Recent Advances in MS

Journal of Pharmaceutical and Biomedical Analysis 69 (2012) 133147
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba
Review
A review of recent advances in mass spectrometric methods for gas-phase chiral

analysis of pharmaceutical and biological compounds
Lianming Wu , Frederick G. Vogt
API Chemistry and Analysis, Product Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e
i n f o
Article history:
Received 20 February 2012
Received in revised form 17 April 2012
Accepted 18 April 2012
Available online 24 April 2012
Keywords:
Tandem mass spectrometry (MS/MS)
Gas-phase enantiomeric separation
Quantitative chiral analysis
Three-point interaction
Pharmaceutical and biological applications
Quality-by-design (QbD)
a b s t r a c t
Chirality has been of great interest in pharmaceutical and biological sciences. The capabilities of mass
spectrometry (MS) for rapid analysis of complex mixtures have encouraged its exploration for gas-phase
chiral differentiation. Although particular instances of successful discrimination between enantiomers
have been reported over the past three decades, a general method of quantitative chiral analysis by MS
has only been demonstrated recently. This review describes the current state of the chiral MS methods without chiral chromatographic separation, which fall into ve main categories: (1) the kinetic
method, (2) hostguest (HG) diastereomeric adduct formation, (3) ion/molecule (equilibrium) reactions, (4) collision-induced dissociation (CID) of diastereomeric adducts, and (5) the emerging technique
for gas-phase separation using ion mobility spectrometry (IMS). It emphasizes tandem mass spectrometry (MS/MS), which provides several unique analytical advantages for quantitative chiral analysis. These
include intrinsically high sensitivity, molecular specicity, and tolerance to impurities as well as the simplicity and speed of the mass spectrometric measurements. Practical prospects and current challenges
in quantitative chiral MS techniques for QbD (quality-by-design)-based pharmaceutical applications are
also discussed.
2012 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fundamental interactions to achieve chiral recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advances in gas-phase mass spectrometric methods for chiral analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
The kinetic method formalism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Dissociation of cluster ions using the kinetic method formalism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Chiral analysis of binary mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Improvements in the kinetic method for chiral analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Hostguest diastereomeric adduct formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Enantiomer labeled-host method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Determination of ee by the EL-host method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Types of host compounds used for the EL-host method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Ion/molecule (equilibrium) reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Collision-induced dissociation of diastereomeric complex ions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recent advances in ion mobility spectrometry for chiral analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
FAIMS technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
DMS technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Traveling-wave IMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmaceutical and biological applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Enantiomeric determination of L-nucleoside analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Enantiomeric quantication of generic chiral drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Method development and validation for chiral purity determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +1 610 270 4936; fax: +1 610 270 6185.
E-mail address: lianming.2.wu@gsk.com (L. Wu).
0731-7085/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2012.04.022
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L. Wu, F.G. Vogt / Journal of Pharmaceutical and Biomedical Analysis 69 (2012) 133147
5.4.
On-line LC-MS/MS chiral analysis by the kinetic method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.
Gas-phase chiral separation by ion mobility spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.
1. Introduction
Life itself has been under the constant inuence of chiral forces,
from the initial chemical processes that led to homochirality in
molecules right through the evolutionary processes that resulted
in the present diversity of biological forms [1,2]. Chirality is an
intrinsic property of the biomolecular building blocks of life, being
incorporated into the essential components that are necessary for
life such as amino acids, sugars, proteins, nucleic acids, and polysaccharides [3,4]. The use of enantiomerically pure compounds as
drugs has continuously attracted much interest in pharmaceutical industry. This is reected by the extensive investigation of
chiral bioactivity of drug molecules including their pharmacology and toxicology [59]. Individual enantiomeric forms of drugs
can produce different therapeutic (or adverse) effects and may be
metabolized at different rate or even by different pathways [10].
As a result, optically pure drugs account for more than 50% of the
total investigational new drugs (INDs) that have been approved by
the US Food and Drug Administration (FDA) since the year of 1999
(Fig. 1) [11]. In addition, among the top ten drugs in sale in the year
of 2010 eight are chiral drugs (Table 1).
Following the current FDA guidelines that recommend the study
of enantioselective identity and stability for the contributions of
individual enantiomers to pharmacological and toxicological activities, it is essential to develop quantitative analytical assays for
qualication of individual enantiomers in samples to support pharmacology and toxicology studies in drug development [79]. These
rules have encouraged pharmaceutical companies to explore new
technologies (in addition to chromatographic methods) for chiral
analysis that are not published in the United States Pharmacopeia
(USP) [12].
Quality-by-design (QbD) is a systematic approach to drug development, which begins with predened objectives, and uses science
and risk management approaches to gain product and process
understanding and ultimately process control [13]. The concept of
QbD has been extended to analytical methods [14]. QbD requires
the denition of a goal for the method, and emphasizes thorough
evaluation and scouting of alternative methods in a systematic
Racemate
Single enanomer
Achiral
80
70
Percentage (%)
60
50
40
30
20
10
0
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
Year
Fig. 1. Drugs approved by the US Food and Drug Administration (FDA) from year
1999 to 2008 (ref. [11]).
144
144
144
145
145
way to obtain optimal method performance. For chiral drug compounds, additional considerations are required for scientic and
regulatory aspects of quality control including (1) selection of a
candidate optical isomer to develop (e.g. a racemic mixture vs. a
single enantiomer); (2) prevention of racemization in vivo; and (3)
quantitation of the levels of stereoisomeric impurities (e.g. safety
assessment by toxicity qualication). The International Conference
on Harmonisation (ICH) guideline (ICH Q6A) requires the use of
robust chiral test methods to obtain proof of absolute chiral congurations and chiral purity. For single enantiomer drugs, chiral
identity is typically a part of the drug substance specication for
batch release. Minor enantiomers that are normally considered as
the critical quality attributes (CQAs) are required to be controlled
in drug substances [15]. In addition, chiral analysis is necessary to
determine if racemization occurs during drug product manufacture
and storage. Consequently, chiral analysis is vital to design space
in terms of (1) determination of critical process parameters (such
as enantiomeric excess (ee) of chiral auxiliary, temperature, crystallization parameters, etc.) that are required in order to control
the minor enantiomers in drug substances during manufacturing
process; and (2) scientic judgments about if enantiomeric quantitation of the upstream intermediates can be sufcient to correlate
with levels in nal drug substances [12].
2. Fundamental interactions to achieve chiral recognition
A well-accepted hypothetical three-point interaction [1618]
between a chiral drug and its binding sites is usually utilized to
elucidate different in vivo behaviors of two enantiomers of a chiral drug [19]. The difference between two enantiomers of a drug
is illustrated in Fig. 2, where one enantiomer is biologically active
while the other enantiomer is not. The portions of the drug labeled
A, B, and C must interact with the corresponding regions of the
binding sites labeled a, b, and c in order to have its pharmacologic
effects. The active enantiomer of the drug has a three-dimensional
structure that can be aligned with the binding sites to allow A to
interact with a, B to interact with b, and C to interact with c. In
contrast, the inactive enantiomer cannot bind in the same way no
matter how it is rotated in space, although the inactive enantiomer
possesses all of the same groups A, B, C, and D as the active enantiomer. Consequently, the inactive enantiomer is prevented from
having a desired biological effect at these binding sites.
The fundamental forces needed to achieve chiral recognition
are traditionally dealt with on a case-by-case basis with collections
of experimental ndings rationalized by qualitative explanations.
Despite the diversity of the interactions and the variety of explanations offered [2024], there are some commonalities that fall
into the concept of the three-point interaction that intrinsically
involves the use of a chiral selector and/or mediator to facilitate
chiral recognition [16]. This resembles the inherent interaction
between a chiral drug and the binding sites, as illustrated in Fig. 2.
The rationale for the three-point rule is that if at least three
active positions of a chiral selector simultaneously interact with
appropriate positions of at least one enantiomer of a chiral drug and
at least one of these interactions is stereochemically dependent,
then the enantiomers can be resolved [19]. This interaction will be
signicantly affected by replacing one enantiomer with the other.
For example, if the SS (S-selector and S-enantiomer) interaction
135
Table 1
Top 10 best-selling small-molecule therapeutics in US in 2010.a
Ranking
Product (Company)
Active Pharmaceutical
Ingredient(s)
Structureb
Form of Active
Ingredient(s)
Nexium (AstraZeneca
Pharmaceuticals)
N
N
H
Esomeprazole
..
O
Single enantiomer
O
N
OH
OH
H
N
2
Lipitor (Pzer Inc)
Atorvastatin
OH
Single enantiomer
Cl
3
Plavix (Bristol-Myers
Squibb Company)
Clopidogrel
Single enantiomer
S
F
O
S
O
HO
H
F
O
4
Advair Diskus
(GlaxoSmithKline)
Fluticasone propionate
and salmeterol
xinafoate
Single enantiomer + racemate
+
OH
HO
N
H
OH
OxyContin (Purdue
Pharma LP)
OH
Oxycodone
Single enantiomer
H
O
Cl
Cl
N
6
Abilify (Bristol-Myers
Squibb Company)
aripiprazole
Achiral
H
N
136
Table 1 (Continued)
Ranking
Product (Company)
Structureb
Active Pharmaceutical
Ingredient(s)
Form of Active
Ingredient(s)
OH
O
7
Singulair (Merck & Co.,

Inc.)
S H
Montelukast
Cl
Single enantiomer
N
HO
HO
N
8
Seroquel (AstraZeneca
Pharmaceuticals)
Quetiapine
Achiral
OH
OH
O
OH
N
9
Crestor (AstraZeneca
Pharmaceuticals)
Rosuvastatin
Single enantiomer
O S
O
10
a
b
Cymbalta (Eli Lilly and

Company)
H
N
O
Duloxetine
Single enantiomer
Information was obtained from webpage: http://www.drugs.com/top200.html.

Structures were obtained from webpage: http://en.wikipedia.org/wiki.html.
Mirror Plane
Acve Enanomer
Inacve Enanomer
Inacve Enanomer
A
Rotaon
B
C
b
a
Drug Binding Site
b
c
Drug Binding Site
Drug Binding Site
Fig. 2. Schematic diagram illustrates the hypothetical three-point interaction between two enantiomers of a chiral drug and its binding sites. The active enantiomer has a
three-dimensional structure that allows drug domain A to interact with binding site domain a, B to interact with b, and C to interact with c. In contrast, the inactive enantiomer
cannot be aligned to bind the same three sites simultaneously. The difference in the three-dimensional structure allows the active enantiomer to bind and have a desired
biological effect, whereas the inactive enantiomer cannot.
is different from the SR (S-selector and R-enantiomer) interaction,

then the free energy difference ((G)) of the two associations will
be non-zero and separation of the enantiomers is possible based
on thermodynamic enantioselectivity [25]. If the association, SS
and SR, is essentially equivalent, there is no free energy difference
and hence no chiral recognition. A chiral selector is an object that
can be used to distinguish two enantiomers. Chiral selectors are
common in nature [26] (e.g. enzymes) or can be prepared articially [25] (e.g. chiral stationary phases, chiral chemical reagents,
etc.).
The three-point model has been successfully used in traditional
wet chiral analysis methods such as chiral liquid chromatography [27] and also is the basis of the current mass spectrometric
analysis methods [28]. Although, the three-point rule is commonly invoked, the fundamental understanding of the nature
of chiral recognition has been pursued on a phenomenological
basis [16,24]. Luckily, MS methods that have been developed
borrow from the framework of the three-point rule despite
our limited understanding of the nature of the interactions in
particular cases. Many chiral selector/analyte combinations are
being explored in MS method development and the potential to
explore many others is very promising as chiral MS develops
[2989].
3. Advances in gas-phase mass spectrometric methods for

chiral analysis
MS began within the eld of fundamental physics. This technique has since diverged from its original discipline and been
directed into the currently popular large biomolecule analysis [90].
This has resulted in a technique that is continually being reinvented, where it encompasses not only fundamental physics but
also organic, analytical, and biological applications. The use of MS
can be seen in all discipline and sub-discipline. In keeping with this
trend chiral analysis via MS is also evolving [29,30]. This can be
seen with the improvements and diversity of MS instrumentation
and ionization, as MS analysis of large biomolecules are currently
emerging with instrumental techniques that offer improved sensitivity [91].
The interest in all aspects of MS is ourishing, especially in pharmaceutical and biological sciences, due to the power and versatility
of this analytical methodology. A natural synergy connecting novel
instrumentation and creative applications has nourished this eld
[92]. With the advent of the sophisticated ionization techniques of
electrospray ionization (ESI) [93,94], atmospheric pressure chemical ionization (APCI) [95], atmospheric pressure photoionization
(APPI) [96], matrix-assisted laser desorption ionization (MALDI)
[97], sonic spray ionization (SSI) [98,99], desorption electrospray
ionization (DESI) [100102], direct analysis at room temperature
(DART) [103] and their derivative ionization techniques [104],
essentially any type of biomolecules can be ionized from either
solution phase or solid state. These techniques are complemented
by exceptional instrumental sensitivity and specicity of various
mass analyzers.
Many MS applications depend on tandem mass spectrometry
(MS/MS) [105,106]. Characterization can be based on collisioninduced dissociation (CID) which yields characteristic fragment
ions or ion/molecule reactions which give rise to characteristic product ions [107]. The instrumentation used for the MS/MS
experiments generally falls into two main categories. The rst
type includes tandem-in-space or beam-type instruments and they
use two or more mass analyzers assembled in sequence. Triple
quadrupole (QqQ) [108,109] and quadrupole/time-of-ight (QTOF) [110] are most commonly employed ones. The second type of
the MS/MS instruments, the tandem-in-time type is an alternative
137
way to perform MS/MS experiments. Quadrupole ion trap [111]

including linear ion trap [112], Fourier transform ion cyclotron
resonance (FT-ICR) [113] and the recent Orbitrap [114,115] mass
analyzer are commonly utilized trapping devices. These mass analyzers have the capability of storing ions of interest and ejecting
unwanted ions. The selected ions can then be activated to cause
dissociation, and the fragment ions are observed using the same
analyzer. This process can be repeated to perform multi-stage mass
spectrometry (MSn ), a useful technique to elucidate the structures
of compounds.
MS is still commonly thought of as a chirally blind technique since each enantiomeric form gives an identical mass
spectrum under most operating conditions. Enantiomeric identication whether done using chemical reactions [116,117] or
instrumental analysis [2931] requires each enantiomer to be subjected to an asymmetric environment. For example, chiral high
performance liquid chromatography (HPLC) is achieved with a chiral stationary phase [118,119], capillary electrophoresis (CE) [120]
and gas chromatography (GC) [121123] as well as subcritical
uid chromatography (SFC) [124,125] are useful alternatives. Circular dichroism (CD) [126] does not use chiral chemical agents but
nuclear magnetic resonance spectroscopy (NMR) [127] and enzymatic technologies [26] as well as the other main methods do
require them.
MS provides a unique method of enantiomeric identication
and quantitation since chiral recognition is studied for specic
molecules of interest in a solvent-free environment [29,30]. This
allows effects of the chiral environment to be probed without any
interference of solvent or other achiral mediators. This might, in
due course, provide a better understating of the nature of chiral
recognition as well as yield improved analytical methods for chiral
analysis. Furthermore, comparisons of gas-phase and condensedphase data provide a powerful means for rationalizing the role of
solvents and counter-ions in determining the outcomes of many
inter-reactions.
Turning to quantitative analysis, one can note that internal standards or external calibration methods can be used with linear,
quadratic, or curve tting procedures in chiral chromatography
[118]. Enantiomers are separated in space due to diastereomeric
interactions with a chiral stationary phase. Optimizing the system
to facilitate chiral recognition is largely dependent on selecting
stationary phases. Chiral chromatography is already a mature technique and many chiral stationary phase options are available.
However, when normal phase is preferred to achieve a successful chiral separation, this makes APCI or APPI as the desired choice
of ionization methods for LC-MS analysis [128]. If a chiral compound were more easily ionized by ESI and this is the case for most
of pharmaceutical and biological compounds, the detection sensitivity would be reduced. While sample derivatization can be used
to avoid this [129], it might be labor-intensive and derivatization
reagents are not always available.
MS was rst demonstrated as a method of chiral discrimination
by Fales and Wright three decades ago [130], but only until the last
decade a strong interest has been seen in improving MS methods
for routine and robust chiral recognition and especially quantication. The gas-phase mass spectrometric methods that have been
extensively developed fall into ve main categories: (1) the kinetic
method formalism [2951], (2) hostguest (HG) diastereomeric
adduct formation [5367], (3) ion/molecule (equilibrium) reactions
[6875], (4) CID of diastereomeric adducts [7678], and (5) the
emerging technique of ion mobility spectrometry (IMS) [131136].
These methods along with other mass spectrometric approaches
[7989] provide a fast means for gas-phase chiral analysis. This
review describes the current state of the chiral MS techniques. It
also notes the major limitations of mass spectrometric chiral analysis.
3.1. The kinetic method formalism
metal-bound trimeric cluster ions corresponding to two enantiomeric forms of an analyte [2951]. The trimeric cluster ions as
opposed to the dimeric cluster ions, as described in the introduction, are used to invoke more sterically hindered effects that have
the potential to magnify (G), thus improving the enantioselectivity. The kinetic method is related to two product ions, as a
result of the loss of either an analyte (An) or a reference (ref*) ligand from the isolated trimeric cluster ions in a MS/MS experiment
(Eq. (3.1.3)).
The kinetic method [137] can be used to recognize and quantify

mixtures of chiral molecules by studying the dissociation kinetics
of trimeric cluster ions, which includes a chiral analyte and a chiral
selector, also called a reference. The technique builds on earlier
work [32] that showed the fragmentation rates of the proton-bound
dimeric cluster ions could be utilized to distinguish enantiomers.
k1
M, ref*
An
[M(An)(ref*)2
3.1.1. Dissociation of cluster ions using the kinetic method

formalism
The use of the competitive gas-phase fragmentation of a cluster
ion to infer thermochemical information has come to be known
as the kinetic method [138,139]. Cluster ions that are bound via a
proton (Eq. 3.1.1), as well as other atomic or polyatomic anions or
cations are isolated and their CID products are recorded in a MS/MS
experiment.
[B 1 H+ B2 ]
k2
H+
(3.1.1)
B1 + B 2
(3.1.1)
In this equation, k1 and k2 represent the rate constants for two

competitive dissociation channels of the proton-bound dimer to
yield B1 H+ and B2 H+ , respectively. The ratio of two rate constants,
viz. the branching ratio of two fragment ion abundances, is related
logarithmically to the gas-phase basicities of B1 and B2 , in the standard form of the kinetic method, as shown in Eq. (3.1.2).
ln
k
1
k2
(G)
=
RTeff
(3.1.3)
k2
Separation of enantiomers was further improved by observing the

relative kinetics of competitive dissociations of the metal-bound
trimeric cluster ions. The ratio of fragment ions varies with the
chirality of the analytes studied. The method has been optimized so
that in principle any chiral species can be quantitatively resolved
[3351].
B 1H + + B 2
H]+ + ref*
H]+
ESI
k1
[M(An)(ref*)
(3.1.2)
In this equation, (G) is the difference in gas-phase basicities of B1 and B2 , R is the gas constant, and Teff is the effective
temperature (the parameter in the kinetic method which connects
the fragmentation of isolated ions to the mean internal energy of
fragmenting ions [138]). Note that this relationship applies when a
number of conditions are fullled.
As demonstrated by the early application of the kinetic method
using 2,3-butanediol for stereoisomeric identication [32], this system was examined by dissociating, in two separate experiments,
both positively and negatively charged proton-bound dimeric
cluster ions. The (G) between the set of diastereomeric complexes can be obtained through the competitive dissociation in
the non-reference channel of the cluster ion, and this represents the fundamental basis for chiral discrimination. Thus, energy
differences <1 kJ/mol for fragmentation result in large changes
in the respective rate constants [139]. This change can be corelated through the fragment ion abundance ratio in a MS/MS
experiment.
3.1.2. Chiral analysis of binary mixtures
The successful extension of the kinetic method for chiral analysis is achieved by examining the dissociation kinetics of the
[M(ref*)2
H]+ + An
(3.1.3)
In this equation, M represent a metal ion (either a divalent transition metal ion such as Mn2+ , Fe2+ , Co2+ , Ni2+ , Cu2+ , Zn2+ , or an
alkali metal ion such as Li+ , Na+ , K+ ). The branching ratio leading to
the formation of two fragment ions is given by Eq. (3.1.4).
R=
[M(An)(ref ) H]
[M(ref )2 H]
(3.1.4)
When the analyte is enantiomerically pure, R becomes RR or RS .

Enantioselectivity, which is dened as Rchiral by Eq. (3.1.5), serves
as a numerical indicator for the degree of chiral recognition.
+
Rchiral =
RR
[M(AnR )(ref ) H] /[M(ref )2 H]
=
+
+
RS
[M(AnS )(ref ) H] /[M(ref )2 H]
(3.1.5)
The larger the difference between Rchiral and unity, the higher
the degree of chiral recognition. When Rchiral is equal to one, the
combination of a metal ion, a reference, and an analyte fails to create a stereochemically dependent interaction. In particular, when
choosing a central metal ion it should be coordinated by a reference
and an analyte simultaneously. The transition metal ions have the
advantage of easy formation of the trimeric cluster ions and large
chiral recognition, especially when aromatic references are used
to promote -cation interactions. There is a logarithmic relationship between the relative ion abundance ratio and the free energy
difference (Eq. (3.1.6)):
ln R =
(G)
RTeff
(3.1.6)
In Eq. (3.1.6), R is the gas constant, Teff is the effective temperature of the activated trimeric complex ion [M(An)(ref*)2 H]+ ,
and (G) is dened as the difference in free energies between
reactions (3.1.7) and (3.1.8) whose reverse barriers are considered
negligible or equal (Fig. 3).
+
[M(An)(ref )2 H] [M(An)(ref ) H] + ref
[M(ref*) 2
H]+ + An Ror S
(3.1.7)
[M (AnS)(ref*)
H]+ + ref*
[M(An R)(ref*)
H]+ + ref*
( G)
Energy
138
[M(An R)(ref*) 2
H]+
[M(An S)(ref*) 2
H]+
Reaction Coordinate
Fig. 3. Potential energy diagram showing two competitive dissociations for chiral
analysis by the kinetic method.
Fixed unit
CID
ligandligand interactions, and hence to maximize chiral recognition. Another advantage of the xed-ligand kinetic method is that
it simplies the dissociation kinetics.
When only one enantiomeric form with known optical purity
is available to develop a chiral method, the quotient-ratio kinetic
method is applicable for chiral analysis provided two independent
measurements for every unknown sample are made [44]. In addition, chiral analysis can be extended to enantiomeric quantitation
of a ternary mixture of a chiral compound [45] and a binary mixture
of different chiral compounds [46].
Dissociaon unit
AnR or S
Lxed
139
ref*
3.2. Hostguest diastereomeric adduct formation

Fig. 4. Schematic diagram of the xed-ligand kinetic method for chiral analysis.
+
[M(An)(ref )2 H] [M(ref )2 H] + An
(3.1.8)
When the analyte consists of a pure R or S-enantiomer, (G)

becomes (G)R or (G)S , respectively, and Eq. (3.1.6) takes the
forms shown in Eqs. (3.1.9) and Eq. (3.1.10):
ln RR =
(G)R
RTeff
(3.1.9)
ln RS =
(G)S
RTeff
(3.1.10)
For an enantiomeric mixture with an enantiomeric excess of the

R-enantiomer given by ee, one can write:
(G) = (G)R
+
[(G)R + (G)S ]
1 + ee
1 ee
+ (G)S
=
2
2
2
[(G)R (G)S ]
ee
2
(3.1.11)
Therefore the relationship between R and ee can be expressed

by combining Eqs. (3.1.5), (3.1.9), (3.1.10), and (3.1.11) to obtain Eq.
(3.1.12):
ln R =
ln(R ) + ln(R ) ln(R

R
S
chiral )
2
ee
(3.1.12)
Eq. (3.1.12) predicts a linear relationship between ln R and ee

based on the applicability of the kinetic method. It is worth to
pointing out that Eq. (3.1.12) gives physical meaning to the calibration curve of ln R against ee, in which the slope is equal to one
half of the natural logarithm of the chiral selectivity and the intercept is the average value of the natural logarithm of the branching
ratio for the pure R- and S-enantiomers. It is clear that the larger
the chiral selectivity, the larger the change in the measured ratio R
with unit change in ee, and hence the higher the accuracy of chiral
analysis. Since the clusters are chosen so that the comparative fragments generated by both diastereomeric ions contain the reference
ligand, this provides an internal reference so that the systems is calibrated and the measured ion abundance ratios have a predictable
(log abundance vs. ee) relationship.
3.1.3. Improvements in the kinetic method for chiral analysis
There are several important improvements in the kinetic
method for chiral analysis. The xed-ligand kinetic method was
developed by the introduction of a variant that replaces one of the
two identical reference ligands with an easily deprotonated compound having high metal afnity, thereafter called the xed ligand
(Lxed ) [4244]. Fragmentation will still occur via two channels,
the loss of the reference ligand and the analyte, but the strongly
chelated xed ligand will not be lost, as illustrated in Fig. 4. The
xed-ligand version of the kinetic method makes it easy to change
the properties of the xed ligand (e.g. size and functionality), to
optimize the chiral interactions resulting from metalligand and
This procedure starts with the formation of diastereomers; however, it is not the properties of the diastereomeric ions that are
examined. Instead, their formation is monitored simply by recording their relative abundances in a mass spectrometer. This method
uses simple single-stage MS to follow chiral-inclusion complex formation through hostguest (HG) interactions [5367]. Like any
single stage MS method it requires that the enantiomeric guest
(analyte) be isotopically labeled such that the HG diastereomeric
adduct can be mass-resolved and the ion abundance ratio can
be utilized for quantitative chiral analysis. This method is readily implemented using various ionization techniques such as fast
atom bombardment (FAB) [5463] and ESI [53,64,66]. However,
the method is best exploited when the examined samples are pure
because MS/MS is not performed. This method is either referred to
as the enantiomer labeled (EL)-host (Scheme 1a) or the enantiomer
labeled-guest method (Scheme 1b), given that either the host or
guest can be isotopically labeled. In practice, the EL-host method
rather than the EL-guest method is more conveniently employed.
Therefore, this review only covers the EL-host method.
3.2.1. Enantiomer labeled-host method
An equimolar mixture of HR and HS-dn (the chiral R and S forms)
is used as the host-pair reagent for ee determination of a chiral
guest (analyte). The host-pair reagent (HR /HS-dn = 1/1) is mixed
with the guest compound and is ionized via FAB-MS or ESI-MS.
Two diastereomeric HG complex ions simultaneously appear in
the single-stage MS spectrum. The ratio of the peak intensities,
called the IRIS value as termed in the original literature [54], reects
the degree of chiral discrimination as shown by Eq. (3.2.1).
I[(HR + G)+ ]
I[(HS-dn + G)+ ]
IR
= IRIS
IS-dn
(3.2.1)
This method focuses on the relative peak intensities of the

diastereomeric HG complex ions as a quantitative measure of chiral composition. The fundamental concept of the EL-host method is
illustrated in Fig. 5. The IRIS values change with the enantiomeric
contents of the guest, showing that the optically pure R-guest
forms a complex with the R-host by an arbitrary factor (e.g. Run 1,
IRIS = 2). Therefore, the optically pure S-guest should complex the
S-host twice as strongly as the R-host (Run 2, IRIS = 0.5) because
of the mirror image relationship during the HG complexation.
Furthermore, the racemic (RS)-guest (0% ee) should provide a pair
of equal peak intensities (Run 3, IRIS = 1) due to the net compensation of a racemic guest/racemic host combination in such
competitive systems. Accordingly, one can expect to determine
the %ee of an unknown analyte from the measured IRIS values.
The concentration ratio of the pre-formed diastereomeric ions in
the matrix is quantitatively or semi-quantitatively reected by the
corresponding relative peak intensity, at least as far as the diastereomeric HG complex ions are concerned. Although it is assumed
that the relative peak intensity reects the relative concentration of the diastereomeric HG complex ions in the matrix, some
140
Scheme 1. Guesthost diastereomeric ion formation for chiral recognition: (a) the host-labeled (EL-host) method; (b) the guest-labeled (EL-guest) method.
discrimination due to isotope effects during the ionization process

might need to be considered for the accurate ee determination.
3.2.2. Determination of ee by the EL-host method

When determining ee of an unknown mixture one must rst
begin by making appropriate standards of known ee from optically pure R and S guest forms. The IRIS values determined by
the EL-host method can then be used to deduce unknown ee
amounts. Since the IRIS values, based on the HG complexation, are
inuenced by the initial concentration of the host and the guest,
preparing equal guest concentrations is important. The EL-host
method was further improved by using double isotope-labeling
with the corresponding deuterium-labeled S- or R-enantiomer of
the guest as the internal standard. However, the isotope-labeled
guest compounds are not always easy to make and these additional
isotope-labeled internal references make the mass spectra more
complicated.
3.2.3. Types of host compounds used for the EL-host method

Since chiral recognition is strongly dependent on the interactions between a given host and a given guest, the size of the guest
and the host cavity must be appropriately matched when designing
host compounds for the purpose of differentiating different types
of chiral guests [140,141]. Cyclic crown ethers, carbohydrates, and
cyclodextrins represent typical host compounds chosen to complex the chiral guests (analytes) in the EL-host method [54]. More
recently, linear and exible gluco- and fructo-oligosaccharides as
well as cyclic antibiotics have been successfully utilized for chiral discrimination of amino acid derivatives by induced-t chiral
recognition [62].
Host
Run No.
3.3. Ion/molecule (equilibrium) reactions

Ion/molecule reactions of the hostguest complexes in the gas
phase have been explored for chiral recognition. Host molecules
such as cyclodextrins (CDs) and crown ethers can form the
hostguest (HG) complexes with a guest (a chiral analyte) via
electrospray or other soft ionization methods. These complex ions
can be mass-selected and characterized by ion/molecule reactions,
e.g. by guest-exchange with a neutral reagent B (either chiral or
non-chiral such as an amine) and the enantioselectivity is obtained
from the different rates of guest-exchanging reactions (Scheme 2)
[6875]. This particular reaction is readily performed in a trapping device such as a quadrupole ion trap [69] or a FT-ICR [70] for
an effective proton-transfer process mediated by a host molecule,
where the exchange rate strongly depends on the chirality of the
guest molecule.
Enantioselectivity, dened by the ratio (S) of the rate constants
kR and kS (kR and kS are the rate constants for the pure R- and Senantiomers, respectively) is shown in Eq. (3.3.1). An S value of
larger or smaller than 1.0 when using the pure enantiomers indicates the enantioselectivity for the HG system in question.
S=
kR
kS
(3.3.1)
This method requires that the host and the guest should form
reasonably stable complexes. It is the cooperative interactions of
several weak forces (including dipoledipole, hydrophobic, and
electrostatic interactions as well as van der Waals and hydrogen
bonding) that lead to chiral differentiation. While the three-point
interaction can be either attractive or repulsive depending on the
functional groups [28], the attractive interaction between a host
and a guest has to be reconciled with the increasing steric repulsion.
Guest
Pattern of MS spectrum
H-G complexes
I R / IS-dn
HR
HS - dn
(R)-100% ee
2.0
HR
HS - dn
(S)-100% ee
0.5
HR
HS - dn
1/1 racemic
1.0
dn
HR
HS - dn
unknown
Fig. 5. Illustration of the EL-host method for chiral analysis.
various
various
141
Scheme 2. Ion/molecule equilibrium reactions of the hostguest complexes.
In addition, the enantioselectivity is enhanced by congruence of the

overall size of the guest and the cavity size of the host. This is often
seen when enantiomers are forced to adopt specic conformations
to promote enantioselectivity.
Calibration curves are generated by directly comparing the relative peak heights using the samples with known values of ee. The
construction of a calibration plot normally involves establishing the
proper conditions for the ion/molecule exchange reaction, which
includes selecting a reagent, optimizing the pressure in the analyzer
chamber, and determining the appropriate reaction time. FT-ICR or
ion traps are typically used in this type of experiments. To construct a calibration curve, three or more analyte standard solutions
are prepared. The quality of the calibration curve is contingent on
the chiral selectivity value (S) which is subject to the selection of
the chiral host and the reagent, where both compounds interact
selectively with the analyte.
different gas-phase ion mobility through the spectrometer and can

be separated in time. In all cases, the addition of the chiral modier
to the drift gas reduced the mobility of both enantiomers; however,
the mobility of one enantiomer is always reduced more than that
of the other in a way that depends on their conformations, enabling
enantiomeric separation.
However, drift-tube IMS has poor sensitivity due to the low duty
cycle of pulse ion injection without ion accumulation. Recently,
several other emerging IMS techniques were developed and commercialized, including (1) high eld asymmetric waveform ion
mobility spectrometry-mass spectrometry (FAIMS) [132] and (2)
differential mobility spectrometry (DMS) in which ions pass
through the drift region at atmospheric pressure [133,134], as well
as (3) traveling-waveTM (T-wave) ion mobility in which ions pass
through the drift region at low pressure [135,136].
4.1. FAIMS technique
3.4. Collision-induced dissociation of diastereomeric complex ions

Diastereomers that are associated with different enantiomeric
forms of an analyte exhibit different stabilities in the gas phase.
As a result, CID of diastereomeric adducts results in differences
in fragmentation patterns which can be utilized to differentiate
chiral molecules [7678]. In principle this procedure is directly
analogous to solution reactions to generate diastereomers but the
method of interrogation of the products is different. Different types
of transition metal (Co2+ , Ni2+ , Cu2+ , and Zn2+ )-bound diastereomeric adducts of chiral compounds can be formed with chiral
auxiliaries (either pre-attached or added directly to the samples).
Analysis of the MS/MS spectra of different diastereomers leads to
the trends that allow the assignments of the absolute stereochemistry of certain functional groups in an unknown compound. The
peak intensity ratio of the product ion to the precursor ion (dissociation efciency) can be measured in a CID experiment and this
ratio depends on the enantiomeric content of an analyte. Therefore, quantitative chiral analysis seems possible, albeit it has not
been established.
4. Recent advances in ion mobility spectrometry for chiral
analysis
The FAIMS technique exploits the properties of gas-phase ions in

an oscillating eld between a high voltage of one polarity and a low
voltage of the opposite polarity at atmospheric pressure [132]. Ion
mobility changes non-linearly with the strength of the electric eld
and is dependent on the charge and collisional cross section of the
ions. The term asymmetric in FAIMS refers to the fact that the higheld portion of the waveform has a signicantly shorter duration
and greater magnitude than the low-eld portion of the opposite
polarity. As a result, a gas-phase ion migrates toward one electrode
during the high-eld portion of the waveform and toward the opposite electrode during the negative portion of the waveform, but the
distance traveled differs between the high-eld and low-eld portions depending on the ion mobility under the conditions within
the FAIMS unit. Oscillating in this fashion, only ions with certain
characteristics entering the FAIMS unit can avoid colliding with one
electrode or the other and actually exit to pass onto the mass analyzer. An additional parameter, a direct current potential referred
to as compensation voltage (CV) applied to the electrodes allows
only certain ions to traverse the gap between electrodes. In this
way, FAIMS acts as a selective mobility lter just as a quadrupole
mass analyzer acts as a mass-to-charge lter as illustrated in Fig. 6.
4.2. DMS technique
Besides the gas-phase chiral analysis methods described above,

IMS is one of the emerging techniques for the gas-phase chiral
separation [131136]. Chiral molecules in the gas phase can be
separated using conventional IMS as a result of differences in collision cross sections when ions travel through a chiral gas-lled drift
tube [131]. Selective interactions occur between the enantiomers
and the chiral modier such that the individual enantiomers have
The DMS technique is another form of ion mobility spectrometry that separates ions at ambient pressure [133,134]. In contrast
to FAIMS, the DMS cell consists of two parallel at plates (instead
of two concentric cylindrical electrodes in FAIMS) that are applied
with asymmetric radiofrequency (RF) voltages. Ions are separated
in trajectory based on differences in their mobility between the
142
Ion mobility electrode

Ion 1
Gas
Ion 2
Ion
source
Mass
Analyzer
Ion 3
Electrode
Voltage (kV)
Compensation Voltage (CV)
Fig. 6. Illustration of the FAIMS for separation of ions in the gas phase.
high-eld and low-eld portions of the RF waveforms. As the ions

migrate towards the DMS plates at different rates, they are separated. By applying the compensation voltage, the trajectory of the
desired ions is corrected along the axis of the cell and towards the
mass analyzer. In comparison with FAIMS, the DMS cell can be readily lled with a volatile reagent along with the gas ow to form
clusters. As the clustered ions move between the high-eld and
low-eld portions of the applied RF, they undergo clustering and
de-clustering. In the high-eld portion, the ions de-cluster due to
the higher energy available. However, in the low-eld portion, the
ions form the clusters again. This interaction dramatically increases
the separation capacity of the DMS cell, taking advantage of chemical properties to add another dimension in the differential mobility
effect.
4.3. Traveling-wave IMS
Traveling-wave (T-wave) is a unique IMS technique [135,136].
The drift cell is made of a series of ring electrodes that have an internal diameter of 7 mm and a center-to-center spacing of 1.5 mm.
Rather than a low electrical eld being applied uniformly across the
cell, a high electrical eld is swept sequentially from one electrode
to the next in the direction of ion migration. This electrical eld
separates the ions according to their mobility (Fig. 7). T-wave IMS
uses a pulsed method for ion injection, resulting in many ions being
discarded while waiting for previously injected ions to separate.
Practically, this duty cycle issue can be resolved by incorporating
an additional T-wave device that traps and transfers the ions with
minimal loss.
5. Pharmaceutical and biological applications
Mass spectrometric methods, including gas-phase ion/molecule
reactions and especially the kinetic method, have been successfully applied to chiral analysis of various types of drug compounds
[36,40,42,44,48,49,70]. This section deals specically with the
kinetic method applications, since the general trend has been established to use the kinetic method to develop chiral MS methods for
ee determination including high-throughput ee analyses for various
chiral drugs [49].
5.1. Enantiomeric determination of L-nucleoside analogs
An increasing number of unnatural L-nucleoside analogs are
being investigated as potent chemotherapeutic agents against
human immunodeciency virus (HIV) [142] and hepatitis B
virus (HBV) [143]. Clevudine (L-FMAU, 2 -uoro-5-methyl-l-arabinofuranosyluracil), for instance, was investigated as a
potent anti-HBV agent [144]. Its enantiomer, D-FMAU, exhibits
unacceptable toxicity in preclinical trials due to its incorporation

into cellular chromosomal and/or mitochondrial DNA [145].
Fig. 8 demonstrates that when the ee values of L-FMAU are varied
the measured branching ratio R values are affected. A relatively simple relationship between R and ee was established and this allowed
chiral mixtures to be rapidly determined by measuring the R values in the MS/MS experiments. A linear relationship between ln(R)
vs. ee was observed with a correlation coefcient (r2 ) of 0.9995,
using N-Acetyl-L-Pro as the reference and Co2+ as the central ion.
The calibration curve was then used to measure the ee values of
unknown samples. It is required that only one measurement in a
single MS/MS spectrum is recorded, nonetheless replicates were
made to increase the precision. An average accuracy of 0.6% ee
was obtained for this particular case. Besides its speed and high
sensitivity, which are the characteristics of most mass spectrometric approaches, this method displays several unique features
including accurate chiral quantication without requiring isotopic
labeling or wet chemical steps. The method is tolerant to matrix
interference and can be implemented using a variety of commercial
instruments.
5.2. Enantiomeric quantication of generic chiral drugs

Besides the chiral anti-viral agent mentioned in the previous section, the kinetic method has been successfully applied
to enantiomeric quantication of several generic drugs including penicillamine, DOPA, norepinephrine, ephedrine, pseudoephedrine, propranolol, isoproterenol, atenolol, and thalidomide
[36,42,44,48,70].
When using the xed-ligand kinetic method, chiral differentiation can be improved as demonstrated by chiral recognition of
R/S-penicillamine that gave rise to chiral selectivity of 4.88 [42].
More interestingly, there are predicted reciprocal relationships
when switching the chirality of the xed/reference ligands since
reversing the chirality of both xed ligand and reference ligand
reverses the sign of the energy difference between the two dimeric
product ions in the MS/MS experiments. This was conrmed by the
experimental results of chiral analysis of D/L-DOPA (Fig. 9) [44]. A
further advantage is to use single-point calibration with the ability
to obtain multiple calibration curves by chirality-switching of the
xed/reference ligands. The value of this option is that it allows one
to cross-check data.
5.3. Method development and validation for chiral purity

determination
Currently, the methods using HPLC with ultraviolet (LC-UV)
and circular dichroism (LC-CD) are being employed for chiral quantication of Flindokalner, a novel potassium channel
opener synthesized at Bristol-Myers Squibb Company (BMS)
[49]. A mass spectrometric kinetic method was developed for
quantitative chiral analysis of Flindokalner by choosing Li+ and (+)5-uorodeoxyuridine as the coordination ion and reference ligand,
respectively. Multiple reaction monitoring (MRM) was performed
on a triplequadrupole mass spectrometer to determine the changes
in the chiral purity of Flindokalner vs. the R values corresponding
to various chiral purity values of Flindokalner at 50.0, 65, 75, 85, 90,
95, and 100.0%. The inter- and intra-day validation procedures were
developed, demonstrating the chiral MS/MS method has comparable linearity, accuracy, and reproducibility to the LC-UV method.
In addition to accuracy and precision, the time for analysis is also
attractive with respect to 5-fold faster benchmarking the chiral
MS/MS method against the conventional methods by chiral LC-UV
and achiral LC-CD.
143
RF (+)
Ion exit
Ion entry
Ring
electrode
RF ()
Ions
Time
Traveling wave voltage pulse

Fig. 7. Illustration of the Traveling-wave IMS to separate ions in the gas phase.
Relative Abundance (%)
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
[CoII(N-Acetyl-L-Pro)2(FMAU) - H]+
632
(a) D-FMAU / L-FMAU = 1:0

(D-FMAU)
372
475 (N-Acetyl-L-Pro)
632
(b) D-FMAU / L-FMAU = 0.5:0.5

(D/L-FMAU)
372
475 (N-Acetyl-L-Pro)
632
(c) D-FMAU / L-FMAU = 0:1

(L-FMAU)
372
475
200
250
300
350
400
450
(N-Acetyl-L-Pro)
500
550
600
650
700
m/z
Fig. 8. MS/MS product ion spectra of [CoII (N-Acetyl-L-Pro)2 (FMAU) H]+ (m/z 632) for the mixtures of D- and L-FMAU in different proportions. Note that the gure was
re-drawn from ref. [40].
144
[Cu2+ + (D-Ala-L-Ala H)(L-Tyr)(D-DOPA)] +

100
80
600
[Cu2+ + (D-Ala-L-Ala H)(L-Tyr)(L-DOPA)] +

100
(a) R fixed = 1.31
80
60
60
40
20
Relative Abundance (%)
600
(b) R fixed = 5.38
403
0
300
419
400
(D-DOPA)
40
(L-Tyr)
20
600
0
300
700
HO
(L-DOPA)
419
500
L-DOPA
(L-Tyr)
NH2 OH
O
HO
403
400
500
600
700
Chirality
Switch
[Cu2+ + (L-Ala-D-Ala H)(D-Tyr)(D-DOPA)]+

100
80
600
100
(c) R fixed = 5.39
60
[Cu2+ + (L-Ala-D-Ala H)(D-Tyr)(L-DOPA)] +
80
0
300
HO
400
(D-Tyr)
500
600
20
0
300
700
NH2 OH
(L-DOPA)
40
419
403
D-DOPA
60
(D-DOPA)
40
20
600
(d) R fixed = 1.29
HO
403 419
400
(D-Tyr)
500
600
700
m/z
Fig. 9. Product ion (MS/MS) spectra of [(CuII + Ala-Ala H)(Tyr)(DOPA)]+ (m/z 600). Two product ions are [(Cu2+ + Ala-Ala H)(DOPA)]+ (m/z 419) and [(Cu2+ + AlaAla H)(Tyr)]+ (m/z 403). (a) Lxed = D-Ala-L-Ala, ref* = L-Tyr, An = D-DOPA; (b) Lxed = D-Ala-L-Ala, ref* = L-Tyr, An = L-DOPA; (c) Lxed = L-Ala-D-Ala, ref* = D-Tyr, An = D-DOPA;
(d) Lxed = L-Ala-D-Ala, ref* = D-Tyr, An = L-DOPA.
Note that the gure was re-drawn from Ref. [44].
5.4. On-line LC-MS/MS chiral analysis by the kinetic method

In practice, enantiomeric quantication solely based on MS can
potentially be challenging from an experimental viewpoint. For
instance, when analyzing real chiral samples the analyte of interest is generally mixed within a complex matrix. The complexity
of the matrix can vary depending on the type of samples being
analyzed, e.g. a matrix can consist of blood, urine, feces or excipients that are formulated with drug substances. Dirty samples can
increase the difculty of any analytical method, e.g. by reducing
sensitivity and selectivity of the methods. Therefore, many analytical procedures require some sample clean up prior to the analysis.
In the case of MS, dirty samples can potentially suppress ion formation and even make peak identication impossible.
A new on-line method involves preliminary reversed-phase
HPLC separation of the chiral analyte from its matrix, in conjunction
with the kinetic method, for the purpose of chiral discriminations
[146]. This has been demonstrated using a commercial linear ion
trap mass spectrometer equipped with an ESI source, where the
trimeric cluster ion was generated by the post-column addition
of a reference ligand and a metal ion after the chiral analyte was
chromatographically separated from its matrix. The chiral selectivity and reproducibility obtained by the data-dependent LC-MS/MS
is similar to the results reported using direct infusion of the pure
enantiomer pairs, corroborating that the on-line LC-MS/MS method
can be used for chiral mixture analysis as well as for high throughput screening of chiral purity. This study showed for the rst time an
on-line gas-phase chiral analysis method utilizing data-dependent
MS/MS.
5.5. Gas-phase chiral separation by ion mobility spectrometry
The concept of chiral ion mobility spectrometry has been
demonstrated to be a promising tool for separation of chiral drugs
such as R/S-atenolol when using (S)-(+)-2-butanol as the chiral

modier [131]. The addition of a chiral modier to the drift gas
reduced the mobility of both enantiomers, but the mobility of
one enantiomer was always reduced more than that of the other,
enabling enantiomeric separation. Further investigation of separation mechanisms and optimization of operating conditions should
result in improving ion mobility resolution [132136]. The data
from these experiments are quite encouraging for the development of novel and rapid analytical methods using different types
of IMS techniques (e.g. FAIMS, DMS, and Traveling-Wave) for the
enantiomeric analysis of chiral drugs in the near future.
6. Concluding remarks and future perspectives
The latest quantitative ee determination methods using MS
have been summarized. The characteristic systems resolve chiral
compounds by single-stage MS methods (e.g. the HG singlestage complexation) and MS/MS methods (e.g. the ion/molecule
equilibrium reactions and the kinetic method). These gas-phase
chiral analysis methods demonstrate a collection of research efforts
spearheaded by many scientists who are interested in expanding the versatility of MS as a tool for chiral quantication, despite
being initially perceived as a chirally blind technique. Albeit the
mass spectrometric methods are still in their infancy as a chirally
resolving tool, there is the potential for this technique to rival the
existing solution-phase methods (e.g. chiral chromatography) for
enantiomeric quantitation.
MS provides a rapid and sensitive means for chiral analysis.
In a clean gas-phase environment, enantio-discrimination mechanisms are easy to understand, the experimental conditions are easy
to optimize and chiral recognition can be readily improved. With
the rapid development of ionization methods and novel instrumentation (offering high mass resolution and mass measurement
accuracy), MS has blossomed into an important technique for chiral
analysis, especially in fast combinatorial screening settings during

drug discovery and clinical diagnostic study [147]. In the future,
developing robust mass spectrometric methods for fast chiral analysis is expected to make great impact on the QbD-based drug
development and manufacturing by accelerating method development and facilitating evaluation of the design space for quality
control.
Although MS has shown promising features for rapid chiral
analysis, several aspects still need improving in order for MS to
become a reliable tool for routine enantiomeric quantitation. Further improvements include lower limits of quantitation, higher
accuracy, increased reproducibility, and reduced ion suppression
of matrix effects during the ionization process. Some experiments have demonstrated that high accuracy and precision can be
obtained but further advances must be made [49]. Since all mass
spectrometric methods share the same feature of using a chiral reference ligand to create an asymmetric environment, the selection
and/or optimization of reference ligands, even the development of
new reference ligands, becomes more important when developing
a more accurate method. Chiral analysis can be further facilitated
by incorporating the concept of chiral morphing, a novel technique
that allows systematic changes in chirality for the purpose of optimizing the properties of a given ligand. For a drug agent, chiral
morphing allows utilization of the spatial diversity of multiple chiral centers to produce drug candidates with improved efciency,
stability, membrane permeability, and oral availability, as well as
decreased toxicity and side effects [148]. The xed-ligand version of
the kinetic method has been incorporated with the chiral morphing
concept (by changing the size and functionality of the xed ligand) to further rene the chiral interactions and hence to maximize
chiral recognition [44].
Lastly, the methodology for gas-phase chiral analysis has the
potential to be extended into many elds. The three-point interaction used for gas-phase chiral recognition is useful in the
exploration of stereoselective ion channel-chiral drug interactions
[149]. Such interactions might give rise to quantitative differences
in stereoselectivity which can be utilized to better understand different pharmacological effects of chiral drugs in different forms of
enantiomers.
Acknowledgment
The authors would like to thank Dr. Gerald J. Teroth and Dr.
Tom D. Roper of GlaxoSmithKline for the critical review of the
manuscript.
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Journal of Pharmaceutical and Biomedical Analysis 69 (2012) 133147

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

A review of recent advances in mass spectrometric methods for gas-phase chiral

Lipitor (Pzer Inc)

Single enantiomer + racemate

Singulair (Merck & Co.,

Cymbalta (Eli Lilly and

Information was obtained from webpage: http://www.drugs.com/top200.html.

Drug Binding Site

Drug Binding Site

Drug Binding Site

is different from the SR (S-selector and R-enantiomer) interaction,

3. Advances in gas-phase mass spectrometric methods for

way to perform MS/MS experiments. Quadrupole ion trap [111]

3.1. The kinetic method formalism

The kinetic method [137] can be used to recognize and quantify

3.1.1. Dissociation of cluster ions using the kinetic method

In this equation, k1 and k2 represent the rate constants for two

Separation of enantiomers was further improved by observing the

When the analyte is enantiomerically pure, R becomes RR or RS .

[M(An)(ref )2 H] [M(An)(ref ) H] + ref

3.2. Hostguest diastereomeric adduct formation

When the analyte consists of a pure R or S-enantiomer, (G)

For an enantiomeric mixture with an enantiomeric excess of the

Therefore the relationship between R and ee can be expressed

ln(R ) + ln(R )  ln(R

Eq. (3.1.12) predicts a linear relationship between ln R and ee

This method focuses on the relative peak intensities of the

discrimination due to isotope effects during the ionization process

3.2.2. Determination of ee by the EL-host method

3.2.3. Types of host compounds used for the EL-host method

3.3. Ion/molecule (equilibrium) reactions

Fig. 5. Illustration of the EL-host method for chiral analysis.

Scheme 2. Ion/molecule equilibrium reactions of the hostguest complexes.

In addition, the enantioselectivity is enhanced by congruence of the

different gas-phase ion mobility through the spectrometer and can

3.4. Collision-induced dissociation of diastereomeric complex ions

The FAIMS technique exploits the properties of gas-phase ions in

Besides the gas-phase chiral analysis methods described above,

Ion mobility electrode

Compensation Voltage (CV)

high-eld and low-eld portions of the RF waveforms. As the ions

unacceptable toxicity in preclinical trials due to its incorporation

5.2. Enantiomeric quantication of generic chiral drugs

5.3. Method development and validation for chiral purity

Traveling wave voltage pulse

Relative Abundance (%)

(a) D-FMAU / L-FMAU = 1:0

(b) D-FMAU / L-FMAU = 0.5:0.5

(c) D-FMAU / L-FMAU = 0:1

[Cu2+ + (D-Ala-L-Ala H)(L-Tyr)(D-DOPA)] +

[Cu2+ + (D-Ala-L-Ala H)(L-Tyr)(L-DOPA)] +

(a) R fixed = 1.31

Relative Abundance (%)

(b) R fixed = 5.38

[Cu2+ + (L-Ala-D-Ala H)(D-Tyr)(D-DOPA)]+

(c) R fixed = 5.39

[Cu2+ + (L-Ala-D-Ala H)(D-Tyr)(L-DOPA)] +

(d) R fixed = 1.29

5.4. On-line LC-MS/MS chiral analysis by the kinetic method

such as R/S-atenolol when using (S)-(+)-2-butanol as the chiral

analysis, especially in fast combinatorial screening settings during

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ln(R ) + ln(R ) ln(R