Mass Spectrometric Quantitation of Chiral Drugs by the Kinetic Method

Anal. Chem. 2001, 73, 1692-1698

Mass Spectrometric Quantitation of Chiral Drugs by the Kinetic Method W. Andy Tao, Fabio C. Gozzo,† and R. Graham Cooks*

Aston Laboratory for Mass Spectrometry, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

A novel mass spectrometric method for rapid, accurate (2-4% ee) quantitation of chiral drugs is described. Copper(II)-bound complexes of seven model drugs (atenolol, DOPA, ephedrine, pseudoephedrine, isoproterenol, norepinephrine, propranolol) with chiral reference compounds (L-amino acids) are generated by electrospray ionization mass spectrometry. The trimeric complex ions (three chiral ligandssone of the analyte and two of the reference compound) are collisionally activated, and they undergo dissociation by competitive loss of either the neutral reference or the neutral drug molecule. The ratio of the two competitive dissociation rates, viz. the product ion branching ratio, is related via the kinetic method to the enantiomeric composition of the drug mixture. A twopoint calibration curve, derived from the kinetic method, allows rapid quantitation of enantiomeric excess of drug mixtures. The chiral sensitivity of the method is such as to allow determination of mixtures with a few percent enantiomeric contamination. There are few topics in biological and pharmaceutical science that have drawn as much interest as the chiral nature of drug molecules. Today, systematic investigation of the biological activity (including pharmacology and toxicology) of individual enantiomers is the rule for all new racemic drug candidates, and an increasing number of optically pure drugs have been approved and marketed. Quantitation of enantiomeric mixtures of drugs and their metabolites has become a requirement in both pharmaceutical discovery and clinical pharmacy.1 The search for new chiral techniques to comply with such demanding requirements has included mass spectrometry, a fast and sensitive analytical tool that plays an increasingly important role in numerous biological applications.2 In contrast to the currently dominant chromatographic approaches,3 mass spectrometry, especially tandem mass spectrometry, is unique by virtue of the fact that it provides information on intrinsic interactions between molecules without matrix interference and accord* To whom correspondence should be addressed. E-mail: [email protected]. Fax: (765)494-9421. † Visiting scientist from State University of CampinassUNICAMP, Campinas, SP Brazil. (1) Stinson, S. C. Chem. Eng. News 2000, 78, 55. (2) McLafferty, F. W.; Fridriksson, E. K.; Horn, D. M.; Lewis, M. A.; Zubarev, R. A. Science 1999, 284, 1289. (3) Menges, R. A.; Armstrong, D. W. In Chiral Separations by Liquid Chromatography; Ahuja, S., Ed.; ACS Symposium Series 471; American Chemical Society: Washington, DC, 1991; p 67.

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ingly allows optimization of intrinsic stereochemical interactions when used for chiral distinction. Important progress has been made during the past few years on chiral recognition and quantification based solely on mass spectrometry. The majority of mass spectrometry-based chiral recognition experiments can be classified into four types. In the first type of experiment, carried out mainly by using chemical ionization (CI)4,5 and fast atom bombardment (FAB) mass spectrometry,6-8 diastereomeric adducts are generated using chiral reference compounds and are investigated in a single-stage mass spectrometry (MS) experiment. One enantiomer of the analyte is isotopically labeled so that the corresponding mixture of diastereomeric adducts can be mass resolved. These adducts are typically of the host-guest type and are bound by such noncovalent forces as hydrogen bonds and van der Waals forces. Very recently, this ionization approach has been extended to include electrospray ionization (ESI)9 and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.10 Chiral recognition based on gas-phase ion/molecule reactions, often exchange reactions, has also been investigated, and this constitutes a second approach to chiral analysis. A diastereomeric adduct, typically generated from a chiral ligand and a chiral host such as β-cyclodextrin (CD), is mass-selected and allowed to exchange the chiral ligand in a reaction with a neutral gas (chiral or achiral).11-13 Chiral distinction is achieved because the exchange rate varies with the chirality of the analyte incorporated into the adduct ion. The third approach for direct chiral recognition is based on collision-induced dissociation (CID) of diastereomeric adducts formed from an analyte and a chiral reference in a tandem mass (4) Nikolaev, E. N.; Denisov, E. V.; Nikolaeva, M. I.; Futrell, J. H.; Rakov, V. S.; Winkler, F. J. Adv. Mass Spectrom. 1998, 14, 279. (5) Nikolaev, E. N.; Denisov, E. V.; Rakov, V. S.; Futrell, J. H. Int. J. Mass Spectrom. 1999, 183, 357. (6) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73. (7) 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. (8) Pocsfalvi, G.; Liptak, M.; Huszthy, P.; Bradshaw, J. S.; Izatt, R. M.; Vekey, K. Anal. Chem. 1996, 68, 792. (9) 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. (10) So, M. P.; Wan, T. S. M.; Chan, T. W. D. Rapid Commun. Mass Spectrom. 2000, 14, 692. (11) Chu, I. H.; Dearden, D. V.; Bradshaw, J. S.; Huszthy, P.; Izatt, R. M. J. Am. Chem. Soc. 1993, 115, 4318. (12) Dearden, D. V.; Dejsupa, C.; Liang, Y. J.; Bradshaw, J. S.; Izatt, R. M. J. Am. Chem. Soc. 1997, 119, 353. (13) Ramirez, J.; He, F.; Lebrilla, C. B. J. Am. Chem. Soc. 1998, 120, 7387. 10.1021/ac001150v CCC: $20.00

© 2001 American Chemical Society Published on Web 03/16/2001

Chart 1

spectrometry (MS/MS) experiment.14-16 Successful experiments are limited to particular molecules, most of which are stereorigid. The fourth approach also uses MS/MS, but it employs the kinetic method to quantify the chiral effects17,18 and is the subject of this study. Recent work using this approach has involved collisioninduced dissociation of trimeric transition metal ion-bound cluster ions which give rise to diastereomeric product ions.19,20 The success of this approach to chiral selectivity of amino acids19,20 and R-hydroxy acids21 is indicated by different relative abundances of the product ions caused by differences in the energy required for their formation. Only a few recent studies have reported quantitative determinations of enantiomeric excess (ee) rather than qualitative chiral recognition using mass spectrometry.9,19,20,22-24 Routine chiral analysis by mass spectrometry has to overcome certain drawbacks present in most current mass spectrometric methods. The method should ideally (i) be made using commercial instruments and a simple protocol. Also, (ii) isotopic labeling should be avoided, and (iii) most importantly, large chiral distinction is preferred to allow accurate enantiomeric quantitation. The present study applies competitive dissociation of copper(II)-bound trimeric cluster ions to the direct enantiomeric quan(14) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293. (15) Ho, Y. H.; Squires, R. R. J. Am. Chem. Soc. 1992, 114, 10961. (16) Tabet, J. C. Tetrahedron 1987, 43, 3413. (17) Shen, W. Y.; Wong, P. S. H.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1997, 11, 71. (18) Vekey, K.; Czira, G. Anal. Chem. 1997, 69, 1700. (19) Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P.; Cooks, R. G. Anal. Chem. 1999, 71, 4427. (20) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598. (21) Tao, W. A.; Wu, L.; Cooks, R. G. Chem. Commun. 2000, 2023. (22) Guo, J.; Wu, J.; Siuzdak, G.; Finn, M. G. Angew. Chem., Int. Ed. 1999, 38, 1755. (23) Liang, Y. J.; Bradshaw, J. S.; Izatt, R. M.; Pope, R. M.; Dearden, D. V. Int. J. Mass Spectrom. 1999, 187, 977. (24) Grigorean, G.; Ramirez, J.; Ahn, S. H.; Lebrilla, C. B. Anal. Chem. 2000, 72, 4275.

titation of model drugs. The study is intended to demonstrate the simplicity and to investigate the reproducibility of the method and its capability of quantifying mixtures in which the enantiomeric contamination is only a few percent. EXPERIMENTAL SECTION A commercial LCQ ion trap mass spectrometer (Finnigan, San Jose, CA), equipped with an ESI source, was operated in the positive ion mode as follows: spray voltage, 5.00 kV; capillary voltage, 3 V; heated capillary temperature, 150 °C; tube lens offset voltage, 20 V; sheath gas (N2) flow rate, 30 units (roughly 0.75 L/min). The sample was infused via a syringe pump at a flow rate of 1-2 µL/min. In the full-scan MS2 and MS3 modes, the parent ion of interest was first isolated by applying an appropriate waveform across the end cap electrodes of the ion trap to resonantly eject all trapped ions, except those ions of the m/z ratio of interest. The isolated ions were then subjected to a supplementary ac signal to resonantly excite them and so cause CID. The Mathieu qz values for resonance excitation and resonance ejection were 0.25 and 0.83, respectively. The excitation time used was 30 ms. The excitation amplitude can be varied from 0 to 100% relative collision energy corresponding to 0-2.5 V zeroto-peak resonant excitation potential; the value was optimized in each experiment, but kept constant for measurements of the R and S enantiomers. Spectra shown represent the average of ∼50 scans, each requiring 0.2 s. Mass/charge ratios (m/z) are reported using the Thomson unit (1 Th ) 1 atomic mass per unit positive charge25). Gas-phase Cu(II) complexes with model drugs were generated by electrospraying 50/50 water/methanol solutions containing a mixture of the drug and the reference compound (an R-amino acid), at a concentration of 100 µM each, and 25 µM copper(II) chloride. Chiral drugs (atenolol, DOPA, ephedrine, pseudoephe(25) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 93.

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drine, isoproterenol, norepinephrine, propranolol) (Chart 1) and amino acids were purchased from Sigma and used without further purification. RESULTS AND DISCUSSION Chiral Mixture Analysis. The kinetic method can be applied in two distinct ways for enantiomeric measurement. These are termed the single ratio method and the quotient ratio method.20 In this report, we only use the simpler single ratio method since this allows mixtures to be analyzed by simply recording a ratio of fragment ion abundances in a single (tandem) mass spectrum. The fundamental concept of the single ratio method is shown in eq 1.

of chiral discrimination, which means that the particular combination of copper(II) ion and reference ligand fails to create stereochemically distinctive interactions with the enantiomers under the conditions employed. The relationship between the relative branching ratio R and the ee of the analyte sample is derived from the kinetic method.26,27 In treating the experimental data using the kinetic method, it is possible either to consider the energies of dissociation20,28 or the corresponding free energies.26,29 Both treatments are useful in kinetic method studies, and previous studies20 on chirality have used the bond energy formalism. In this following, we choose to use the free energy form of the kinetic method. The natural logarithm of the ratio (R) of rate constants is proportional to the differences between the free energies changes for the competitive dissociations to yield the two dimeric products in eq 1, i.e.

ln R )

Singly charged trimeric cluster ions [CuII(ref*)2(A) - H]+ are generated by electrospraying an aqueous methanol solution containing an analyte (chiral drug, A, present as a mixture of the enantiomers AR and AS), a chiral reference compound (chiral amino acid, ref*), and a transition metal ion (in this case, Cu(II)). The diastereomeric cluster ions are mass-selected and collisionally excited in a quadrupole ion trap; they dissociate competitively to form the dimeric complexes [CuII(A)(ref*) - H]+ and [CuII(ref*)2 - H]+ by the loss of neutral reference compound, ref*, and analyte, A, respectively. (Note that the trimeric complexes of DOPA and norepinephrine display a distinctive feature by showing the loss of an analyte radical to form the [Cu(ref*)2]+ ions upon CID, while that of isoproterenol shows the loss of both neutral analyte and its radical. These features are discussed further below.) The differences in energy required to generate the diastereomeric forms of the fragment ions [CuII(A)(ref*) - H]+, due to the two configurations of the analyte A, result in differences in their abundances, measured relative to the abundance of [CuII(ref*)2 - H]+. The relative branching ratio R (eq 2) depends on

R ) [CuII(A)(ref*) - H]+/[CuII(ref*)2 - H]+

(2)

the enantiomeric composition of the analyte, A. In the special cases of DOPA and norepinephrine, the relative branching ratio R is set as [CuII(A)(ref*) - H]+/[Cu(ref*)2]+. When the analyte is enantiomerically pure, R is given as RR or RS and the chiral selectivity, the ratio of RR to RS, defined as Rchiral, measures the degree of chiral distinction:

Rchiral ) RR/RS ) [CuII(AR)(ref*) - H]+/[CuII(ref*)2 - H]+ [CuII(AS)(ref*) - H]+/[CuII(ref*)2 - H]

(3)

Rchiral is equivalently defined as the ratio of RD to RL since the D,L nomenclature is commonly used for many types of chiral molecules. The more different the Rchiral value is from unity, the higher the degree of chiral recognition. Thus, Rchiral ) 1 indicates a lack 1694

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∆(∆G) RTeff

(4)

where R in the denominator is the gas constant, Teff is the effective temperature of the activated trimer (the average temperature of the two activated complexes for the two competitive reactions), and ∆(∆G) is defined as the difference in free energies between the reactions 5 and 6 whose reverse barriers are considered negligible.

[CuII(ref*)2 (A) - H]+ f [CuII(A)(ref*) - H]+ + ref* (5) [CuII(ref*)2 (A) - H]+ f [CuII(ref*)2 - H]+ + A (6) When the analyte consists of a pure R or S enantiomer, ∆(∆G) becomes ∆(∆G)R or ∆(∆G)S. For an enantiomeric mixture with enantiomeric excess of the R enantiomer given by ee, by analogy with the earlier enthalpy case,20 one can write

1 + ee 1 - ee + ∆(∆G)S ) 2 2 [∆(∆G)R + ∆(∆G)S] [∆(∆G)R - ∆(∆G)S] + ee (7) 2 2

∆(∆G) ) ∆(∆G)R

and therefore the relationship between R and ee can be expressed by combining eq 4 and eq 7 to obtain

ln R )

∆(∆G)R + ∆(∆G)S ∆(∆G)R - ∆(∆G)S + ee (8) 2RTeff 2RTeff

Equation 8 predicts a linear relationship between ln R and ee, and previous studies on chiral amino acids confirmed the existence of such a relationship20 although expressed in terms of energy rather than free energy as done here. The calibration curves are constructed in this study on the basis of a linear relationship between ln R and ee. In theory, two-point calibrations can be (26) Cooks, R. G.; Wong, P. S. H. Acc. Chem. Res. 1998, 31, 379. (27) Cooks, R. G.; Patrick, J. S.; Kotiaho, T.; McLuckey, S. A. Mass Spectrom. Rev. 1994, 13, 287. (28) Nemirovskiy, O. V.; Gross, M. L. J. Am Soc. Mass Spectrom. 2000, 11, 770. (29) Armentrout, P. B. J. Am. Soc. Mass Spectrom. 2000, 11, 371.

Figure 1. ESI mass spectrum of copper(II)/atenolol/L-abrine solution. Major cluster ions are indicated. The spectrum was obtained for a 50/50 water/methanol solution of 100 µM atenolol (A), 100 µM L-abrine (ref*), and 25 µM CuCl2‚2H2O.

performed using a racemic sample and a sample of known ee, or the calibration curve can be constructed using pure R enantiomers and S enantiomers, respectively. Unknown enantiomeric mixtures are analyzed by measuring the ratio of two fragment ions in a single spectrum. The calibration data depend on the internal energy of the activated ions and hence on the activation condition chosen. Even though it has been found that the calibration curve might be valid for several months, we recommend that a fresh calibration curve be constructed prior to routine measurements, since this only takes a few minutes. Chiral Recognition of Model Drugs. Seven drugs (Chart 1), ranging from β-blockers to decongestants, were selected to investigate the analytical applications of the kinetic method of chiral determination. Chiral references were chosen for their capability to produce large steric interactions and for structural similarity to particular analytes. Such similarity allows the complexes to form easily and it also allows accurate relative abundance ratios to be measured; otherwise, dissociation proceeds overwhelmingly to form the more stable product. R-Amino acids have structures similar to that of the analyte and the 19 natural chiral R-amino acids, plus numerous other amino acids, provide an array of choices. Aromatic amino acids often provide the greatest chiral distinction.20 Cluster ions [CuII(ref*)2(A) - H]+ can be generated efficiently in the gas phase by electrospray ionization of aqueous methanol solutions of Cu(II)/amino acid/drug mixtures, typified by the case of a Cu(II) salt/abrine/atenolol mixture, the ESI mass spectrum of which is illustrated in Figure 1. In addition to the Cu(II)-bound deprotonated trimeric cluster, other ions, including proton and sodium-based cluster ions, also occur. CID of [CuII(ref*)2(A) H]+ cluster ions typically yields dimeric complexes [CuII(A)(ref*) - H]+ and [CuII(ref*)2 - H]+ by competitive loss of the neutral reference compound, ref*, and the analyte, A, respectively. The branching ratio of these fragment ions depends strongly on the stereochemistry of the ligands, and when the same reference ligand is used, it depends specifically on the chirality of the analyte drug. Figure 2 shows the product mass spectra of the trimeric cluster ions [CuII(ref*)2(A) - H]+ (ref* ) L-Trp, A ) enantiomeric mixtures of ephedrine with ee of the (-)-enantiomer equal to -100, 0, 100%). Chiral selectivity, Rchiral (eq 3), is found to 3.40

Figure 2. Product ion (MS/MS) spectra of [63CuII(ephedrine)(L-Tyr)2 - H]+ (m/z 635) for mixtures of (+) and (-)-ephedrine. The CID activation level is chosen as 11%, corresponding to ∼275 mV ac. Table 1. Chiral Analysis of Drugsa,b [CuII(ref*)(A) - H]+/ [ CuII(ref*)2 - H]+ c A

ref*

R isomer

S isomer

Rchiral

atenolol DOPA Ephedrine ψ-ephedrine isoproterenol norepinephrine propranolol

L-abrine

37.6 0.906 0.895 14.2 1.77 0.522 41.3

21.6 0.164 0.253 6.89 1.15 0.420 95.2

1.74 5.52 3.40 2.05 1.54 1.24 0.43

L-Tyr L-Trp L-Trp L-abrine L-Phe L-His

a R b chiral is defined in eq 3. CID activation level is optimized in each experiment and then kept constant for the measurements of enantiomers. c For DOPA and norepinephrine, the ratio is measured as [CuII(ref*)(A) - H]+/[CuII(ref*)2]+. See related text for further explanation.

for ephedrine when L-tryptophan is used as chiral reference using the activation conditions described in the Experimental Section using 11% CID. Chiral recognition is achieved for all seven drugs, indicated by the chiral selectivity values Rchiral, summarized in Table 1. The DOPA/L-tyrosine chiral reference combination shows the largest chiral distinction (Rchiral ) 5.52). Norepionephrine shows a relatively low chiral selectivity (Rchiral ) 1.24) using L-phenylalanine as the reference. However, previous studies on R-hydroxy acids shown that, even with such low selectivity, precise chiral quantitation is still achievable.21 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

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Figure 3. Two-point calibration curves constructed using pure enantiomers (filled circles) for (a) pseudoephedrine and (b) DOPA. Data for test samples are shown as open symbols.

Analysis of Enantiomeric Test Mixtures. Pseudoephedrine (ψ-ephedrine) and DOPA were selected to explore further these enantiomeric measurements since they show different chiral selectivities; i.e., DOPA has a large chiral selectivity while pseudoephedrine shows a relatively low chiral selectivity. They are also both important drugs: pseudoephedrine is a decongestant, while L-DOPA (levodopa) is used for the treatment of Parkinson’s disease. By measuring R values of pure enantiomers (ee ) -100 and +100%), a two-point calibration curve can be constructed according to the semilog relationship between R and ee (eq 8). Panels a and b of Figure 3 show the calibration curves for pseudoephedrine and DOPA, respectively, constructed by measurements made on the pure enantiomers (filled circles in figures). The corresponding linear equations were used to measure percent ee of “unknown” samples. The analysis of each sample only requires one measurement in a single MS/MS spectrum, although replicates were made to increase precision. Table 2 lists the actual ee values of the mixtures and the values obtained through use of the calibration curves. The measured R values fit well on with the calibration curves (open symbols in Figure 3). The measured average ee values of the mixtures also correlate well with the actual ee values, as shown in Figure 4. The overall correlations (r2) are 0.9998 and 0.9992 for measurements of pseudoephedrine and DOPA, respectively. Each point shown in Figure 4 is the averaged result of three samples and the error bar is indicated as the overall averaged difference 1696 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

Figure 4. Plots of actual versus the measured ee for (a) pseudoephedrine and (b) DOPA. Table 2. Enantiomeric Excess (ee) for Mixtures of Pseudoephedrine and DOPA enantiomeric excess (%) experimental sample ψ-ephedrine I II III IV V VI VII VIII IX

actual

(1)

(2)

(3)

av diff

-98.0 -95.0 -90.0 -50.0 0.00 50.0 90.0 95.0 98.0

-96.6 -94.1 -92.3 -50.5 4.9 44.4 84.5 94.3 95.8

-100.3 -92.5 -84.2 -50.9 0.8 50.6 88.4 96.0 96.8

-99.8 -93.3 -94.0 -50.6 2.8 53.2 93.8 96.1 99.8

1.9 1.7 4.1 0.7 2.9 3.1 3.6 0.9 1.7

overall av error DOPA I II III IV V VI VII VIII IX overall av error

2.3 -98.0 -95.0 -90.0 -50.0 0.00 50.0 90.0 95.0 98.0

-99.7 -99.0 -92.8 -43.5 4.8 56.2 87.7 91.7 94.5

-102.0 -100 -85.3 -47.7 6.4 53.3 91.0 93.6 94.9

-100.8 -91.3 -93.7 -54.6 5.5 45.3 88.2 97.5 101.2

2.9 4.5 3.8 4.4 5.6 4.8 1.7 2.4 3.3 3.7

between the actual and the measured values. Two conclusions can be drawn directly from the data. One is that the ee of pseudoephedrine and DOPA can be determined quite accurately

(overall average error for pseudoephedrine is 2.3 ee%, and 3.7 ee% for DOPA), even though the chiral selectivities (Rchiral) of the two are quite different (Rchiral for pseudoephedrine is 2.05, and 5.52 for DOPA). Another is that it is straightforward to determine ee values even for samples that only contain a few percent of one enantiomer. Note that all experimental ee values were obtained from the two-point calibration curve and that accuracy should be improved by construction of a calibration curve with multiple measurements of samples at different ee. It is particularly advantageous to be able to make chiral determinations of samples that only contain a few percent of one enantiomer. While current asymmetric syntheses and combinatorial techniques aim at high enantiomeric purity (>90% ee), most chiral techniques (chromatographic and NMR approaches) have difficulty in quantifying at levels below 1% ee. Most chiral resolution methods discriminate between two enantiomers on the basis of their different reaction rates toward a common chiral selector. Chiral analysis, when the enantiomeric contaminant is present at only a few percent or less, is limited by the selectivity factor s (the ratio of competing rate constants). Distinction of more reactive enantiomers, present in only a few percent from less reactive enantiomers present at very high ee, requires extraordinarily high selectivity. For example, s needs to be ∼200 to quantify samples at 96% ee in particular cases, and at 98%ee, s needs to be as high as 500.30 Such high selectivity is currently beyond the reach of most condensed-phase reactions. This is a particularly a challenge for gas-phase chiral analysis, since most reported mass spectrometric reactions display lower selectivities.6,31 The present report on chiral analysis is performed on the basis of two independent ion/molecule and subsequent dissociation reactions. For this reason, high selectivity is not required in order to quantify enantiomers at low ee. The two enantiomers (AR and AS) react equally toward the chiral reference (ref*) and copper(II) ion, and therefore, the relative abundances of [CuII(AR)(ref*)2 - H]+ and [CuII(AS)(ref*)2 - H]+ reflect the enantiomeric composition of the analyte A in solution. While a discussion of the reason the formation of cluster ions [CuII(AR)(ref*)2 - H]+ and [CuII(AS)(ref*)2 - H]+ is not stereochemically discriminatory is beyond the subject of this report,32 chiral distinction is determined to occur only at the dissociation step in the systems examined here. Since there is no competition between the dissociations of cluster ions [CuII(AR)(ref*)2 - H]+ and [CuII(AS)(ref*)2 - H]+, in other words, the dissociations of cluster ions [CuII(AR)(ref*)2 - H]+ and [CuII(AS)(ref*)2 - H]+occur as two independent parallel reactions, chiral distinction is governed only by the selectivity of dissociation and enantiomeric analysis can be measured over a wide range of enantiomeric excess in the initial solution. This approach is analogous to a condensed-phase method termed “parallel kinetic resolution” that was introduced recently by Vedejs et al.33,34 Concentration Effects on R Values. In real mixture analysis, the total concentration of an analyte is usually unknown. There(30) Kagan, H. B.; Fiaud, J. C. Top. Stereochem. 1988, 18, 249. (31) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, M. Int. J. Mass Spectrom. 2000, 198, 137. (32) Tao, W. A.; Cooks, R. G. Angew. Chem., Int. Ed. 2001, 40, 757. (33) Vedejs, E.; Chen, X. J. Am. Chem. Soc. 1997, 119, 2584. (34) Eames, J. Angew. Chem., Int. Ed. 2000, 39, 885.

Table 3. Relative Abundance Ratio [CuII(ref*)(A) H]+/[CuII(ref*)2 - H]+ for Various Ratios of A/ref* (A ) (+)-ephedrine; ref* ) L-Trp) [A]/[ref*] 5:1 4:1 3:1 2:1 1:1 1:2 1:3 1:4 1:5

[CuII(ref*)(A) - H]+/ [CuII(ref*)2 - H]+ 0.267 0.270 0.272 0.272 0.270 0.270 0.276 0.274 0.271

fore, it is important to characterize the effect of addition of different concentrations of chiral selector (the reference compound ref* in this study) and the metal ion. A systematic study of the influence of the change in [A]/[ref*] concentration ratios in solution on the value of [CuII(A)(ref*) - H]+/[CuII(ref*)2 - H]+ was carried out, and the results are summarized in Table 3. The study shows that, at least for the case examined, the relative abundance ratios are virtually independent of the relative concentration ratio of the analyte and the reference in solution. As a consequence, it is clear that the addition of different concentrations of chiral reference compounds to the unknown sample has little effect on the accuracy of quantitation, a practical advantage for this approach. Structures of Cluster Ions. A complete understanding of the structures of the copper(II)-bound cluster ions is not crucial to the analytical application of this method, and therefore, a detailed investigation of the structures of copper cluster ions on chiral selectivity will be the subject of a future report. Previous studies on copper(II)-amino acid cluster ions suggested that the trimeric cluster ions are tetracoordinate, with two monodentate ligands and one bidentate ligand. This conclusion was based on the ease of simple ligand loss from the trimeric cluster ions.20 Both ligands in the dimeric cluster ions, in contrast, are polydentate and simple ligand loss is not observed. Multiple interactions between the two ligands mediated by copper(II) are assumed responsible for the observed efficient chiral distinction, according to the long-standing three-point rule.35 Copper(II) ion readily forms square planer structures and this structure shows distinctive differences when one chiral center in one ligand is reversed. In the present system, many observations on copper(II)drug-amino acid cluster ions parallel those made previously for copper(II)-amino acid clusters and suggest similarities in structure. However, when the analyte is DOPA, norepinephrine, or isoproterenol, CID of their trimeric cluster ions shows a distinctive feature. The ion [CuII(A)(ref*)2 - H]+ (A ) DOPA or norepinephrine; ref* ) chiral amino acid) fragments upon low collision energy by competitive loss of an intact neutral amino acid (ref*) or an analyte radical (A - H). When the analyte is isoproterenol, loss of an intact isoproterenol molecule or a radical, along with the competitive loss of the intact reference compound, is observed from the CID of its trimeric cluster ion. Such an unexpected feature does not influence the observation of chiral distinction, exemplified by chiral quantitation of DOPA, in which R was (35) Salem, L.; Chapuisat, X.; Segal, G.; Hiberty, P. C.; Minot, C.; Leforrestier, C.; Sautet, P. J. Am. Chem. Soc. 1987, 109, 2887.

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measured by the value of [CuII(A)(ref*) - H]+/[Cu(ref*)2]+ (Figure 3b). In the case of isoproterenol, CID of its trimeric cluster ion [CuII(A)(ref*)2 - H]+ (A ) isoprotereonol; ref* ) L-abrine) generated a constant relative abundance ratio of product ions [CuII(ref*)2]+ and [CuII(ref*)2 - H]+. Therefore, the chiral selectivity, Rchiral, is unchanged when the reference ion is either [Cu(ref*)2]+, [CuII(ref*)2 - H]+, or both. The special structural aspects that lead to such unexpected CID behavior are still under investigation and will not be reported here, even though it seems clear that the 3,4-dihydroxyphenyl group is involved. DOPA, norepinephrine, and isoproterenol all contain a 3,4-dihydroxyphenyl group while other analytes do not. CONCLUSIONS Tandem mass spectrometry of copper(II)-bound complexes provides a rapid, sensitive, and precise method for direct quantitation of chiral drugs. 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 experiment, along with the previous successful chiral analysis of other types of chiral compounds by the kinetic method, suggests that this may

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represent a general and practical mass spectrometric method for direct chiral analysis. The method does not rely on condensed-phase derivatization or chromatographic separation and, therefore, may be particularly useful for cases in which chromatography and derivatization are ineffective or inconvenient. A distinctive feature of this method is its high precision and accuracy, even for samples with enantiomeric contamination of a few percent, a feature that may make it practically useful for routine measurements of enantiomeric products from combinatorial synthesis. Extension of this work to perform on-line routine analysis is in progress. ACKNOWLEDGMENT This work was supported by the U.S. Department of Energy, Office of Energy Research. A fellowship from Triangle Pharmaceuticals (to W.A.T.) is gratefully acknowledged. F.C.G. acknowledges a fellowship from the Research Support Foundation of the State of Sao Paulo (FAPESP), Brazil. Received for review September 26, 2000. Accepted February 5, 2001. AC001150V