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 Parkinsons 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.

Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 663

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.

664

Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

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 -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 M each, with the metal

salt present at 25 M 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.

Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

665

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

Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

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- 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

667

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

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.

668

Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

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

D-Ala-D-Ala
L-Ala-L-Ala

D-Tyr
L-Tyr

D-Ala-D-Ala
L-Ala-L-Ala

L-Tyr
D-Tyr

D-Ala-L-Ala
L-Ala-D-Ala

D-Tyr
L-Tyr

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

669

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 M), the dipeptide L-Ala-L-Ala (100 M), the reference L-Tyr
(100 M), and (a) L-DOPA (100 M) and carbidopa (25 M) and (b)
L-DOPA (100 M).

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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671