligand or the reference ligand, the
Figure 2. Product ion (MS/MS) spectra of [(CuII + Ala-Ala - H)(Tyr)(DOPA)]+ (m/z 600). Two product ions are [(CuII + Ala-Ala - H)(DOPA)]+ (m/z 419) and [(CuII + Ala-Ala - H)(Tyr)(DOPA)]+ (m/z 403). (a) Lfixed ) D-Ala-L-Ala, ref* ) L-Tyr, A ) D-DOPA; (b) Lfixed ) D-Ala-L-Ala, ref* ) L-Tyr, A ) L-DOPA; (c) Lfixed ) L-Ala-D-Ala, ref* ) D-Tyr, A ) D-DOPA; (d) Lfixed ) L-Ala-D-Ala, ref* ) D-Tyr, A ) L-DOPA. The CID activation level is chosen as 10.5%, corresponding to ∼267 mV AC. Table 2. Chiral Recognition of DOPA Measured with the Fixed and Reference Ligand of Different Chiralitya-c [CuII(Lfixed-H)(An)]+/ [CuII(Lfixed-H)(ref*)]+ entry
Lfixed
ref*
D-DOPA
L-DOPA
(RR)fixed
1/(RR)fixed
1
D-Ala-D-Ala L-Ala-L-Ala
D-Tyr L-Tyr
2
D-Ala-D-Ala L-Ala-L-Ala
L-Tyr D-Tyr
3
D-Ala-L-Ala L-Ala-D-Ala
D-Tyr L-Tyr
4
D-Ala-L-Ala L-Ala-D-Ala
L-Tyr D-Tyr
4.47 7.29 3.42 5.08 3.98 21.9 1.31 5.39
7.27 4.46 5.02 3.45 22.2 3.99 5.38 1.29
0.615 1.63 0.681 1.47 0.179 5.49 0.243 4.18
1.63 0.612 1.47 0.679 5.59 0.182 4.11 0.239
fixed (RR)Chiral
0.377 0.463 0.0326 0.0581
a CuII as the central metal ion. b (RR)fixed is defined in eq 2. c CID activation level is optimized and kept constant for all measurements of chiral enantiomers.
calibration curves constructed using the quotient ratios also give a linear relationship but they do not pass through the origin. The intrinsic linear relationship between ln(RR)fixed and ee was demonstrated further in a series of measurements on the chiral drug DOPA. Four representative pairs of the fixed ligand and the reference ligand were selected: (i) D-Ala-D-Ala/D-Tyr and L-AlaL-Ala/L-Tyr; (ii) D-Ala-D-Ala/L-Tyr and L-Ala-L-Ala/D-Tyr; (iii) D-AlaL-Ala/D-Tyr and L-Ala-D-Ala/L-Tyr; (iv) D-Ala-L-Ala/L-Tyr and L-AlaD-Ala/D-Tyr. In these experiments, the analyte DOPA was used in mixtures of various proportions to generate the corresponding trimeric clusters. In this QRfixed method, every calibration point was obtained by two consecutive measurements using optically pure fixed ligands and reference ligands. As expected, (RR)fixed was largest when pure D- or L-analytes were examined, and as also expected, the (RR)fixed value was equal to 1 (ln(RR)fixed ) 0) when the racemic analytes were examined. Linear relationships of ln(RR)fixed versus the ee of the L-isomer were obtained, and they showed correlation coefficients (r2) from 0.9992 to 0.9996, as illustrated in Figure 3. Note that this fit was not forced through
Figure 3. Calibration curves for chiral analysis of D/L-DOPA using the fixed-ligand QRfixed method with four combinations of chirality of the fixed ligands and reference ligands.
the origin, and the data confirm the effect predicted by eq 10. After verifying the linear relationship between ln(RR)fixed and ee predicted by eq 9, calibration curves could be constructed using only one single enantiomer. Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
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Figure 5. Single-point calibration curves constructed using pure enantiomer as the calibrant (filled circle) for DOPA. Data for test samples are shown as symbols with error bars. Each unknown sample shows error bars and represents the average of five individual measurements. Table 3. Enantiomeric Excess Measurements on L-DOPA in the Presence of Co-Drug Compound L-Carbidopa sample [DOPA]/ [CarbiDOPA]
Figure 4. ESI-MS spectra of a solution containing copper(II) chloride salt (25 µM), the dipeptide L-Ala-L-Ala (100 µM), the reference L-Tyr (100 µM), and (a) L-DOPA (100 µM) and carbidopa (25 µM) and (b) L-DOPA (100 µM).
Analysis of Enantiomeric Test Mixtures of DOPA in Matrixes of Carbidopa. Chiral analysis of DOPA was performed based on two practical Sinemet formulations: a mixture of L-DOPA and L-carbidopa in 10:1 or 4:1 ratio. Since chiral analysis using the kinetic method can tolerate impurities (matrix) interference, as shown by a study of chiral mixture analysis of different amino acids,42 it is expected that quantitation of L-DOPA in the presence of the co-drug carbidopa can be achieved. Figure 4a shows a typical ESI-MS spectrum of a solution containing a fixed ligand L-Ala-L-Ala, a reference ligand L-Tyr, and an analyte L-DOPA (with 20% carbidopa) in the presence of copper(II) chloride. In comparison, Figure 4b shows a typical ESI-MS spectrum obtained by electrospraying an aqueous methanolic solution containing a fixed ligand L-Ala-L-Ala, a reference ligand L-Tyr, and an analyte L-DOPA in the presence of copper(II) chloride. The spectra are composed of several types of ions, including relatively abundant protonated and transition metal-bound clusters, especially those involving the fixed ligand, which has high proton and metal ion affinities. Comparing panels a and b of Figure 4, carbidopa does not obviously affect the peak distribution except to yield one additional peak of protonated carbidopa at 227 Th. When the ratio of L-DOPA and carbidopa is 10:1, the protonated carbidoap peak decreases proportionally but other peaks do not change significantly (figure not shown). The chirality switch of the fixed ligand, the reference ligand, and the analyte do not affect the peak distributions in the ESI-MS spectrum either. This is consistent with the results of previous isotopically labeled experiments showing no discrimination when changing the chirality of analyte.31,35 670 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
actual
ee % of L-DOPA experimental (1) (2) (3)
average (% ee)
relative error (%)
4:1
entry 1 entry 2 entry 3 entry 4 entry 5
20 50 80 90 98
19.3 50.6 79.5 91.9 98.8
19.7 50.8 78.7 89.6 99.6
20.2 50.3 80.3 92.2 99.7
19.7 50.6 79.5 91.2 99.4 average
1.5 1.2 0.6 1.3 1.4 1.2
10:1
entry 1 entry 2 entry 3 entry 4 entry 5
20 50 80 90 98
19.8 48.8 81.7 91.1 99.3
20.5 48.1 80.8 91.8 99.2
20.3 50.6 79.9 89.7 98.6
20.2 49.2 80.8 90.9 99.0 average
1.0 1.6 1.0 1.0 1.0 1.1
overall average error
1.2
As illustrated in Figure 5, two calibration curves (1 and 2), although four calibration curves are possible, for the analysis of DOPA with 20% carbidopa are constructed by using only pure L-DOPA with two chiral combinations of the fixed ligands and reference ligands. Using Cartesian coordinates, the calibration lines are constructed by simply connecting the origin and the measured point (100, 0.483) and (100, 1.68). The corresponding linear equations, obtained using the single-point calibration QRfixed method, were used to measure the ee of L-DOPA of “unknown” samples. Table 3 lists five representative actual ee values of the mixtures and the values obtained through use of the calibration curves 1 and 2 when the ratio of DOPA/carbidopa is 4:1. The differences between actual input values and the experimentally measured results obtained from Figure 5 curve 1 are from 0.2 to 1.4% with the relative values from 0.6 to 1.5%; whereas the differences obtained from Figure 5 curve 2 are from 0.2 to 1.0% with the relative errors of from 1.0 to 1.6%. The overall average values of the relative errors are very similar: 1.2 and 1.1%. It is clear that quantification of L-DOPA can be successfully achieved in matrixes including the co-drug carbidopa. In comparison with the early quantification results (2.4% relative error) obtained using the QR method,44 improved accuracy in chiral quantitation of DOPA is attributed to the fixed-ligand approach to simplify the dissociation kinetics and improve chiral selectivity.43
CONCLUSIONS The QRfixed method presented here is a novel variant of the kinetic method for direct chiral analysis. It requires two consecutive measurements and assesses the ratio of branching ratios in two tandem mass spectra; i.e., it uses the double ratio given in eq 1. The major advantage of the method is that the necessary calibration curves can be constructed using only one single optically pure sample. In comparison with the standard QR method, the introduction of a fixed ligand replaces one of two reference ligands giving the QRfixed method the advantages of (i) improved quantitation accuracy due to simplification of the dissociation kinetics and improving chiral selectivity (by changing the properties such as the size and functionality of the fixed ligands); (ii) multiple possible ways (e.g., eight possible calibration curves in the case of the tripeptide Ala-Ala-Ala as the fixed ligand) to construct single-point calibration curves which can be used to cross-check data, an important issue in pharmaceutical applications; (iii) application of the chiral morphing technique, that is by making changes in the chirality of the fixed or reference ligands, the chiral interactions in the cluster ion are refined allowing one to maximize chiral differentiation. The chiral morphing concept is used to refine the chiral interactions between the metal ion and the binding ligands
Tandem mass spectrometry of transition metal ion-bound complexes provides a rapid, sensitive, and precise method for direct quantitation of chiral molecules. The measurements are carried out on a commercial instrument, they use standard ESI mass spectrometry and tandem mass spectrometry, and they require only very small amounts of sample for analysis. The present experiment solves a significant problem in applying this approach when the analyte is only available in one optical purity. ACKNOWLEDGMENT This work was supported by the National Science Foundation, CHE 97-32670, and the U.S. Department of Energy, Office of Energy Research. L.W. expresses appreciation for the 2003 American Chemical Society, Division of Analytical Chemistry Graduate Fellowship Award sponsored by Johnson & Johnson Pharmaceutical Research and Development. The authors acknowledge the contributions of Professor Marcos N. Eberlin to the program of study reported here.
Received for review August 4, 2003. Accepted November 10, 2003. AC0349072
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